AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis

PubMed Central

Delevoye, Cédric; Hurbain, Ilse; Tenza, Danièle; Sibarita, Jean-Baptiste; Uzan-Gafsou, Stéphanie; Ohno, Hiroshi; Geerts, Willie J.C.; Verkleij, Arie J.; Salamero, Jean; Marks, Michael S.

2009-01-01

Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type–specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1– and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type–specific positioning of endosomes that facilitate endosome–LRO contacts and are required for organelle maturation. PMID:19841138

Endocytosis and Endosomal Trafficking in Plants.

PubMed

Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

2016-04-29

Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants.

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

PubMed Central

Phillips-Krawczak, Christine A.; Singla, Amika; Starokadomskyy, Petro; Deng, Zhihui; Osborne, Douglas G.; Li, Haiying; Dick, Christopher J.; Gomez, Timothy S.; Koenecke, Megan; Zhang, Jin-San; Dai, Haiming; Sifuentes-Dominguez, Luis F.; Geng, Linda N.; Kaufmann, Scott H.; Hein, Marco Y.; Wallis, Mathew; McGaughran, Julie; Gecz, Jozef; van de Sluis, Bart; Billadeau, Daniel D.; Burstein, Ezra

2015-01-01

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling. PMID:25355947

Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Padilla, Benjamin E; Muller, Laurent; Eckman, Christopher B; Bunnett, Nigel W; Steinhoff, Martin

2007-07-10

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with beta-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of beta-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide-receptor-arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of beta-arrestins followed by trafficking of the SP-NK1R-beta-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; beta-arrestins returned to the cytosol, and the NK1R, freed from beta-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide-receptor-arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control beta-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Regulation of vesicular traffic at the T cell immune synapse: lessons from the primary cilium.

PubMed

Finetti, Francesca; Onnis, Anna; Baldari, Cosima T

2015-03-01

The signals that orchestrate the process of T cell activation are coordinated at the specialized interface that forms upon contact with an antigen presenting cell displaying a specific MHC-associated peptide ligand, known as the immune synapse. The central role of vesicular traffic in the assembly of the immune synapse has emerged only in recent years with the finding that sustained T-cell receptor (TCR) signaling involves delivery of TCR/CD3 complexes from an intracellular pool associated with recycling endosomes. A number of receptors as well as membrane-associated signaling mediators have since been demonstrated to exploit this process to localize to the immune synapse. Here, we will review our current understanding of the mechanisms responsible for TCR recycling, with a focus on the intraflagellar transport system, a multimolecular complex that is responsible for the assembly and function of the primary cilium which we have recently implicated in polarized endosome recycling to the immune synapse. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila.

PubMed

Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

2016-06-02

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.

Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

PubMed Central

Liu, Ou; Grant, Barth D.

2015-01-01

The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity*

PubMed Central

van der Kant, Rik; Jonker, Caspar T. H.; Wijdeven, Ruud H.; Bakker, Jeroen; Janssen, Lennert; Klumperman, Judith; Neefjes, Jacques

2015-01-01

Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway. PMID:26463206

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

PubMed

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-05-18

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

PubMed

Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila

PubMed Central

Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

2016-01-01

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.14226.001 PMID:27253064

Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

PubMed Central

Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

2012-01-01

Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes. PMID:22825447

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

PubMed Central

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-01-01

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

PubMed Central

Lee, Seongju; Chang, Jaerak; Blackstone, Craig

2016-01-01

The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27–retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27–retromer–WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi. PMID:26956659

FAM21 directs SNX27-retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus.

PubMed

Lee, Seongju; Chang, Jaerak; Blackstone, Craig

2016-03-09

The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27-retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27-retromer-WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi.

Parkinson Disease-linked Vps35 R524W Mutation Impairs the Endosomal Association of Retromer and Induces α-Synuclein Aggregation.

PubMed

Follett, Jordan; Bugarcic, Andrea; Yang, Zhe; Ariotti, Nicholas; Norwood, Suzanne J; Collins, Brett M; Parton, Robert G; Teasdale, Rohan D

2016-08-26

Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia.

PubMed

Khanal, Ichha; Elbediwy, Ahmed; Diaz de la Loza, Maria Del Carmen; Fletcher, Georgina C; Thompson, Barry J

2016-07-01

In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli. © 2016. Published by The Company of Biologists Ltd.

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling.

PubMed

Bai, Zhiyong; Grant, Barth D

2015-03-24

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1-positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42-associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

PubMed Central

Bai, Zhiyong; Grant, Barth D.

2015-01-01

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling. PMID:25775511

Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons.

PubMed

Cai, Qian; Lu, Li; Tian, Jin-Hua; Zhu, Yi-Bing; Qiao, Haifa; Sheng, Zu-Hang

2010-10-06

Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis. Copyright © 2010 Elsevier Inc. All rights reserved.

RhoB-dependent modulation of postendocytic traffic in polarized Madin-Darby canine kidney cells.

PubMed

Rondanino, Christine; Rojas, Raul; Ruiz, Wily G; Wang, Exing; Hughey, Rebecca P; Dunn, Kenneth W; Apodaca, Gerard

2007-07-01

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein-tagged wild-type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin-Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant-negative RhoBN19 or dominant-active RhoBV14 altered postendocytic traffic of ligand-receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral-to-apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.

Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK*

PubMed Central

Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A.

2014-01-01

β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET178P), but not rat β-arrestin-1 (PER177P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK178P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species. PMID:25016018

Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

PubMed Central

Meister, M; Bänfer, S; Gärtner, U; Koskimies, J; Amaddii, M; Jacob, R; Tikkanen, R

2017-01-01

Ubiquitin-dependent sorting of membrane proteins in endosomes directs them to lysosomal degradation. In the case of receptors such as the epidermal growth factor receptor (EGFR), lysosomal degradation is important for the regulation of downstream signalling. Ubiquitinated proteins are recognised in endosomes by the endosomal sorting complexes required for transport (ESCRT) complexes, which sequentially interact with the ubiquitinated cargo. Although the role of each ESCRT complex in sorting is well established, it is not clear how the cargo is passed on from one ESCRT to the next. We here show that flotillin-1 is required for EGFR degradation, and that it interacts with the subunits of ESCRT-0 and -I complexes (hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Tsg101). Flotillin-1 is required for cargo recognition and sorting by ESCRT-0/Hrs and for its interaction with Tsg101. In addition, flotillin-1 is also required for the sorting of human immunodeficiency virus 1 Gag polyprotein, which mimics ESCRT-0 complex during viral assembly. We propose that flotillin-1 functions in cargo transfer between ESCRT-0 and -I complexes. PMID:28581508

Aggregation of endosomal-vacuolar compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tatsumi, Akinori; Shoji, Jun-ya; Kikuma, Takashi

2007-10-19

Previously, we found that deletion of Aovps24, an ortholog of Saccharomyces cerevisiae VPS24, that encodes an ESCRT (endosomal sorting complex required for transport)-III component required for late endosomal function results in fragmented and aggregated vacuoles. Although defective late endosomal function is likely responsible for this phenotype, critical lack of our knowledge on late endosomes in filamentous fungi prevented us from further characterization. In this study, we identified late endosomes of Aspergillus oryzae, by expressing a series of fusion proteins of fluorescent proteins with orthologs of late endosomal proteins. Using these fusion proteins as markers, we observed late endosomes in themore » wild type strain and the Aovps24 disruptant and demonstrated that late endosomes are aberrantly aggregated in the Aovps24 disruptant. Moreover, we revealed that the aggregated late endosomes have features of vacuoles as well. As deletion of another ESCRT-III component-encoding gene, Aovps2, resulted in similar phenotypes to that in the Aovps24 disruptant, phenotypes of the Aovps24 disruptant are probably due to defective late endosomal function.« less

Erythroid cell mitochondria receive endosomal iron by a "kiss-and-run" mechanism.

PubMed

Hamdi, Amel; Roshan, Tariq M; Kahawita, Tanya M; Mason, Anne B; Sheftel, Alex D; Ponka, Prem

2016-12-01

In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe 2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59 Fe by reticulocytes and diminishes 59 Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles. Copyright © 2016 Elsevier B.V. All rights reserved.

Determinants of [Cl−] in recycling and late endosomes and Golgi complex measured using fluorescent ligands

PubMed Central

Sonawane, N.D.; Verkman, A.S.

2003-01-01

Chloride concentration ([Cl−]) was measured in defined organellar compartments using fluorescently labeled transferrin, α2-macroglobulin, and cholera toxin B-subunit conjugated with Cl−-sensitive and -insensitive dyes. In pulse-chase experiments, [Cl−] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over ∼10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl−] just after internalization (compared with 137 mM solution [Cl−]) was prevented by reducing the interior-negative Donnan potential. [Cl−] in α2-macroglobulin–labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1–45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl− accumulation was prevented by bafilomycin but restored by valinomycin. A Cl− channel inhibitor slowed endosomal acidification and Cl− accumulation by ∼2.5-fold. [Cl−] was 49 mM and pH was 6.42 in cholera toxin B subunit–labeled Golgi complex in Vero cells; Golgi compartment Cl− accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl− is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl−] early after internalization. We propose that reduced [Cl−] and volume in early endosomes permits endosomal acidification and [Cl−] accumulation without lysis. PMID:12668661

A biomimetic pH-responsive polymer directs endosomal release and intracellular delivery of an endocytosed antibody complex.

PubMed

Lackey, Chantal A; Press, Oliver W; Hoffman, Allan S; Stayton, Patrick S

2002-01-01

Poly(propylacrylic acid) (PPAAc) is a synthetic pH-responsive polymer that has been shown to disrupt cell membranes at low pH values typical of the endosome, but not at physiological pH, suggesting its use as an endosomal-releasing agent [Murthy et al. J. Controlled Release 61, 137-43]. We have constructed an antibody-targeted biotherapeutic model to investigate whether PPAAc can enhance intracellular trafficking of proteins to the cytoplasm. A ternary complex composed of a biotinylated anti-CD3 antibody, streptavidin, and biotinylated PPAAc was fluorescently labeled, and its intracellular fate was analyzed by confocal microscopy, flow cytometry, and quantitative western blotting of cell fractionates. The 64.1 anti-CD3 antibody was previously shown to direct receptor-mediated endocytosis in the Jurkat T-cell lymphoma cell line and was rapidly trafficked from the endosome to the lysosomal compartment. The antibody-streptavidin complex was also rapidly internalized to the endosomal/lysosomal compartment and retained there, as evidenced by punctate regions of fluorescence observed by confocal fluorescence microscopy. In samples containing the ternary complex of antibody, streptavidin, and PPAAc-biotin, diffuse fluorescence in the cytoplasm was observed, indicating that PPAAc enhanced translocation to the cytoplasm. This was confirmed by western blotting analysis of the isolated cytoplasm. Flow cytometry results demonstrated that neither streptavidin nor PPAAc caused nonspecific uptake of the complex, nor did they inhibit antibody-mediated endocytosis. The striking enhancement of protein delivery to the cytoplasm by complexed PPAAc suggests that this polymer could provide a new delivery agent for therapeutic, vaccine, and diagnostics development.

BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia

PubMed Central

2017-01-01

Primary cilia perceive the extracellular environment through receptors localized in the ciliary membrane, but mechanisms directing specific proteins to this domain are poorly understood. To address this question, we knocked down proteins potentially important for ciliary membrane targeting and determined how this affects the ciliary trafficking of fibrocystin, polycystin-2, and smoothened. Our analysis showed that fibrocystin and polycystin-2 are dependent on IFT20, GMAP210, and the exocyst complex, while smoothened delivery is largely independent of these components. In addition, we found that polycystin-2, but not smoothened or fibrocystin, requires the biogenesis of lysosome-related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with the role of BLOC-1 in sorting from the endosome, we find that disrupting the recycling endosome reduces ciliary polycystin-2 and causes its accumulation in the recycling endosome. This is the first demonstration of a role for BLOC-1 in ciliary assembly and highlights the complexity of pathways taken to the cilium. PMID:28576874

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

PubMed Central

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. PMID:26041286

The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin

PubMed Central

Arnold, Miranda; Cross, Rebecca; Singleton, Kaela S.; Zlatic, Stephanie; Chapleau, Christopher; Mullin, Ariana P.; Rolle, Isaiah; Moore, Carlene C.; Theibert, Anne; Pozzo-Miller, Lucas; Faundez, Victor; Larimore, Jennifer

2016-01-01

AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function. PMID:27713690

Integration of two RAB5 groups during endosomal transport in plants

PubMed Central

Ebine, Kazuo; Choi, Seung-won; Ichinose, Sakura; Uemura, Tomohiro; Nakano, Akihiko

2018-01-01

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells. PMID:29749929

DNA internalized via caveolae requires microtubule-dependent, Rab7-independent transport to the late endocytic pathway for delivery to the nucleus.

PubMed

Wong, Athena W; Scales, Suzie J; Reilly, Dorothea E

2007-08-03

Using cationic liposomes to mediate gene delivery by transfection has the advantages of improved safety and simplicity of use over viral gene therapy. Understanding the mechanism by which cationic liposome:DNA complexes are internalized and delivered to the nucleus should help identify which transport steps might be manipulated in order to improve transfection efficiencies. We therefore examined the endocytosis and trafficking of two cationic liposomes, DMRIE-C and Lipofectamine LTX, in CHO cells. We found that DMRIE-C-transfected DNA is internalized via caveolae, while LTX-transfected DNA is internalized by clathrin-mediated endocytosis, with both pathways converging at the late endosome or lysosome. Inhibition of microtubule-dependent transport with nocodazole revealed that DMRIE-C:DNA complexes cannot enter the cytosol directly from caveosomes. Lysosomal degradation of transfected DNA has been proposed to be a major reason for poor transfection efficiency. However, in our system dominant negatives of both Rab7 and its effector RILP inhibited late endosome to lysosome transport of DNA complexes and LDL, but did not affect DNA delivery to the nucleus. This suggests that DNA is able to escape from late endosomes without traversing lysosomes and that caveosome to late endosome transport does not require Rab7 function. Lysosomal inhibition with chloroquine likewise had no effect on transfection product titers. These data suggest that DMRIE-C and LTX transfection complexes are endocytosed by separate pathways that converge at the late endosome or lysosome, but that blocking lysosomal traffic does not improve transfection product yields, identifying late endosome/lysosome to nuclear delivery as a step for future study.

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network.

PubMed

Raub, T J; Koroly, M J; Roberts, R M

1990-04-01

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

PubMed

Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

2018-03-01

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein, LMP1, into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here, we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate a key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insights into the complex intersection of cellular secretory and degradative mechanisms and the implications of these processes in viral replication. Copyright © 2018 American Society for Microbiology.

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

PubMed Central

McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

2016-01-01

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

PubMed Central

Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

2006-01-01

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

Structural Basis for Endosomal Targeting by the Bro1 Domain

PubMed Central

Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M.; Odorizzi, Greg; Hurley, James H.

2010-01-01

Summary Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. PMID:15935782

Endosomal-sorting complexes required for transport (ESCRT) pathway-dependent endosomal traffic regulates the localization of active Src at focal adhesions.

PubMed

Tu, Chun; Ortega-Cava, Cesar F; Winograd, Paul; Stanton, Marissa Jo; Reddi, Alagarsamy Lakku; Dodge, Ingrid; Arya, Ranjana; Dimri, Manjari; Clubb, Robert J; Naramura, Mayumi; Wagner, Kay-Uwe; Band, Vimla; Band, Hamid

2010-09-14

Active Src localization at focal adhesions (FAs) is essential for cell migration. How this pool is linked mechanistically to the large pool of Src at late endosomes (LEs)/lysosomes (LY) is not well understood. Here, we used inducible Tsg101 gene deletion, TSG101 knockdown, and dominant-negative VPS4 expression to demonstrate that the localization of activated cellular Src and viral Src at FAs requires the endosomal-sorting complexes required for transport (ESCRT) pathway. Tsg101 deletion also led to impaired Src-dependent activation of STAT3 and focal adhesion kinase and reduced cell migration. Impairment of the ESCRT pathway or Rab7 function led to the accumulation of active Src at aberrant LE/LY compartments followed by its loss. Analyses using fluorescence recovery after photo-bleaching show that dynamic mobility of Src in endosomes is ESCRT pathway-dependent. These results reveal a critical role for an ESCRT pathway-dependent LE/LY trafficking step in Src function by promoting localization of active Src to FAs.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport

PubMed Central

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-01-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. PMID:25657322

Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to MelanosomesD⃞

PubMed Central

Theos, Alexander C.; Tenza, Danièle; Martina, José A.; Hurbain, Ilse; Peden, Andrew A.; Sviderskaya, Elena V.; Stewart, Abigail; Robinson, Margaret S.; Bennett, Dorothy C.; Cutler, Daniel F.; Bonifacino, Juan S.; Marks, Michael S.; Raposo, Graça

2005-01-01

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies. PMID:16162817

Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes.

PubMed

Sonawane, N D; Szoka, Francis C; Verkman, A S

2003-11-07

The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.

Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

PubMed Central

Ehrlich, Kenneth C.; Mack, Brian M.; Wei, Qijian; Li, Ping; Roze, Ludmila V.; Dazzo, Frank; Cary, Jeffrey W.; Bhatnagar, Deepak; Linz, John E.

2012-01-01

Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export. PMID:23342682

The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis.

PubMed

Zheng, Xiudan; Zhang, Jing; Liao, Kan

2014-07-08

During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.

PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

PubMed Central

Hong, Nan Hyung; Qi, Aidong

2015-01-01

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

Vps33B is required for delivery of endocytosed cargo to lysosomes.

PubMed

Galmes, Romain; ten Brink, Corlinda; Oorschot, Viola; Veenendaal, Tineke; Jonker, Caspar; van der Sluijs, Peter; Klumperman, Judith

2015-12-01

Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA-gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal-lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal-lysosomal fusion events. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

PubMed

Edler, Eileen; Stein, Matthias

2017-10-25

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

NASA Astrophysics Data System (ADS)

Matsuda, Shinji; Kakegawa, Wataru; Budisantoso, Timotheus; Nomura, Toshihiro; Kohda, Kazuhisa; Yuzaki, Michisuke

2013-11-01

Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin’s interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors—transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes—through its sequential binding to AP-2 and AP-3A.

Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

PubMed Central

Lladó, Anna; Timpson, Paul; Vilà de Muga, Sandra; Moretó, Jemina; Pol, Albert; Grewal, Thomas; Daly, Roger J.

2008-01-01

The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase Cδ (PKCδ). On inhibition of CaM, PKCδ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCδ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCδ-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCδ. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCδ, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCδ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR. PMID:17959830

Retriever, a multiprotein complex for retromer-independent endosomal cargo recycling

PubMed Central

McNally, Kerrie E.; Faulkner, Rebecca; Steinberg, Florian; Gallon, Matthew; Ghai, Rajesh; Pim, David; Langton, Paul; Pearson, Neil; Danson, Chris M.; Nägele, Heike; Morris, Lindsey M; Singla, Arnika; Overlee, Brittany L; Heesom, Kate J.; Sessions, Richard; Banks, Lawrence; Collins, Brett M; Berger, Imre; Billadeau, Daniel D.; Burstein, Ezra; Cullen, Peter J.

2018-01-01

Following endocytosis and entry into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are alternatively retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multi-protein complex which orchestrates cargo retrieval and recycling and importantly, is biochemically and functionally distinct to the established retromer pathway. Composed of a heterotrimer of DSCR3, C16orf62 and VPS29, and bearing striking similarity with retromer, we have called this complex ‘retriever’. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to the CCC and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1-integrin. Through quantitative proteomic analysis we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, which require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major new endosomal retrieval and recycling pathway. PMID:28892079

Retrogradely Transported TrkA Endosomes Signal Locally within Dendrites to Maintain Sympathetic Neuron Synapses.

PubMed

Lehigh, Kathryn M; West, Katherine M; Ginty, David D

2017-04-04

Sympathetic neurons require NGF from their target fields for survival, axonal target innervation, dendritic growth and formation, and maintenance of synaptic inputs from preganglionic neurons. Target-derived NGF signals are propagated retrogradely, from distal axons to somata of sympathetic neurons via TrkA signaling endosomes. We report that a subset of TrkA endosomes that are transported from distal axons to cell bodies translocate into dendrites, where they are signaling competent and move bidirectionally, in close proximity to synaptic protein clusters. Using a strategy for spatially confined inhibition of TrkA kinase activity, we found that distal-axon-derived TrkA signaling endosomes are necessary within sympathetic neuron dendrites for maintenance of synapses. Thus, TrkA signaling endosomes have unique functions in different cellular compartments. Moreover, target-derived NGF mediates circuit formation and synapse maintenance through TrkA endosome signaling within dendrites to promote aggregation of postsynaptic protein complexes. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

PubMed

Campagne, Cécile; Ripoll, Léa; Gilles-Marsens, Floriane; Raposo, Graça; Delevoye, Cédric

2018-02-14

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis

PubMed Central

Campagne, Cécile; Ripoll, Léa; Gilles-Marsens, Floriane; Raposo, Graça

2018-01-01

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease. PMID:29443872

EGF receptor lysosomal degradation is delayed in the cells stimulated with EGF-Quantum dot bioconjugate but earlier key events of endocytic degradative pathway are similar to that of native EGF

PubMed Central

Leontieva, Ekaterina A.; Kornilova, Elena S.

2017-01-01

Quantum dots (QDs) complexed to ligands recognizing surface receptors undergoing internalization are an attractive tool for live cell imaging of ligand-receptor complexes behavior and for specific tracking of the cells of interest. However, conjugation of quasi-multivalent large QD-particle to monovalent small growth factors like EGF that bound their tyrosine-kinase receptors may affect key endocytic events tightly bound to signaling. Here, by means of confocal microscopy we have addressed the key endocytic events of lysosomal degradative pathway stimulated by native EGF or EGF-QD bioconjugate. We have demonstrated that the decrease in endosome number, increase in mean endosome integrated density and the pattern of EEA1 co-localization with EGF-EGFR complexes at early stages of endocytosis were similar for the both native and QD-conjugated ligands. In both cases enlarged hollow endosomes appeared after wortmannin treatment. This indicates that early endosomal fusions and their maturation proceed similar for both ligands. EGF-QD and native EGF similarly accumulated in juxtanuclear region, and live cell imaging of endosome motion revealed the behavior described elsewhere for microtubule-facilitated motility. Finally, EGF-QD and the receptor were found in lysosomes. However, degradation of receptor part of QD-EGF-EGFR-complex was delayed compared to native EGF, but not inhibited, while QDs fluorescence was detected in lysosomes even after 24 hours. Importantly, in HeLa and A549 cells the both ligands behaved similarly. We conclude that during endocytosis EGF-QD behaves as a neutral marker for degradative pathway up to lysosomal stage and can also be used as a long-term cell marker. PMID:28574831

The Siderocalin/Enterobactin Interaction: A Link between Mammalian Immunity and Bacterial Iron Transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meux, Susan C.

2008-05-12

The siderophore enterobactin (Ent) is produced by enteric bacteria to mediate iron uptake. Ent scavenges iron and is taken up by the bacteria as the highly stable ferric complex [Fe{sup III}(Ent)]{sup 3-}. This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an anti-bacterial agent by specifically sequestering siderophores and their ferric complexes during infection. Recent literature suggesting that Scn may also be involved in cellular iron transport has increased the importance of understanding the mechanism of siderophore interception and clearance by Scn; Scn is observed to release iron in acidicmore » endosomes and [Fe{sup III}(Ent)]{sup 3-} is known to undergo a change from catecholate to salicylate coordination in acidic conditions, which is predicted to be sterically incompatible with the Scn binding pocket (also referred to as the calyx). To investigate the interactions between the ferric Ent complex and Scn at different pH values, two recombinant forms of Scn with mutations in three residues lining the calyx were prepared: Scn-W79A/R81A and Scn-Y106F. Binding studies and crystal structures of the Scn-W79A/R81A:[Fe{sup III}(Ent)]{sup 3-} and Scn-Y106F:[Fe{sup III}(Ent)]{sup 3-} complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [Fe{sup III}(Ent)]{sup 3-}. Fluorescence, UV-Vis and EXAFS spectroscopies were used to determine Scn/siderophore dissociation constants and to characterize the coordination mode of iron over a wide pH range, in the presence of both mutant proteins and synthetic salicylate analogs of Ent. While Scn binding hinders salicylate coordination transformation, strong acidification results in the release of iron and degraded siderophore. Iron release may therefore result from a combination of Ent degradation and coordination change.« less

The Siderocalin/Enterobactin Interaction: a Link Between Mammalian Immunity And Bacterial Iron Transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abergel, R.J.; Clifton, M.C.; Pizarro, J.C.

2009-05-12

The siderophore enterobactin (Ent) is produced by enteric bacteria to mediate iron uptake. Ent scavenges iron and is taken up by the bacteria as the highly stable ferric complex [Fe{sup III}(Ent)]{sup 3-}. This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an antibacterial agent by specifically sequestering siderophores and their ferric complexes during infection. Recent literature suggesting that Scn may also be involved in cellular iron transport has increased the importance of understanding the mechanism of siderophore interception and clearance by Scn; Scn is observed to release iron in acidicmore » endosomes and [Fe{sup III}(Ent)]{sup 3-} is known to undergo a change from catecholate to salicylate coordination in acidic conditions, which is predicted to be sterically incompatible with the Scn binding pocket (also referred to as the calyx). To investigate the interactions between the ferric Ent complex and Scn at different pH values, two recombinant forms of Scn with mutations in three residues lining the calyx were prepared: Scn-W79A/R81A and Scn-Y106F. Binding studies and crystal structures of the Scn-W79A/R81A:[Fe{sup III}(Ent)]{sup 3-} and Scn-Y106F:[Fe{sup III}(Ent)]{sup 3-} complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [Fe{sup III}(Ent)]{sup 3-}. Fluorescence, UV-vis, and EXAFS spectroscopies were used to determine Scn/siderophore dissociation constants and to characterize the coordination mode of iron over a wide pH range, in the presence of both mutant proteins and synthetic salicylate analogues of Ent. While Scn binding hinders salicylate coordination transformation, strong acidification results in the release of iron and degraded siderophore. Iron release may therefore result from a combination of Ent degradation and coordination change.« less

Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval

PubMed Central

Hirst, Jennifer; Itzhak, Daniel N.; Antrobus, Robin; Borner, Georg H. H.

2018-01-01

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5–associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders. PMID:29381698

Role of the Retromer Complex in Neurodegenerative Diseases

PubMed Central

Li, Chaosi; Shah, Syed Zahid Ali; Zhao, Deming; Yang, Lifeng

2016-01-01

The retromer complex is a protein complex that plays a central role in endosomal trafficking. Retromer dysfunction has been linked to a growing number of neurological disorders. The process of intracellular trafficking and recycling is crucial for maintaining normal intracellular homeostasis, which is partly achieved through the activity of the retromer complex. The retromer complex plays a primary role in sorting endosomal cargo back to the cell surface for reuse, to the trans-Golgi network (TGN), or alternatively to specialized endomembrane compartments, in which the cargo is not subjected to lysosomal-mediated degradation. In most cases, the retromer acts as a core that interacts with associated proteins, including sorting nexin family member 27 (SNX27), members of the vacuolar protein sorting 10 (VPS10) receptor family, the major endosomal actin polymerization-promoting complex known as Wiskott-Aldrich syndrome protein and scar homolog (WASH), and other proteins. Some of the molecules carried by the retromer complex are risk factors for neurodegenerative diseases. Defects such as haplo-insufficiency or mutations in one or several units of the retromer complex lead to various pathologies. Here, we summarize the molecular architecture of the retromer complex and the roles of this system in intracellular trafficking related the pathogenesis of neurodegenerative diseases. PMID:26973516

The endosomal recycling of FgSnc1 by FgSnx41-FgSnx4 heterodimer is essential for polarized growth and pathogenicity in Fusarium graminearum.

PubMed

Zheng, Wenhui; Lin, Yahong; Fang, Wenqin; Zhao, Xu; Lou, Yi; Wang, Guanghui; Zheng, Huawei; Liang, Qifu; Abubakar, Yakubu Saddeeq; Olsson, Stefan; Zhou, Jie; Wang, Zonghua

2018-04-20

Endosomal sorting machineries regulate the transport of their cargoes among intracellular compartments. However, the molecular nature of such intracellular trafficking processes in pathogenic fungal development and pathogenicity remains unclear. Here, we dissect the roles and molecular mechanisms of two sorting nexin proteins and their cargoes in endosomal recycling in Fusarium graminearum using high-resolution microscopy and high-throughput co-immunoprecipitation strategies. We show that the sorting nexins, FgSnx41 and FgSnx4, interact with each other and assemble into a functionally interdependent heterodimer through their respective BAR domains. Further analyses demonstrate that the dimer localizes to the early endosomal membrane and coordinates endosomal sorting. The small GTPase FgRab5 regulates the correct localization of FgSnx41-FgSnx4 and is consequently required for its trafficking function. The protein FgSnc1 is a cargo of FgSnx41-FgSnx4 and regulates the fusion of secreted vesicles with the fungal growing apex and plasma membrane. In the absence of FgSnx41 or FgSnx4, FgSnc1 is mis-sorted and degraded in the vacuole, and null deletion of either component causes defects in the fungal polarized growth and virulence. Overall, for the first time, our results reveal the mechanism of FgSnc1 endosomal recycling by FgSnx41-FgSnx4 heterodimer which is essential for polarized growth and pathogenicity in F. graminearum. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

How PI3K-derived lipids control cell division.

PubMed

Campa, Carlo C; Martini, Miriam; De Santis, Maria C; Hirsch, Emilio

2015-01-01

To succeed in cell division, intense cytoskeletal and membrane remodeling are required to allow accurate chromosome segregation and cytoplasm partitioning. Spatial restriction of the actin dynamics and vesicle trafficking define the cell symmetry and equivalent membrane scission events, respectively. Protein complexes coordinating mitosis are recruited to membrane microdomains characterized by the presence of the phosphatidylinositol lipid members (PtdIns), like PtdIns(3,4,5)P 3,PtdIns(4,5)P 2, and PtdIns(3)P. These PtdIns represent a minor component of cell membranes, defining membrane domain identity, ultimately controlling cytoskeleton and membrane dynamics during mitosis. The coordinated presence of PtdIns(3,4,5)P 3 at the cell poles and PtdIns(4,5)P 2 at the cleavage furrow controls the polarity of the actin cytoskeleton leading to symmetrical cell division. In the endosomal compartment, the trafficking of PtdIns(3)P positive vesicles allows the recruitment of the protein machinery required for the abscission.

The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

PubMed

Lizundia, Regina; Chaussepied, Marie; Naissant, Bernina; Masse, Guillemette X; Quevillon, Emmanuel; Michel, Fréderique; Monier, Solange; Weitzman, Jonathan B; Langsley, Gordon

2007-08-01

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis

PubMed Central

2014-01-01

Background During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Results Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. Conclusions As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation. PMID:25005938

The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

PubMed

Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

2016-09-01

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

Phosphatidylinositol-4-kinase type II alpha contains an AP-3-sorting motif and a kinase domain that are both required for endosome traffic.

PubMed

Craige, Branch; Salazar, Gloria; Faundez, Victor

2008-04-01

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.

The ocular albinism type 1 (OA1) GPCR is ubiquitinated and its traffic requires endosomal sorting complex responsible for transport (ESCRT) function

PubMed Central

Giordano, Francesca; Simoes, Sabrina; Raposo, Graça

2011-01-01

The function of signaling receptors is tightly controlled by their intracellular trafficking. One major regulatory mechanism within the endo-lysosomal system required for receptor localization and down-regulation is protein modification by ubiquitination and downstream interactions with the endosomal sorting complex responsible for transport (ESCRT) machinery. Whether and how these mechanisms operate to regulate endosomal sorting of mammalian G protein-coupled receptors (GPCRs) remains unclear. Here, we explore the involvement of ubiquitin and ESCRTs in the trafficking of OA1, a pigment cell-specific GPCR, target of mutations in Ocular Albinism type 1, which localizes intracellularly to melanosomes to regulate their biogenesis. Using biochemical and morphological methods in combination with overexpression and inactivation approaches we show that OA1 is ubiquitinated and that its intracellular sorting and down-regulation requires functional ESCRT components. Depletion or overexpression of subunits of ESCRT-0, -I, and -III markedly inhibits OA1 degradation with concomitant retention within the modified endosomal system. Our data further show that OA1 ubiquitination is uniquely required for targeting to the intralumenal vesicles of multivesicular endosomes, thereby regulating the balance between down-regulation and delivery to melanosomes. This study highlights the role of ubiquitination and the ESCRT machinery in the intracellular trafficking of mammalian GPCRs and has implications for the physiopathology of ocular albinism type 1. PMID:21730137

Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

PubMed

Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

2015-01-01

During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

PubMed Central

Dennis, Megan K.; Mantegazza, Adriana R.; Snir, Olivia L.; Tenza, Danièle; Acosta-Ruiz, Amanda; Delevoye, Cédric; Zorger, Richard; Sitaram, Anand; de Jesus-Rojas, Wilfredo; Ravichandran, Keerthana; Rux, John; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Setty, Subba Rao Gangi

2015-01-01

Hermansky–Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2–deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2–deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation. PMID:26008744

Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance

PubMed Central

Naik, Subhashchandra; Brock, Susan; Akkaladevi, Narahari; Tally, Jon; Mcginn-Straub, Wesley; Zhang, Na; Gao, Phillip; Gogol, E. P.; Pentelute, B. L.; Collier, R. John; Fisher, Mark T.

2013-01-01

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions. PMID:23964683

Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

PubMed

Naik, Subhashchandra; Brock, Susan; Akkaladevi, Narahari; Tally, Jon; McGinn-Straub, Wesley; Zhang, Na; Gao, Phillip; Gogol, E P; Pentelute, B L; Collier, R John; Fisher, Mark T

2013-09-17

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å β barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

The ESCRT-III Subunit hVps24 Is Required for Degradation but Not Silencing of the Epidermal Growth Factor Receptor

PubMed Central

Bache, Kristi G.; Stuffers, Susanne; Malerød, Lene; Slagsvold, Thomas; Raiborg, Camilla; Lechardeur, Delphine; Wälchli, Sébastien; Lukacs, Gergely L.; Brech, Andreas; Stenmark, Harald

2006-01-01

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling. PMID:16554368

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor.

PubMed

Bache, Kristi G; Stuffers, Susanne; Malerød, Lene; Slagsvold, Thomas; Raiborg, Camilla; Lechardeur, Delphine; Wälchli, Sébastien; Lukacs, Gergely L; Brech, Andreas; Stenmark, Harald

2006-06-01

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.

Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.

PubMed

Tornieri, Karine; Zlatic, Stephanie A; Mullin, Ariana P; Werner, Erica; Harrison, Robert; L'hernault, Steven W; Faundez, Victor

2013-12-20

Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke

Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss ofmore » p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.« less

Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions ▿

PubMed Central

Ai, Li-Shuang; Lee, Yu-Wen; Chen, Steve S.-L.

2009-01-01

The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis. PMID:19605478

Structural Insights into the Phospholipid Binding Specificity of Human Evectin-2

NASA Astrophysics Data System (ADS)

Okazaki, Seiji; Kato, Ryuichi; Wakatsuki, Soichi; Uchida, Yasunori; Taguchi, Tomohiko; Arai, Hiroyuki

Evectin-2 is a recycling endosomal protein and plays an essential role in retrograde transport from recycling endosomes to the trans-Golgi network. The pleckstrin homology (PH) domain of Evectin-2 can specifically binds to phosphatidylserine (PS), which is enriched in recycling endosomes. To elucidate the molecular mechanism how it specifically binds to PS, we solved the crystal structures of human Evectin-2 PH domain for apo and O-phospho-L-serine complexed forms at 1.75 and 1.00 Å resolution, respectively. These structural analyses clearly show that PS-induced conformational change of Evectin-2 PH domain effectively explains the strict phospholipid binding specificity.

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

PubMed Central

Pelayo, Juan-Carlos; Poole, Daniel P; Steinhoff, Martin; Cottrell, Graeme S; Bunnett, Nigel W

2011-01-01

Abstract Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK1R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nm, 10 min) induced interaction of NK1R and β-arrestin at the plasma membrane, and the SP–NK1R–β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK1R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H+ATPase inhibitor bafilomycin A1, which prevent endosomal SP degradation, suppressed NK1R recycling by >50%. Preincubation of neurones with SP (10 nm, 5 min) desensitized Ca2+ transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK1R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP–NK1R–β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK1R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling. PMID:21878523

Retromer association with membranes: plants have their own rules!

PubMed

Zelazny, Enric; Santambrogio, Martina; Gaude, Thierry

2013-09-01

The retromer is an endosome-localized complex involved in protein trafficking. To better understand its function and regulation in plants, we recently investigated how Arabidopsis retromer subunits assemble and are targeted to endosomal membranes and highlighted original features compared with mammals. We characterized Arabidopsis vps26 null mutant and showed that it displays severe developmental defaults similar to those observed in vps29 mutant. Here, we go further by describing new phenotypic defects associated with loss of VPS26 function, such as inhibition of lateral root initiation. Recently, we showed that VPS35 subunit plays a crucial role in the recruitment of the plant retromer to endosomes, probably through an interaction with the Rab7 homolog RABG3f. In this work, we now show that contrary to mammals, Arabidopsis Rab5 homologs do not seem to be necessary for the recruitment of the core retromer to endosomal membranes, which highlights a new specificity of the plant retromer.

Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.

PubMed

Merdan, Thomas; Kunath, Klaus; Fischer, Dagmar; Kopecek, Jindrich; Kissel, Thomas

2002-02-01

Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.

Rab coupling protein mediated endosomal recycling of N-cadherin influences cell motility.

PubMed

Lindsay, Andrew J; McCaffrey, Mary W

2017-12-01

Rab coupling protein (RCP) is a Rab GTPase effector that functions in endosomal recycling. The RCP gene is frequently amplified in breast cancer, leading to increased cancer aggressiveness. Furthermore, RCP enhances the motility of ovarian cancer cells by coordinating the recycling of α5β1 integrin and EGF receptor to the leading edge of migrating cells. Here we report that RCP also influences the motility of lung adenocarcinoma cells. Knockdown of RCP inhibits the motility of A549 cells in 2D and 3D migration assays, while its overexpression enhances migration in these assays. Depletion of RCP leads to a reduction in N-cadherin protein levels, which could be restored with lysosomal inhibitors. Trafficking assays revealed that RCP knockdown inhibits the return of endocytosed N-cadherin to the cell surface. We propose that RCP regulates the endosomal recycling of N-cadherin, and in its absence N-cadherin is diverted to the degradative pathway. The increased aggressiveness of tumour cells that overexpress RCP may be due to biased recycling of N-cadherin in metastatic cancer cells.

Lysosomal degradation of membrane lipids.

PubMed

Kolter, Thomas; Sandhoff, Konrad

2010-05-03

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

IL-1β impairs retrograde flow of BDNF signaling by attenuating endosome trafficking.

PubMed

Carlos, Anthony J; Tong, Liqi; Prieto, G Aleph; Cotman, Carl W

2017-02-02

Pro-inflammatory cytokines accumulate in the brain with age and Alzheimer's disease and can impair neuron health and cognitive function. Brain-derived neurotrophic factor (BDNF) is a key neurotrophin that supports neuron health, function, and synaptic plasticity. The pro-inflammatory cytokine interleukin-1β (IL-1β) impairs BDNF signaling but whether it affects BDNF signaling endosome trafficking has not been studied. This study uses an in vitro approach in primary hippocampal neurons to evaluate the effect of IL-1β on BDNF signaling endosome trafficking. Neurons were cultured in microfluidic chambers that separate the environments of the cell body and its axon terminal, enabling us to specifically treat in axon compartments and trace vesicle trafficking in real-time. We found that IL-1β attenuates BDNF signaling endosomes throughout networks in cultures. In IL-1β-treated cells, overall BDNF endosomal density was decreased, and the colocalization of BDNF endosomes with presynaptic terminals was found to be more than two times higher than in control cultures. Selective IL-1β treatment to the presynaptic compartment in microfluidic chamber attenuated BDNF endosome flux, as measured by reduced BDNF-GFP endosome counts in the somal compartment. Further, IL-1β decreased the BDNF-induced phosphorylation of Erk5, a known BDNF retrograde trafficking target. Mechanistically, the deficiency in trafficking was not due to impaired endocytosis of the BDNF-TrkB complex, or impaired transport rate, since BDNF endosomes traveled at the same rate in both control and IL-1β treatment groups. Among the regulators of presynaptic endosome sorting is the post-translational modification, ubiquitination. In support of this possibility, the IL-1β-mediated suppression of BDNF-induced Erk5 phosphorylation can be rescued by exogenous ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that regulates ubiquitin and endosomal trafficking. We observed a state of neurotrophic resistance whereby, in the prolonged presence of IL-1β, BDNF is not effective in delivering long-distance signaling via the retrograde transport of signaling endosomes. Since IL-1β accumulation is an invariant feature across many neurodegenerative diseases, our study suggest that compromised BDNF retrograde transport-dependent signaling may have important implications in neurodegenerative diseases.

Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1.

PubMed

Perrin, Laura; Laura, Perrin; Lacas-Gervais, Sandra; Sandra, Lacas-Gervais; Gilleron, Jérôme; Jérôme, Gilleron; Ceppo, Franck; Franck, Ceppo; Prodon, François; François, Prodon; Benmerah, Alexandre; Alexandre, Benmerah; Tanti, Jean-François; Jean-François, Tanti; Cormont, Mireille; Mireille, Cormont

2013-11-01

The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.

Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromer-mediated endosome-to-TGN transport

PubMed Central

Carlton, Jez G.; Bujny, Miriam V.; Peter, Brian J.; Oorschot, Viola M. J.; Rutherford, Anna; Arkell, Rebecca S.; Klumperman, Judith; McMahon, Harvey T.; Cullen, Peter J.

2006-01-01

Summary Sorting nexins are a large family of phox-homology-domain-containing proteins that have been implicated in the control of endosomal sorting. Sorting nexin-1 is a component of the mammalian retromer complex that regulates retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network. In yeast, retromer is composed of Vps5p (the orthologue of sorting nexin-1), Vps17p (a related sorting nexin) and a cargo selective subcomplex composed of Vps26p, Vps29p and Vps35p. With the exception of Vps17p, mammalian orthologues of all yeast retromer components have been identified. For Vps17p, one potential mammalian orthologue is sorting nexin-2. Here we show that, like sorting nexin-1, sorting nexin-2 binds phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,5-bisphosphate, and possesses a Bin/Amphiphysin/Rvs domain that can sense membrane curvature. However, in contrast to sorting nexin-1, sorting nexin-2 could not induce membrane tubulation in vitro or in vivo. Functionally, we show that endogenous sorting nexin-1 and sorting nexin-2 co-localise on high curvature tubular elements of the 3-phosphoinositide-enriched early endosome, and that suppression of sorting nexin-2 does not perturb the degradative sorting of receptors for epidermal growth factor or transferrin, nor the steady-state distribution of the cation-independent mannose 6-phosphate receptor. However, suppression of sorting nexin-2 results in a subtle alteration in the kinetics of cation-independent mannose 6-phosphate receptor retrieval. These data suggest that although sorting nexin-2 may be a component of the retromer complex, its presence is not essential for the regulation of endosome-to-trans Golgi network retrieval of the cation-independent mannose 6-phosphate receptor. PMID:16179610

Cholesterol transfer at endosomal-organelle membrane contact sites.

PubMed

Ridgway, Neale D; Zhao, Kexin

2018-06-01

Cholesterol is delivered to the limiting membrane of late endosomes by Niemann-Pick Type C1 and C2 proteins. This review summarizes recent evidence that cholesterol transfer from endosomes to the endoplasmic reticulum and other organelles is mediated by lipid-binding proteins that localize to membrane contact sites (MCS). LDL-cholesterol in the late endosomal/lysosomes is exported to the plasma membrane, where most cholesterol resides, and the endoplasmic reticulum, which harbors the regulatory complexes and enzymes that control the synthesis and esterification of cholesterol. A major advance in dissecting these cholesterol transport pathways was identification of frequent and dynamic MCS between endosomes and the endoplasmic reticulum, peroxisomes and plasma membrane. Positioned at these MCS are members of the oxysterol-binding protein (OSBP) and steroidogenic acute regulatory protein-related lipid-transfer family of lipid transfer proteins that bridge the opposing membranes and directly or indirectly mediate cholesterol transfer. OSBP-related protein 1L (ORP1L), ORP5 and ORP6 mediate cholesterol transfer to the endoplasmic reticulum that regulates cholesterol homeostasis. ORP1L and STARD3 also move cholesterol from the endoplasmic reticulum-to-late endosomal/lysosomes under low-cholesterol conditions to facilitate intraluminal vesicle formation. Cholesterol transport also occurs at MCS with peroxisomes and possibly the plasma membrane. Frequent contacts between organelles and the endo-lysosomal vesicles are sites for bidirectional transfer of cholesterol.

The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

PubMed Central

Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

2015-01-01

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

Overcoming endosomal barrier by amphotericin B-loaded dual pH-responsive PDMA-b-PDPA micelleplexes for siRNA delivery.

PubMed

Yu, Haijun; Zou, Yonglong; Wang, Yiguang; Huang, Xiaonan; Huang, Gang; Sumer, Baran D; Boothman, David A; Gao, Jinming

2011-11-22

The endosomal barrier is a major bottleneck for the effective intracellular delivery of siRNA by nonviral nanocarriers. Here, we report a novel amphotericin B (AmB)-loaded, dual pH-responsive micelleplex platform for siRNA delivery. Micelles were self-assembled from poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-b-PDPA) diblock copolymers. At pH 7.4, AmB was loaded into the hydrophobic PDPA core, and siRNA was complexed with a positively charged PDMA shell to form the micelleplexes. After cellular uptake, the PDMA-b-PDPA/siRNA micelleplexes dissociated in early endosomes to release AmB. Live cell imaging studies demonstrated that released AmB significantly increased the ability of siRNA to overcome the endosomal barrier. Transfection studies showed that AmB-loaded micelleplexes resulted in significant increase in luciferase (Luc) knockdown efficiency over the AmB-free control. The enhanced Luc knockdown efficiency was abolished by bafilomycin A1, a vacuolar ATPase inhibitor that inhibits the acidification of the endocytic organelles. These data support the central hypothesis that membrane poration by AmB and increased endosomal swelling and membrane tension by a "proton sponge" polymer provided a synergistic strategy to disrupt endosomes for improved intracellular delivery of siRNA. © 2011 American Chemical Society

Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pH-sensitive disruption of endosomal vesicles.

PubMed

Jones, Rachel A; Cheung, Charles Y; Black, Fiona E; Zia, Jasmine K; Stayton, Patrick S; Hoffman, Allan S; Wilson, Mark R

2003-05-15

The permeability barrier posed by cell membranes represents a challenge for the delivery of hydrophilic molecules into cells. We previously proposed that poly(2-alkylacrylic acid)s are endocytosed by cells into acidified vesicles and are there triggered by low pH to disrupt membranes and release the contents of endosomes/lysosomes to the cytosol. If this hypothesis is correct, these polymers could be valuable in drug-delivery applications. The present paper reports functional comparisons of a family of three poly(2-alkylacrylic acid)s. Poly(2-propylacrylic acid) (PPAA), poly(2-ethylacrylic acid) (PEAA) and poly(2-methylacrylic acid) (PMAA) were compared in red-blood-cell haemolysis assays and in a lipoplex (liposome-DNA complex) assay. We also directly examined the ability of these polymers to disrupt endosomes and lysosomes in cultured human cells. Our results show that: (i) unlike membrane-disruptive peptides, the endosomal-disruptive ability of poly(2-alkylacrylic acid)s cannot necessarily be predicted from their haemolytic activity at low pH, (ii) PPAA (but not PEAA or PMAA) potently facilitates gene transfection by cationic lipoplexes and (iii) endocytosed poly(2-alkylacrylic acid)s are triggered by luminal acidification to selectively disrupt endosomes (not lysosomes) and release their contents to the cytosol. These results will facilitate the rational design of future endosomal-disrupting polymers for drug delivery.

bicoid RNA localization requires specific binding of an endosomal sorting complex

PubMed Central

Irion, Uwe; St Johnston, Daniel

2007-01-01

Summary paragraph: bicoid mRNA localises to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first identified localised cytoplasmic determinant1-4, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that it is recognised by a complex of many redundant proteins, each of which binds to the localisation element in its 3'UTR with little or no specificity5. Indeed, the only known RNA-binding protein that co-localises with bicoid mRNA is Staufen, which binds non-specifically to dsRNA in vitro6, 7. Here we show that mutants in all subunits of the ESCRT-II complex (Vps22, Vps25 and Vps36) abolish the final Staufen-dependent step in bcd RNA localisation. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles8, 9, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm10, 11. However, the role of ESCRT-II in bicoid localisation appears to be independent of endosomal sorting, because mutations in ESCRT-I and III components have no effect of the targeting of bicoid mRNA. Instead, Vps36 functions by binding directly and specifically to stem-loop V of the bicoid 3'UTR through its N-terminal GLUE domain12, making it the first example of a sequence specific RNA-binding protein that recognises the bicoid localisation signal. Furthermore, Vps36 localises to the anterior of the oocyte in a bicoid mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This novel function of ESCRT-II as an RNA-binding complex is conserved in vertebrates, and may explain some of its roles that are independent of endosomal sorting. PMID:17268469

Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation

PubMed Central

Corrotte, Matthias; Fernandes, Maria Cecilia; Tam, Christina; Andrews, Norma W.

2012-01-01

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca2+-dependent manner. Resealing involves Ca2+-dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes. PMID:22212686

The Last Enzyme of the De Novo Purine Synthesis Pathway 5-aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC) Plays a Central Role in Insulin Signaling and the Golgi/Endosomes Protein Network*

PubMed Central

Boutchueng-Djidjou, Martial; Collard-Simard, Gabriel; Fortier, Suzanne; Hébert, Sébastien S.; Kelly, Isabelle; Landry, Christian R.; Faure, Robert L.

2015-01-01

Insulin is internalized with its cognate receptor into the endosomal apparatus rapidly after binding to hepatocytes. We performed a bioinformatic screen of Golgi/endosome hepatic protein fractions and found that ATIC, which is a rate-limiting enzyme in the de novo purine biosynthesis pathway, and PTPLAD1 are associated with insulin receptor (IR) internalization. The IR interactome (IRGEN) connects ATIC to AMPK within the Golgi/endosome protein network (GEN). Forty-five percent of the IR Golgi/endosome protein network have common heritable variants associated with type 2 diabetes, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalized within the plasma membrane (PM) and Golgi/endosome fractions after insulin stimulation and that ATIC later accumulates in the Golgi/endosome fraction. Using an in vitro reconstitution system and siRNA-mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPK-Thr172 phosphorylation in IR complexes. We observed that IR internalization was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. These results suggest the presence of a signaling mechanism that senses adenylate synthesis, ATP levels, and IR activation states and that acts in regulating IR autophosphorylation and endocytosis. PMID:25687571

Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

PubMed

Ejzykowicz, Daniele E; Locken, Kristopher M; Ruiz, Fiona J; Manandhar, Surya P; Olson, Daniel K; Gharakhanian, Editte

2017-06-01

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks

PubMed Central

Samson, Edward B.; Tsao, David S.; Zimak, Jan; McLaughlin, R. Tyler; Trenton, Nicholaus J.; Mace, Emily M.; Orange, Jordan S.; Schweikhard, Volker

2017-01-01

ABSTRACT IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks. PMID:28455356

EARP, a multisubunit tethering complex involved in endocytic recycling

PubMed Central

Schindler, Christina; Chen, Yu; Pu, Jing; Guo, Xiaoli; Bonifacino, Juan S.

2015-01-01

Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, it is unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. Herein we describe a tethering complex named Endosome-Associated Recycling Protein (EARP) that is structurally related to the previously described Golgi-Associated Retrograde Protein (GARP) complex. Both complexes share the Ang2, Vps52 and Vps53 subunits, but EARP comprises an uncharacterized protein, Syndetin, in place of the Vps54 subunit of GARP. This change determines differential localization of EARP to recycling endosomes and GARP to the Golgi complex. EARP interacts with the target-SNARE Syntaxin 6 and various cognate SNAREs. Depletion of Syndetin or Syntaxin 6 delays recycling of internalized transferrin to the cell surface. These findings implicate EARP in canonical membrane-fusion events in the process of endocytic recycling. PMID:25799061

Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasan, Neil; Hutagalung, Alex; Novick, Peter

2010-08-13

The Golgi-associated retrograde protein (GARP) complex is a membrane-tethering complex that functions in traffic from endosomes to the trans-Golgi network. Here we present the structure of a C-terminal fragment of the Vps53 subunit, important for binding endosome-derived vesicles, at a resolution of 2.9 {angstrom}. We show that the C terminus consists of two {alpha}-helical bundles arranged in tandem, and we identify a highly conserved surface patch, which may play a role in vesicle recognition. Mutations of the surface result in defects in membrane traffic. The fold of the Vps53 C terminus is strongly reminiscent of proteins that belong to threemore » other tethering complexes - Dsl1, conserved oligomeric Golgi, and the exocyst - thought to share a common evolutionary origin. Thus, the structure of the Vps53 C terminus suggests that GARP belongs to this family of complexes.« less

Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method.

PubMed

Lehtinen, Julia; Hyvönen, Zanna; Subrizi, Astrid; Bunjes, Heike; Urtti, Arto

2008-10-21

Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

Hemolytic activity of pH-responsive polymer-streptavidin bioconjugates.

PubMed

Lackey, C A; Murthy, N; Press, O W; Tirrell, D A; Hoffman, A S; Stayton, P S

1999-01-01

Drug delivery systems that increase the rate and/or quantity of drug release to the cytoplasm are needed to enhance cytosolic delivery and to circumvent nonproductive cell trafficking routes. We have previously demonstrated that poly(2-ethylacrylic acid) (PEAAc) has pH-dependent hemolytic properties, and more recently, we have found that poly(2-propylacrylic acid) (PPAAc) displays even greater pH-responsive hemolytic activity than PEAAc at the acidic pHs of the early endosome. Thus, these polymers could potentially serve as endosomal releasing agents in immunotoxin therapies. In this paper, we have investigated whether the pH-dependent membrane disruptive activity of PPAAc is retained after binding to a protein. We did this by measuring the hemolytic activity of PPAAc-streptavidin model complexes with different protein to polymer stoichiometries. Biotin was conjugated to amine-terminated PPAAc, which was subsequently bound to streptavidin by biotin complexation. The ability of these samples to disrupt red blood cell membranes was investigated for a range of polymer concentrations, a range of pH values, and two polymer-to-streptavidin ratios of 3:1 and 1:1. The results demonstrate that (a) the PPAAc-streptavidin complex retains the ability to lyse the RBC lipid bilayers at low pHs, such as those existing in endosomes, and (b) the hemolytic ability of the PPAAc-streptavidin complex is similar to that of the free PPAAc.

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport.

PubMed

Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

2015-09-01

The importance of endosome-to-trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51-VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. © 2015 Hirata, Fujita, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL

PubMed Central

Romano-Moreno, Miguel; Rojas, Adriana L.; Williamson, Chad D.; Lucas, María; Isupov, Michail N.; Bonifacino, Juan S.; Machner, Matthias P.; Hierro, Aitor

2017-01-01

Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29–VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes. PMID:29229824

Structural and functional insights into sorting nexin 5/6 interaction with bacterial effector IncE.

PubMed

Sun, Qingxiang; Yong, Xin; Sun, Xiaodong; Yang, Fan; Dai, Zhonghua; Gong, Yanqiu; Zhou, Liming; Zhang, Xia; Niu, Dawen; Dai, Lunzhi; Liu, Jia-Jia; Jia, Da

2017-01-01

The endosomal trafficking pathways are essential for many cellular activities. They are also important targets by many intracellular pathogens. Key regulators of the endosomal trafficking include the retromer complex and sorting nexins (SNXs). Chlamydia trachomatis effector protein IncE directly targets the retromer components SNX5 and SNX6 and suppresses retromer-mediated transport, but the exact mechanism has remained unclear. We present the crystal structure of the PX domain of SNX5 in complex with IncE, showing that IncE binds to a highly conserved hydrophobic groove of SNX5. The unique helical hairpin of SNX5/6 is essential for binding, explaining the specificity of SNX5/6 for IncE. The SNX5/6-IncE interaction is required for cellular localization of IncE and its inhibitory function. Mechanistically, IncE inhibits the association of CI-MPR cargo with retromer-containing endosomal subdomains. Our study provides new insights into the regulation of retromer-mediated transport and illustrates the intricate competition between host and pathogens in controlling cellular trafficking.

On the role of methacrylic acid copolymers in the intracellular delivery of antisense oligonucleotides.

PubMed

Yessine, Marie-Andrée; Meier, Christian; Petereit, Hans-Ulrich; Leroux, Jean-Christophe

2006-05-01

The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.

Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited

PubMed Central

1995-01-01

We have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-1, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila phagosome rapidly clears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophila phagosomes, phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit phagosome-lysosome fusion, is heterogeneous. PMID:7807006

Mechanisms of Entry and Endosomal Pathway of African Swine Fever Virus

PubMed Central

G. Sánchez, Elena; Pérez-Núñez, Daniel; Revilla, Yolanda

2017-01-01

African Swine Fever Virus (ASFV) causes a serious swine disease that is endemic in Africa and Sardinia and presently spreading in Russia and neighboring countries, including Poland and recently, the Czech Republic. This uncontrolled dissemination is a world-wide threat, as no specific protection or vaccine is available. ASFV is a very complex icosahedral, enveloped virus about 200 nm in diameter, which infects several members of pigs. The virus enters host cells by receptor-mediated endocytosis that depends on energy, vacuolar pH and temperature. The specific receptor(s) and attachment factor(s) involved in viral entry are still unknown, although macropinocytosis and clathrin-dependent mechanisms have been proposed. After internalization, ASFV traffics through the endolysosomal system. The capsid and inner envelope are found in early endosomes or macropinosomes early after infection, colocalizing with EEA1 and Rab5, while at later times they co-localize with markers of late endosomes and lysosomes, such as Rab7 or Lamp 1. A direct relationship has been established between the maturity of the endosomal pathway and the progression of infection in the cell. Finally, ASFV uncoating first involves the loss of the outer capsid layers, and later fusion of the inner membrane with endosomes, releasing the nude core into the cytosol. PMID:29117102

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay.

PubMed

Karim, Mahmoud Abdul; Samyn, Dieter Ronny; Mattie, Sevan; Brett, Christopher Leonard

2018-02-01

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

PubMed Central

Yang, Xi; Arines, Felichi Mae; Zhang, Weichao

2018-01-01

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors. PMID:29355480

Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

PubMed

Siddiqa, Abida; Massimi, Paola; Pim, David; Broniarczyk, Justyna; Banks, Lawrence

2018-03-15

Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of VAP blocks HPV infectious entry at a step after capsid uncoating but prior to localization at the trans -Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry. Copyright © 2018 American Society for Microbiology.

Mechanisms of polarized membrane trafficking in neurons – focusing in on endosomes

PubMed Central

Lasiecka, Zofia M.; Winckler, Bettina

2011-01-01

Neurons are polarized cells that have a complex and unique morphology: long processes (axons and dendrites) extending far from the cell body. In addition, the somatodendritic and axonal domains are further divided into specific subdomains, such as synapses (pre- and postsynaptic specializations), proximal and distal dendrites, axon initial segments, nodes of Ranvier, and axon growth cones. The striking asymmetry and complexity of neuronal cells is necessary for their function in receiving, processing and transferring electrical signals, with each domain playing a precise function in these processes. In order to establish and maintain distinct neuronal domains, mechanisms must exist for protein delivery to specific neuronal compartments, such that each compartment has the correct functional molecular composition. How polarized membrane domains are established and maintained is a long-standing question. Transmembrane proteins, such as receptors and adhesion molecules, can be transported to their proper membrane domains by several pathways. The biosynthetic secretory system delivers newly synthesized transmembrane proteins from the ER-Golgi via the trans-Golgi network (TGN) to the plasma membrane. In addition, the endosomal system is critically involved in many instances in ensuring proper (re)targeting of membrane components because it can internalize and degrade mislocalized proteins, or recycle proteins from one domain to another. The endosomal system is thus crucial for establishing and maintaining neuronal polarity. In this review, we focus mainly on the intracellular compartments that serve as sorting stations for polarized transport, with particular emphasis on the emerging roles of endosomes. PMID:21762782

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.

PubMed

Carette, Jan E; Raaben, Matthijs; Wong, Anthony C; Herbert, Andrew S; Obernosterer, Gregor; Mulherkar, Nirupama; Kuehne, Ana I; Kranzusch, Philip J; Griffin, April M; Ruthel, Gordon; Dal Cin, Paola; Dye, John M; Whelan, Sean P; Chandran, Kartik; Brummelkamp, Thijn R

2011-08-24

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

PubMed

Girada, Shravan Babu; Kuna, Ramya S; Bele, Shilpak; Zhu, Zhimeng; Chakravarthi, N R; DiMarchi, Richard D; Mitra, Prasenjit

2017-10-01

Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Regulation of podocalyxin trafficking by Rab small GTPases in epithelial cells

PubMed Central

Mrozowska, Paulina S.; Fukuda, Mitsunori

2016-01-01

ABSTRACT The characteristic feature of polarity establishment in MDCK II cells is transcytosis of apical glycoprotein podocalyxin (PCX) from the outer plasma membrane to the newly formed apical domain. This transcytotic event consists of multiple steps, including internalization from the plasma membrane, transport through early endosomes and Rab11-positive recycling endosomes, and delivery to the apical membrane. These steps are known to be tightly coordinated by Rab small GTPases, which act as molecular switches cycling between active GTP-bound and inactive GDP-bound states. However, our knowledge regarding which sets of Rabs regulate particular steps of PCX trafficking was rather limited. Recently, we have performed a comprehensive analysis of Rab GTPase engagement in the transcytotic pathway of PCX during polarity establishment in 2-dimensional (2D) and 3-dimensional (3D) MDCK II cell cultures. In this Commentary we summarize our findings and set them in the context of previous reports. PMID:27463697

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

PubMed

Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

2009-06-17

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

Spatial Relationships between Markers for Secretory and Endosomal Machinery in Human Cytomegalovirus-Infected Cells versus Those in Uninfected Cells▿†

PubMed Central

Das, Subhendu; Pellett, Philip E.

2011-01-01

Human cytomegalovirus (HCMV) induces extensive remodeling of the secretory apparatus to form the cytoplasmic virion assembly compartment (cVAC), where virion tegumentation and envelopment take place. We studied the structure of the cVAC by confocal microscopy to assess the three-dimensional distribution of proteins specifically associated with individual secretory organelles. In infected cells, early endosome antigen 1 (EEA1)-positive vesicles are concentrated at the center of the cVAC and, as previously seen, are distinct from structures visualized by markers for the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). EEA1-positive vesicles can be strongly associated with markers for recycling endosomes, to a lesser extent with markers associated with components of the endosomal sorting complex required for transport III (ESCRT III) machinery, and then with markers of late endosomes. In comparisons of uninfected and infected cells, we found significant changes in the structural associations and colocalization of organelle markers, as well as in net organelle volumes. These results provide new evidence that the HCMV-induced remodeling of the membrane transport apparatus involves much more than simple relocation and expansion of preexisting structures and are consistent with the hypothesis that the shift in identity of secretory organelles in HCMV-infected cells results in new functional profiles. PMID:21471245

Selective endosomal microautophagy is starvation-inducible in Drosophila.

PubMed

Mukherjee, Anindita; Patel, Bindi; Koga, Hiroshi; Cuervo, Ana Maria; Jenny, Andreas

2016-11-01

Autophagy delivers cytosolic components to lysosomes for degradation and is thus essential for cellular homeostasis and to cope with different stressors. As such, autophagy counteracts various human diseases and its reduction leads to aging-like phenotypes. Macroautophagy (MA) can selectively degrade organelles or aggregated proteins, whereas selective degradation of single proteins has only been described for chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI). These 2 autophagic pathways are specific for proteins containing KFERQ-related targeting motifs. Using a KFERQ-tagged fluorescent biosensor, we have identified an eMI-like pathway in Drosophila melanogaster. We show that this biosensor localizes to late endosomes and lysosomes upon prolonged starvation in a KFERQ- and Hsc70-4- dependent manner. Furthermore, fly eMI requires endosomal multivesicular body formation mediated by ESCRT complex components. Importantly, induction of Drosophila eMI requires longer starvation than the induction of MA and is independent of the critical MA genes atg5, atg7, and atg12. Furthermore, inhibition of Tor signaling induces eMI in flies under nutrient rich conditions, and, as eMI in Drosophila also requires atg1 and atg13, our data suggest that these genes may have a novel, additional role in regulating eMI in flies. Overall, our data provide the first evidence for a novel, starvation-inducible, catabolic process resembling endosomal microautophagy in the Drosophila fat body.

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors*

PubMed Central

Stahlschmidt, Wiebke; Robertson, Mark J.; Robinson, Phillip J.; McCluskey, Adam; Haucke, Volker

2014-01-01

Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane. PMID:24407285

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

PubMed Central

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

PubMed

Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

PubMed

Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal-Like Compartments and the Golgi Complex

PubMed Central

Jackson, Allison J; Clucas, Caroline; Mamczur, Nicola J; Ferguson, David J; Meissner, Markus

2013-01-01

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway. PMID:23962112

Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome.

PubMed

Killisch, I; Steinlein, P; Römisch, K; Hollinshead, R; Beug, H; Griffiths, G

1992-09-01

We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42 degrees C. Biochemical analysis showed that most (approximately 90%) of the internalized 125I-Tf recycled within approximately 30 min while a smaller fraction of 125I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (FITC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface. The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.

Engagement of the small GTPase Rab31 protein and its effector, early endosome antigen 1, is important for trafficking of the ligand-bound epidermal growth factor receptor from the early to the late endosome.

PubMed

Chua, Christelle En Lin; Tang, Bor Luen

2014-05-02

Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.

Engagement of the Small GTPase Rab31 Protein and Its Effector, Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome*

PubMed Central

Chua, Christelle En Lin; Tang, Bor Luen

2014-01-01

Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5. PMID:24644286

Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

PubMed Central

Caviston, Juliane P.; Zajac, Allison L.; Tokito, Mariko; Holzbaur, Erika L.F.

2011-01-01

Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles. PMID:21169558

Sorting of Clathrin-Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin-Mediated Endocytosis.

PubMed

Dutta, Dipannita; Donaldson, Julie G

2015-09-01

Clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) co-exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6-associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6-GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6-GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin-coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6-GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Nanoconjugation prolongs endosomal signaling of the epidermal growth factor receptor and enhances apoptosis

NASA Astrophysics Data System (ADS)

Wu, L.; Xu, F.; Reinhard, B. M.

2016-07-01

It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex.It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex. Electronic supplementary information (ESI) available: DLS data of NP-EGF in growth medium; MTT cell viability assay; validation of MW-NP uptake; positive controls for pharmacological inhibitors; EEA1 background for NP-EGF incubated with cell lysate; phosphorylation for EGF-Alexa647; live cell dynamic colocalization movie of MDA-MB-468 cells expressing Rab5a-GFP (green) 4.5 h after exposure to 8 pM NP-EGF (red). See DOI: 10.1039/c6nr02974d

The balance of protein expression and degradation: an ESCRTs point of view.

PubMed

Babst, Markus; Odorizzi, Greg

2013-08-01

Endosomal sorting complexes required for transport (ESCRTs) execute the biogenesis of late endosomal multivesicular bodies (MVBs). The ESCRT pathway has traditionally been viewed as a means by which transmembrane proteins are degraded in vacuoles/lysosomes. More recent studies aimed at understanding the broader functions of ESCRTs have uncovered unexpected links with pathways that control cellular metabolism. Central to this communication is TORC1, the kinase complex that controls many of the catabolic and anabolic systems. The connection between TORC1 activity and ESCRTs allows cells to quickly adapt to the stress of nutrient limitations until the longer-term autophagic pathway is activated. Increasing evidence also points to ESCRTs regulating RNA interference (RNAi) pathways that control translation. Copyright © 2013 Elsevier Ltd. All rights reserved.

WNK4 inhibits plasma membrane targeting of NCC through regulation of syntaxin13 SNARE formation.

PubMed

Chung, Woo Young; Park, Hyun Woo; Han, Jung Woo; Lee, Min Goo; Kim, Joo Young

2013-12-01

WNK4, a serine/threonine kinase, plays a critical role in the expression of membrane proteins in the cell surface; however, the underlying mechanism of WNK4 is not clear. Here, we demonstrate that WNK4 inhibits the fusion of plasma membrane delivering vesicle with sorting/recycling endosome through disrupting SNARE formation of syntaxin13, an endosomal t-SNARE and VAMP2, the v-SNARE in plasma membrane delivering vesicle. Their interaction and co-localization were enhanced by hyperosmotic stimulation which is known for WNK4 activation. The kinase domain of WNK4 interacts with the transmembrane domain (TM) of syntaxin13 and this interaction was abolished when the TM was replaced with that of syntaxin16. Interestingly, cell fractionation using sucrose gradients revealed that WNK4 inhibited the formation of the syntaxin13/VAMP2 SNARE complex in the endosomal compartment, but not syntaxin16/VAMP2 or syntaxin13/VAMP7. Syntaxin13 was not phosphorylated by WNK4 and WNK4KI also showed the same binding strength and similar inhibitory regulation on SNARE formation of syntaxin13. Physiological relevance of this mechanism was proved with the expression of NCC (Na(+) C1(-) co-transporter) in the cell surface. The inhibiting activity of WNK4 on surface expression of NCC was abolished by syntaxin13 siRNA transfection. These results suggest that WNK4 attenuates PM targeting of NCC proteins through regulation of syntaxin13 SNARE complex formation with VAMP2 in recycling and sorting endosome. © 2013.

Polyethylenimine-mediated gene delivery: a mechanistic study.

PubMed

Kichler, A; Leborgne, C; Coeytaux, E; Danos, O

2001-01-01

Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes. To investigate the importance of the acidification of endosomes during PEI-mediated DNA transfer we used proton pump inhibitors such as bafilomycin A1 and concanamycin A. Moreover, we tested whether PEI is able to destabilize natural membranes per se at neutral or acidic pH by performing erythrocyte lysis assays. PEI-mediated transfection in the presence of bafilomycin A1 resulted in a 7-74-fold decrease in reporter gene expression depending on the cell line used. In contrast, the efficiency of the monocationic lipid, DOTAP, was not importantly altered in the presence of the drug. Furthermore, the present data show that PEI cannot destabilize erythrocyte membranes, even at acidic pH, and that PEI, complexed or not to DNA, can increase the transfection efficiency of the cationic polymer, polylysine, when added at the same time to the cells. The transfection efficiency of PEIs partially relies on their ability to capture the protons which are transferred into the endosomes during their acidification. In addition, PEI is able to deliver significant amounts of DNA into cells and the DNA complexes involved in the expression of the transgene escape within 4 h from the endosomes.

African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

PubMed Central

Hernáez, Bruno; Guerra, Milagros; Salas, María L.

2016-01-01

African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

Signaling pathways coordinating the alkaline pH response confer resistance to the hevein-type plant antimicrobial peptide Pn-AMP1 in Saccharomyces cerevisiae.

PubMed

Kwon, Youngho; Chiang, Jennifer; Tran, Grant; Giaever, Guri; Nislow, Corey; Hahn, Bum-Soo; Kwak, Youn-Sig; Koo, Ja-Choon

2016-12-01

Genome-wide screening of Saccharomyces cerevisiae revealed that signaling pathways related to the alkaline pH stress contribute to resistance to plant antimicrobial peptide, Pn-AMP1. Plant antimicrobial peptides (AMPs) are considered to be promising candidates for controlling phytopathogens. Pn-AMP1 is a hevein-type plant AMP that shows potent and broad-spectrum antifungal activity. Genome-wide chemogenomic screening was performed using heterozygous and homozygous diploid deletion pools of Saccharomyces cerevisiae as a chemogenetic model system to identify genes whose deletion conferred enhanced sensitivity to Pn-AMP1. This assay identified 44 deletion strains with fitness defects in the presence of Pn-AMP1. Strong fitness defects were observed in strains with deletions of genes encoding components of several pathways and complex known to participate in the adaptive response to alkaline pH stress, including the cell wall integrity (CWI), calcineurin/Crz1, Rim101, SNF1 pathways and endosomal sorting complex required for transport (ESCRT complex). Gene ontology (GO) enrichment analysis of these genes revealed that the most highly overrepresented GO term was "cellular response to alkaline pH". We found that 32 of the 44 deletion strains tested (72 %) showed significant growth defects compared with their wild type at alkaline pH. Furthermore, 9 deletion strains (20 %) exhibited enhanced sensitivity to Pn-AMP1 at ambient pH compared to acidic pH. Although several hundred plant AMPs have been reported, their modes of action remain largely uncharacterized. This study demonstrates that the signaling pathways that coordinate the adaptive response to alkaline pH also confer resistance to a hevein-type plant AMP in S. cerevisiae. Our findings have broad implications for the design of novel and potent antifungal agents.

Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting

PubMed Central

Di Giovanni, Jerome; Sheng, Zu-Hang

2015-01-01

Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin−/− neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin−/− phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca2+ sensor, we demonstrate that snapin–dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca2+ sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting. PMID:26108535

Dual-responsive polyplexes with enhanced disassembly and endosomal escape for efficient delivery of siRNA.

PubMed

Zhu, Jia; Qiao, Mingxi; Wang, Qi; Ye, Yuqing; Ba, Shuang; Ma, Jingjing; Hu, Haiyang; Zhao, Xiuli; Chen, Dawei

2018-04-01

Despite the extracellular barriers for siRNA delivery have been overcome by utilizing advanced nanoparticle delivery systems, the key intracellular barriers after internalization including efficient disassembly of siRNA and endosomal escape still remains challenging. To address the issues, we developed a unique pH- and redox potential-responsive polyplex delivery system based on the copolymer of mPEG-b-PLA-PHis-ssPEI1.8 k, which is composed of a pH-responsive copolymer of PEG-b-PLA-PHis (Mw 5 k) and a branched PEI (Mw1.8 k) linked with redox cleavable disulfide bond. The copolymer showed excellent siRNA complexation and protection abilities against endogenous substances at the relatively low N/P ratio of 6. The siRNA release from the polyplexes (N/P 6) was markedly increased from 13.62% to 58.67% under conditions simulating the endosomal microenvironment. Fluorescence resonance energy transfer (FRET) test also indicated a higher disassembly extent of siRNA from the copolymer. The accelerated siRNA release from the polyplexes was markedly restrained when the N/P ratio was raised above 10 due to the increasing of electrostatic interactions. The efficient endosomal escape of siRNA after internalization was confirmed by confocal microscopy, which was attributed to the cleavaged PEI chains inducing membrane destabilization, the "proton sponge effect" of PHis and PEI as well as the relative small size of after disassembly. The enhanced disassembly and endosomal escape were elucidated as the leading cause for polyplexes (N/P 6) showed more efficient Bcl-2 silencing (85.45%) than those polyplexes with higher N/P ratios (N/P 10 and 15). In vivo results further demonstrated that polyplexes (N/P 6) delivery of siBcl-2 significantly inhibited the MCF-7 breast tumor growth as compared to its counterparts. The incorporation of convertible non-electrical interactions at a balance with electrostatic interactions in complexation siRNA has been demonstrated as an effective strategy to achieve efficient disassembly from stable polyplexes. Moreover, polyplexes equipped with the enhanced disassembly and endosomal escape provides a new potential way to tackle the intracellular delivery bottleneck for siRNA delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

PubMed Central

Woo, Sang Su; James, Declan J.; Martin, Thomas F. J.

2017-01-01

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

Cortactin promotes exosome secretion by controlling branched actin dynamics

PubMed Central

Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Seiki, Motoharu; Tyska, Matthew J.

2016-01-01

Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

Cortactin promotes exosome secretion by controlling branched actin dynamics.

PubMed

Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

2016-07-18

Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. © 2016 Sinha et al.

The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis.

PubMed

Coonrod, Emily M; Stevens, Tom H

2010-12-01

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication

PubMed Central

Sindhwani, Aastha; Kaur, Harmeet; Tuli, Amit

2017-01-01

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor—HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication. PMID:29084291

Efficient Endocytic Uptake and Maturation in Drosophila Oocytes Requires Dynamitin/p50

PubMed Central

Liu, Guojun; Sanghavi, Paulomi; Bollinger, Kathryn E.; Perry, Libby; Marshall, Brendan; Roon, Penny; Tanaka, Tsubasa; Nakamura, Akira; Gonsalvez, Graydon B.

2015-01-01

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. In this study, we examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of Yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. We therefore propose that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor colocalized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation. PMID:26265702

The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain.

PubMed

Shi, Hang; Rojas, Raul; Bonifacino, Juan S; Hurley, James H

2006-06-01

The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29 and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1-A resolution reveals two curved beta-sandwich domains connected by a polar core and a flexible linker. Vps26 has an unpredicted structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a glycine in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.

CC2D1A and CC2D1B regulate degradation and signaling of EGFR and TLR4.

PubMed

Deshar, Rakesh; Cho, Eun-Bee; Yoon, Sungjoo Kim; Yoon, Jong-Bok

2016-11-11

Signaling through many transmembrane receptors is terminated by their sorting to the intraluminal vesicles (ILVs) of multivescular bodies (MVBs) and subsequent lysosomal degradation. ILV formation requires the endosomal sorting complex required for transport (ESCRT) machinery. CC2D1A and CC2D1B interact with the CHMP4 family of proteins, the major subunit of the ESCRT-III complex, however, their roles in receptor degradation and signaling are poorly defined. Here, we report that CC2D1A binds to CHMP4B polymers formed on endosomes to regulate the endosomal sorting pathway. We show that depletion of CC2D1A and B accelerates degradation of EGFR and elicits rapid termination of its downstream signaling through ERK1 and 2. Depletion of CC2D1A and B promotes sorting of EGFR to ILV leading to its rapid lysosomal degradation. In addition, we show that knockdown of CC2D1A and B has similar effects on degradation and downstream signaling of another membrane receptor, TLR4. Thus, these findings suggest that CC2D1A and B may have broad effects on transmembrane receptors by preventing premature ILV sorting and termination of signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

PubMed

Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

2016-08-31

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

PubMed

Dolnik, Olga; Stevermann, Lea; Kolesnikova, Larissa; Becker, Stephan

2015-01-01

Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding. Copyright © 2015 Elsevier GmbH. All rights reserved.

Endothelin-converting Enzyme 1 and β-Arrestins Exert Spatiotemporal Control of Substance P-induced Inflammatory Signals*

PubMed Central

Jensen, Dane D.; Halls, Michelle L.; Murphy, Jane E.; Canals, Meritxell; Cattaruzza, Fiore; Poole, Daniel P.; Lieu, TinaMarie; Koon, Hon-Wai; Pothoulakis, Charalabos; Bunnett, Nigel W.

2014-01-01

Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins β-arrestin (βARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and βARR1/2 terminate plasma membrane Ca2+ signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. βARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from βARRs, to recycle. Thus, both ECE-1 and βARRs mediate the resensitization of NK1R Ca2+ signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or βARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of βARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation. PMID:24898255

Signal dependent transport of a membrane cargo from early endosomes to recycling endosomes.

PubMed

Mahmoud, Ismail S; Louber, Jade; Dower, Steve K; Verhagen, Anne M; Gleeson, Paul A

2017-08-01

Many membrane cargoes undergo endocytosis and intracellular recycling to the plasma membrane via the early endosomes and the recycling endosomes. However whether specific sorting signals are required for transport from early endosomes to recycling endosomes is not known and the current view is that transport to the recycling endosomes is by a passive default process. Here we show that the cytoplasmic tail of the neonatal Fc receptor (FcRn) contains discrete signals for endocytosis and for sorting to the recycling endosomes. The FcRn cytoplasmic tail has previously been shown to contain the unusual WISL motif for AP2/clathrin-mediated endocytosis. By analysing FcRn mutants and CD8/FcRn chimeric molecules, we have identified an extended WISL sequence (GLPAPWISL) which promotes sorting from the early endosomes to the recycling endosomes. The insertion of GLPAPWISL into the cytoplasmic tail of CD8 resulted in efficient endocytosis and trafficking to the recycling endosomes, with only low levels detected in the late endosomes. Replacement of the highly conserved GLAPAP sequence within the GLPAPWISL motif with alanine residues resulted in endocytosis of the CD8/FcRn chimera to the early endosomes which was then trafficked predominantly to the late endosomes rather than the recycling endosomes. These studies demonstrate that signals within the cytoplasmic domains of membrane cargo can mediate active transport from early to recycling endosomes. Copyright © 2017 Elsevier GmbH. All rights reserved.

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

PubMed Central

McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

2012-01-01

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

PubMed Central

Lippé, Roger; Miaczynska, Marta; Rybin, Vladimir; Runge, Anja; Zerial, Marino

2001-01-01

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases. PMID:11452015

WASH and WAVE actin regulators of the Wiskott-Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes.

PubMed

Jia, Da; Gomez, Timothy S; Metlagel, Zoltan; Umetani, Junko; Otwinowski, Zbyszek; Rosen, Michael K; Billadeau, Daniel D

2010-06-08

We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

PubMed Central

Prabhu, Yogikala; Burgos, Patricia V.; Schindler, Christina; Farías, Ginny G.; Magadár, Javier G.; Bonifacino, Juan S.

2012-01-01

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway. PMID:22553349

Two barcodes encoded by the type-1 PDZ and by phospho-Ser312 regulate retromer/WASH-mediated sorting of the ß1-adrenergic receptor from endosomes to the plasma membrane.

PubMed

Nooh, Mohammed M; Bahouth, Suleiman W

2017-01-01

Recycling of the majority of agonist-internalized GPCR is dependent on a type I-PDZ "barcode" in their C-tail. The recycling of wild-type (WT) ß 1 -AR is also dependent on its default "type-1 PDZ barcode", but trafficking of the ß 1 -AR is inhibited when PKA or its substrate serine at position 312 (Ser 312 ) are inactivated. We tested the hypothesis that phospho-Ser 312 provided a second barcode for ß 1 -AR sorting from endosomes to the plasma membrane by determining the role of retromer/WASH complexes in ß 1 -AR trafficking. Recycling of WT ß 1 -AR or WT ß 2 -AR was dependent on targeting the retromer to endosomal membranes via SNX3 and rab7a, and on complexing the retromer to the WASH pentamer via the C-tail of FAM21 (FAM21 C ). These maneuvers however, did not inhibit the recycling of a phospho-Ser 312 ß 1 -AR mimic ((S312D) ß 1 -AR). Knockdown of the trans-acting PDZ protein sorting nexin27 (SNX27) inhibited the recycling of WT ß 1 -AR and WT ß 2 -AR, but had no effect on (S312D) ß 1 -AR∆PDZ or on phosphorylation of WT ß 1 -AR by PKA at Ser 312 . However, depletion of FKBP15, a FAM21 C -binding endosomal protein, selectively inhibited WT ß 1 -AR but not ß 2 -AR recycling, suggesting divergence might exist in GPCR trafficking roadmaps. These results indicate that two barcodes are involved in sorting WT ß 1 -AR out of early endosomes. The first and antecedent "barcode" was the "type-1 PDZ", followed by a second reversible "phospho-Ser 312 " verification "barcode". This organization allows tight regulation of ß 1 -AR density to signaling intensity in conditions associated with aberrant ß 1 -AR signaling such as in hypertension and heart failure. Copyright © 2016 Elsevier Inc. All rights reserved.

Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

PubMed

Vandenbulcke, F; Nouel, D; Vincent, J P; Mazella, J; Beaudet, A

2000-09-01

The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

Endosomal Interactions during Root Hair Growth.

PubMed

von Wangenheim, Daniel; Rosero, Amparo; Komis, George; Šamajová, Olga; Ovečka, Miroslav; Voigt, Boris; Šamaj, Jozef

2015-01-01

The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes-termed herein as dancing-endosomes-which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth.

From synapse to nucleus and back again--communication over distance within neurons.

PubMed

Fainzilber, Mike; Budnik, Vivian; Segal, Rosalind A; Kreutz, Michael R

2011-11-09

How do neurons integrate intracellular communication from synapse to nucleus and back? Here we briefly summarize aspects of this topic covered by a symposium at Neuroscience 2011. A rich repertoire of signaling mechanisms link both dendritic terminals and axon tips with neuronal soma and nucleus, using motor-dependent transport machineries to traverse the long intracellular distances along neuronal processes. Activation mechanisms at terminals include localized translation of dendritic or axonal RNA, proteolytic cleavage of receptors or second messengers, and differential phosphorylation of signaling moieties. Signaling complexes may be transported in endosomes, or as non-endosomal complexes associated with importins and dynein. Anterograde transport of RNA granules from the soma to neuronal processes, coupled with retrograde transport of proteins translated locally at terminals or within processes, may fuel ongoing bidirectional communication between soma and synapse to modulate synaptic plasticity as well as neuronal growth and survival decisions.

Protoparvovirus Knocking at the Nuclear Door.

PubMed

Mäntylä, Elina; Kann, Michael; Vihinen-Ranta, Maija

2017-10-02

Protoparvoviruses target the nucleus due to their dependence on the cellular reproduction machinery during the replication and expression of their single-stranded DNA genome. In recent years, our understanding of the multistep process of the capsid nuclear import has improved, and led to the discovery of unique viral nuclear entry strategies. Preceded by endosomal transport, endosomal escape and microtubule-mediated movement to the vicinity of the nuclear envelope, the protoparvoviruses interact with the nuclear pore complexes. The capsids are transported actively across the nuclear pore complexes using nuclear import receptors. The nuclear import is sometimes accompanied by structural changes in the nuclear envelope, and is completed by intranuclear disassembly of capsids and chromatinization of the viral genome. This review discusses the nuclear import strategies of protoparvoviruses and describes its dynamics comprising active and passive movement, and directed and diffusive motion of capsids in the molecularly crowded environment of the cell.

The Recycling Endosome of Madin-Darby Canine Kidney Cells Is a Mildly Acidic Compartment Rich in Raft Components

PubMed Central

Gagescu, Raluca; Demaurex, Nicolas; Parton, Robert G.; Hunziker, Walter; Huber, Lukas A.; Gruenberg, Jean

2000-01-01

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway. PMID:10930469

Effectiveness of a dynein team in a tug of war helped by reduced load sensitivity of detachment: evidence from the study of bidirectional endosome transport in D. discoideum.

PubMed

Bhat, Deepak; Gopalakrishnan, Manoj

2012-08-01

Bidirectional cargo transport by molecular motors in cells is a complex phenomenon in which the cargo (usually a vesicle) alternately moves in retrograde and anterograde directions. In this case, teams of oppositely pulling motors (e.g., kinesin and dynein) bind to the cargo, simultaneously, and 'coordinate' their activity such that the motion consists of spells of positively and negatively directed segments, separated by pauses of varying duration. A set of recent experiments have analyzed the bidirectional motion of endosomes in the amoeba D. discoideum in detail. It was found that in between directional switches, a team of five to six dyneins stall a cargo against a stronger kinesin in a tug of war, which lasts for almost a second. As the mean detachment time of a kinesin under its stall load was also observed to be ∼1 s, we infer that the collective detachment time of the dynein assembly must also be similar. Here, we analyze this inference from a modeling perspective, using experimentally measured single-molecule parameters as inputs. We find that the commonly assumed exponential load-dependent detachment rate is inconsistent with observations, as it predicts that a five-dynein assembly will detach under its combined stall load in less than a hundredth of a second. A modified model where the load-dependent unbinding rate is assumed to saturate at stall-force level for super-stall loads gives results which are in agreement with experimental data. Our analysis suggests that the load-dependent detachment of a dynein in a team is qualitatively different at sub-stall and super-stall loads, a conclusion which is likely to have implications in other situations involving collective effects of many motors.

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

PubMed

Bahouth, Suleiman W; Nooh, Mohammed M

2017-08-01

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction pathway in membranes. Copyright © 2017. Published by Elsevier Inc.

Intracellular trafficking pathways for nuclear delivery of plasmid DNA complexed with highly efficient endosome escape polymers.

PubMed

Gillard, Marianne; Jia, Zhongfan; Hou, Jeff Jia Cheng; Song, Michael; Gray, Peter P; Munro, Trent P; Monteiro, Michael J

2014-10-13

Understanding the pathways for nuclear entry could see vast improvements in polymer design for the delivery of genetic materials to cells. Here, we use a novel diblock copolymer complexed with plasmid DNA (pDNA) to determine both its cellular entry and nuclear pathways. The diblock copolymer (A-C3) is specifically designed to bind and protect pDNA, release it at a specific time, but more importantly, rapidly escape the endosome. The copolymer was taken up by HEK293 cells preferentially via the clathrin-mediated endocytosis (CME) pathway, and the pDNA entered the nucleus to produce high gene expression levels in all cells after 48 h, a similar observation to the commercially available polymer transfection agent, PEI Max. This demonstrates that the polymers must first escape the endosome and then mediate transport of pDNA to the nucleus for occurrence of gene expression. The amount of pDNA within the nucleus was found to be higher for our A-C3 polymer than PEI Max, with our polymer delivering 7 times more pDNA than PEI Max after 24 h. We further found that entry into the nucleus was primarily through the small nuclear pores and did not occur during mitosis when the nuclear envelope becomes compromised. The observation that the polymers are also found in the nucleus supports the hypothesis that the large pDNA/polymer complex (size ~200 nm) must dissociate prior to nucleus entry and that cationic and hydrophobic monomer units on the polymer may facilitate active transport of the pDNA through the nuclear pore.

A transport and retention mechanism for the sustained distal localization of Spn-F-IKKε during Drosophila bristle elongation.

PubMed

Otani, Tetsuhisa; Oshima, Kenzi; Kimpara, Akiyo; Takeda, Michiko; Abdu, Uri; Hayashi, Shigeo

2015-07-01

Stable localization of the signaling complex is essential for the robust morphogenesis of polarized cells. Cell elongation involves molecular signaling centers that coordinately regulate intracellular transport and cytoskeletal structures. In Drosophila bristle elongation, the protein kinase IKKε is activated at the distal tip of the growing bristle and regulates the shuttling movement of recycling endosomes and cytoskeletal organization. However, how the distal tip localization of IKKε is established and maintained during bristle elongation is unknown. Here, we demonstrate that IKKε distal tip localization is regulated by Spindle-F (Spn-F), which is stably retained at the distal tip and functions as an adaptor linking IKKε to cytoplasmic dynein. We found that Javelin-like (Jvl) is a key regulator of Spn-F retention. In jvl mutant bristles, IKKε and Spn-F initially localize to the distal tip but fail to be retained there. In S2 cells, particles that stain positively for Jvl or Spn-F move in a microtubule-dependent manner, whereas Jvl and Spn-F double-positive particles are immobile, indicating that Jvl and Spn-F are transported separately and, upon forming a complex, immobilize each other. These results suggest that polarized transport and selective retention regulate the distal tip localization of the Spn-F-IKKε complex during bristle cell elongation. © 2015. Published by The Company of Biologists Ltd.

The FgVps39-FgVam7-FgSso1 Complex Mediates Vesicle Trafficking and Is Important for the Development and Virulence of Fusarium graminearum.

PubMed

Li, Bing; Liu, Luping; Li, Ying; Dong, Xin; Zhang, Haifeng; Chen, Huaigu; Zheng, Xiaobo; Zhang, Zhengguang

2017-05-01

Vesicle trafficking is an important event in eukaryotic organisms. Many proteins and lipids transported between different organelles or compartments are essential for survival. These processes are mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Rab-GTPases, and multisubunit tethering complexes such as class C core vacuole or endosome tethering and homotypic fusion or vacuole protein sorting (HOPS). Our previous study has demonstrated that FgVam7, which encodes a SNARE protein involving in vesicle trafficking, plays crucial roles in growth, asexual or sexual development, deoxynivalenol production, and pathogenicity in Fusarium graminearum. Here, the affinity purification approach was used to identify FgVam7-interacting proteins to explore its regulatory mechanisms during vesicle trafficking. The orthologs of yeast Vps39, a HOPS tethering complex subunit, and Sso1, a SNARE protein localized to the vacuole or endosome, were identified and selected for further characterization. In yeast two-hybrid and glutathione-S-transferase pull-down assays, FgVam7, FgVps39, and FgSso1 interacted with each other as a complex. The ∆Fgvps39 mutant generated by targeted deletion was significantly reduced in vegetative growth and asexual development. It failed to produce sexual spores and was defective in plant infection and deoxynivalenol production. Further cellular localization and cytological examinations suggested that FgVps39 is involved in vesicle trafficking from early or late endosomes to vacuoles in F. graminearum. Additionally, the ∆Fgvps39 mutant was defective in vacuole morphology and autophagy, and it was delayed in endocytosis. Our results demonstrate that FgVam7 interacts with FgVps39 and FgSso1 to form a unique complex, which is involved in vesicle trafficking and modulating the proper development of infection-related morphogenesis in F. graminearum.

Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities.

PubMed

Gauthier, Sébastien A; Pérez-González, Rocío; Sharma, Ajay; Huang, Fang-Ke; Alldred, Melissa J; Pawlik, Monika; Kaur, Gurjinder; Ginsberg, Stephen D; Neubert, Thomas A; Levy, Efrat

2017-08-29

A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer's disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.

The Arabidopsis USL1 controls multiple aspects of development by affecting late endosome morphology.

PubMed

Yuan, Rongrong; Lan, Jingqiu; Fang, Yuxing; Yu, Hao; Zhang, Jinzhe; Huang, Jiaying; Qin, Genji

2018-06-13

The polar transport of auxin controls many aspects of plant development. However, the molecular mechanisms underlying auxin tranport regulation remain to be further elucidated. We identified a mutant named as usl1 (unflattened and small leaves) in a genetic screen in Arabidopsis thaliana. The usl1 displayed multiple aspects of developmental defects in leaves, embryogenesis, cotyledons, silique phyllotaxy and lateral roots in addition to abnormal leaves. USL1 encodes a protein orthologous to the yeast vacuolar protein sorting (Vps) 38p and human UV RADIATION RESISTANCE-ASSOCIATED GENE (UVRAG). Cell biology, Co-IP/MS and yeast two-hybrid were used to identify the function of USL1. USL1 colocalizes at the subcellular level with VPS29, a key factor of the retromer complex that controls auxin transport. The morphology of the VPS29-associated late endosomes (LE) is altered from small dots in the wild-type to aberrant enlarged circles in the usl1 mutants. The usl1 mutant synergistically interacts with vps29. We also found that USL1 forms a complex with AtVPS30 and AtVPS34. We propose that USL1 controls multiple aspects of plant development by affecting late endosome morphology and by regulating the PIN1 polarity. Our findings provide a new layer of the understanding on the mechanisms of plant development regulation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Specific recycling receptors are targeted to the immune synapse by the intraflagellar transport system

PubMed Central

Finetti, Francesca; Patrussi, Laura; Masi, Giulia; Onnis, Anna; Galgano, Donatella; Lucherini, Orso Maria; Pazour, Gregory J.; Baldari, Cosima T.

2014-01-01

ABSTRACT T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5+ endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis. PMID:24554435

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

NASA Astrophysics Data System (ADS)

Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

2013-08-01

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles.

PubMed

Somiya, Masaharu; Yoshimoto, Nobuo; Iijima, Masumi; Niimi, Tomoaki; Dewa, Takehisa; Jung, Joohee; Kuroda, Shun'ichi

2012-06-15

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs.

PubMed

Chen, Mo; Qiu, Tao; Wu, Jiajie; Yang, Yang; Wright, Graham D; Wu, Min; Ge, Ruowen

2018-03-09

Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein.

PubMed

Nabhan, Joseph F; Hu, Ruoxi; Oh, Raymond S; Cohen, Stanley N; Lu, Quan

2012-03-13

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.

Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion

PubMed Central

Mankouri, Jamel; Walter, Cheryl; Stewart, Hazel; Bentham, Matthew; Park, Wei Sun; Heo, Won Do; Fukuda, Mitsunori

2016-01-01

ABSTRACT The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins. IMPORTANCE The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition, we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects a unique modification to cellular trafficking pathways. PMID:27226379

The pH-Triggered Triblock Nanocarrier Enabled Highly Efficient siRNA Delivery for Cancer Therapy.

PubMed

Du, Lili; Zhou, Junhui; Meng, Lingwei; Wang, Xiaoxia; Wang, Changrong; Huang, Yuanyu; Zheng, Shuquan; Deng, Liandong; Cao, Huiqing; Liang, Zicai; Dong, Anjie; Cheng, Qiang

2017-01-01

Small interfering RNA (siRNA) therapies have been hampered by lack of delivery systems in the past decades. Nowadays, a few promising vehicles for siRNA delivery have been developed and it is gradually revealed that enhancing siRNA release from endosomes into cytosol is a very important factor for successful delivery. Here, we designed a novel pH-sensitive nanomicelle, PEG-PTTMA-P(GMA-S-DMA) (PTMS), for siRNA delivery. Owing to rapid hydrolysis in acidic environment, PTMS NPs underwent hydrophobic-to-hydrophilic transition in endosomes that enabled combination of proton sponge effect and raised osmotic pressure in endosomes, resulting in vigorous release of siRNAs from endosomes into cytosol. In vitro results demonstrated that PTMS/siRNA complexes exhibited excellent gene silencing effects in several cell lines. Their gene silencing efficiency could reach ~91%, ~87% and ~90% at the N/P ratio of 50/1 in MDA-MB-231, A549 and Hela cells respectively, which were better than that obtained with Lipofectamine 2000. The highly efficient gene silencing was then proven from enhanced siRNA endosomal release, which is mainly attributed to pH-triggered degradation of polymer and acid-accelerated siRNA release. In vivo experiments indicated that NPs/siRNA formulation rapidly accumulated in tumor sites after i.v. injection. Tumor growth was effectively inhibited and ~45% gene knockdown efficacy was determined at the siRRM2 dose of 1mg/kg. Meanwhile, no significant toxicity was observed during the whole treatment. We also found that PTMS/siRNA formulations could lead to significant gene silencing effects in liver (~63%) and skin (~80%) when injected by i.v. and s.c., respectively. This research work gives a rational strategy to optimize siRNA delivery systems for tumor treatments.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

PubMed

Woo, Sang Su; James, Declan J; Martin, Thomas F J

2017-03-15

Munc13-4 is a Ca 2+ -dependent SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca 2+ -evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca 2+ -binding C2 domains functions as a Ca 2+ sensor for SG exocytosis. Unexpectedly, Ca 2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4 + /Rab7 + /Rab11 + endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4 + /Rab7 + SGs, followed by a merge with Rab11 + endosomes, and depended on Ca 2+ binding to Munc13-4. Munc13-4 promoted the Ca 2+ -stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca 2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. © 2017 Woo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Rab11 in Recycling Endosomes Regulates the Sorting and Basolateral Transport of E-CadherinV⃞

PubMed Central

Lock, John G.; Stow, Jennifer L.

2005-01-01

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion. PMID:15689490

HIV-1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and recycling

PubMed Central

Dirk, Brennan S.; Pawlak, Emily N.; Johnson, Aaron L.; Van Nynatten, Logan R.; Jacob, Rajesh A.; Heit, Bryan; Dikeakos, Jimmy D.

2016-01-01

A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the cell. Furthermore, super-resolution imaging identified that the Nef:MHC-I BiFC complex transits through both early and late endosomes before ultimately residing at the trans-Golgi network. Together we demonstrate the importance of the early stages of the endocytic network in the removal of MHC-I from the cell surface and its re-localization within the cell, which allows HIV-1 to optimally evade host immune responses. PMID:27841315

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.

PubMed

Groppelli, Elisabetta; Levy, Hazel C; Sun, Eileen; Strauss, Mike; Nicol, Clare; Gold, Sarah; Zhuang, Xiaowei; Tuthill, Tobias J; Hogle, James M; Rowlands, David J

2017-02-01

Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes

PubMed Central

Vonderheit, Andreas

2005-01-01

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles. PMID:15954801

Endosomal Interactions during Root Hair Growth

PubMed Central

von Wangenheim, Daniel; Rosero, Amparo; Komis, George; Šamajová, Olga; Ovečka, Miroslav; Voigt, Boris; Šamaj, Jozef

2016-01-01

The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes—termed herein as dancing-endosomes—which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth. PMID:26858728

A Unique N-Terminal Sequence in the Carnation Italian ringspot virus p36 Replicase-Associated Protein Interacts with the Host Cell ESCRT-I Component Vps23

PubMed Central

Richardson, Lynn G. L.; Clendening, Eric A.; Sheen, Hyukho; Gidda, Satinder K.; White, K. Andrew

2014-01-01

ABSTRACT Like most positive-strand RNA viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. The tombusvirus Tomato bushy stunt virus (TBSV) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (ESCRT). ESCRT is comprised of a series of protein subcomplexes (i.e., ESCRT-0 -I, -II, and -III) that normally participate in late endosome biogenesis and some of which are also hijacked by certain enveloped retroviruses (e.g., HIV) for viral budding from the plasma membrane. Here we show that the replication of Carnation Italian ringspot virus (CIRV), a tombusvirus that replicates at mitochondrial membranes also relies on ESCRT. In plant cells, CIRV recruits the ESCRT-I protein, Vps23, to mitochondria through an interaction that involves a unique region in the N terminus of the p36 replicase-associated protein that is not conserved in TBSV or other peroxisome-targeted tombusviruses. The interaction between p36 and Vps23 also involves the Vps23 C-terminal steadiness box domain and not its N-terminal ubiquitin E2 variant domain, which in the case of TBSV (and enveloped retroviruses) mediates the interaction with ESCRT. Overall, these results provide evidence that CIRV uses a unique N-terminal sequence for the recruitment of Vps23 that is distinct from those used by TBSV and certain mammalian viruses for ESCRT recruitment. Characterization of this novel interaction with Vps23 contributes to our understanding of how CIRV may have evolved to exploit key differences in the plant ESCRT machinery. IMPORTANCE Positive-strand RNA viruses replicate their genomes in association with specific host cell membranes. To accomplish this, cellular components responsible for membrane biogenesis and modeling are appropriated by viral proteins and redirected to assemble membrane-bound viral replicase complexes. The diverse pathways leading to the formation of these replication structures are poorly understood. We have determined that the cellular ESCRT system that is normally responsible for mediating late endosome biogenesis is also involved in the replication of the tombusvirus Carnation Italian ringspot virus (CIRV) at mitochondria. Notably, CIRV recruits ESCRT to the mitochondrial outer membrane via an interaction between a unique motif in the viral protein p36 and the ESCRT component Vps23. Our findings provide new insights into tombusvirus replication and the virus-induced remodeling of plant intracellular membranes, as well as normal ESCRT assembly in plants. PMID:24672030

Endosomal Redox Signaling in the Antiphospholipid Syndrome.

PubMed

Lackner, Karl J; Manukyan, Davit; Müller-Calleja, Nadine

2017-04-01

It is well established that the antiphospholipid syndrome (APS) is caused by antiphospholipid antibodies (aPL). While several underlying mechanisms have been described in the past, many open questions remain. Here, we will review data on endosomal signaling and, in particular, redox signaling in APS. Endosomal redox signaling has been implicated in several cellular processes including signaling of proinflammatory cytokines. We have shown that certain aPL can activate endosomal NADPH-oxidase (NOX) in several cell types followed by induction of proinflammatory and procoagulant cellular responses in vitro. Involvement of endosomes in aPL signaling has also been reported by others. In wild-type mice but not in NOX-deficient mice, aPL accelerate venous thrombus formation underscoring the relevance of endosomal NOX. Furthermore, hydroxychloroquine (HCQ) inhibits activation of endosomal NOX and prevents thrombus formation in aPL-treated mice. Endosomal redox signaling is an important novel mechanism involved in APS pathogenesis. This makes endosomes a potential target for future treatment approaches of APS.

UNC93B1 mediates differential trafficking of endosomal TLRs

PubMed Central

Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

2013-01-01

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.00291.001 PMID:23426999

ESCRT-II/Vps25 constrains digit number by endosome-mediated selective modulation of FGF-SHH signaling.

PubMed

Handschuh, Karen; Feenstra, Jennifer; Koss, Matthew; Ferretti, Elisabetta; Risolino, Maurizio; Zewdu, Rediet; Sahai, Michelle A; Bénazet, Jean-Denis; Peng, Xiao P; Depew, Michael J; Quintana, Laura; Sharpe, James; Wang, Baolin; Alcorn, Heather; Rivi, Roberta; Butcher, Stephen; Manak, J Robert; Vaccari, Thomas; Weinstein, Harel; Anderson, Kathryn V; Lacy, Elizabeth; Selleri, Licia

2014-10-23

Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here, we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis, we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25(ENU)). Unlike Vps25-null embryos we generated, Vps25(ENU/ENU) mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyperactivation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25(ENU/ENU) Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however, SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

PubMed Central

Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

2013-01-01

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm. PMID:23947891

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes.

PubMed

Vetrivel, Kulandaivelu S; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C; Xu, Huaxi; Thinakaran, Gopal

2004-10-22

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes*

PubMed Central

Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C.; Xu, Huaxi; Thinakaran, Gopal

2005-01-01

Alzheimer’s disease-associated β-amyloid peptides (Aβ) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major γ-secretase in neurons is a palmi-toylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the γ-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1−/−/PS2−/− and NCT−/− fibroblasts, γ-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires γ-secretase complex assembly. Biochemical evidence shows that subunits of the γ-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of γ-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP. PMID:15322084

Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells

PubMed Central

Eichholz, Karsten; Bru, Thierry; Tran, Thi Thu Phuong; Fernandes, Paulo; Mennechet, Franck J. D.; Manel, Nicolas; Alves, Paula; Perreau, Matthieu

2016-01-01

Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles containing a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health standards. Following repeat exposure to multiple HAdV types, we develop robust and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and persistent HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein containing a caspase activation/recruitment domain) aggregation, inflammasome formation, caspase 1 activation, and IL-1β and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs. PMID:27636895

Endothelin-converting enzyme 1 and β-arrestins exert spatiotemporal control of substance P-induced inflammatory signals.

PubMed

Jensen, Dane D; Halls, Michelle L; Murphy, Jane E; Canals, Meritxell; Cattaruzza, Fiore; Poole, Daniel P; Lieu, TinaMarie; Koon, Hon-Wai; Pothoulakis, Charalabos; Bunnett, Nigel W

2014-07-18

Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins β-arrestin (βARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and βARR1/2 terminate plasma membrane Ca(2+) signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. βARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from βARRs, to recycle. Thus, both ECE-1 and βARRs mediate the resensitization of NK1R Ca(2+) signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or βARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of βARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Polyamidoamine-Decorated Nanodiamonds as a Hybrid Gene Delivery Vector and siRNA Structural Characterization at the Charged Interfaces.

PubMed

Lim, Dae Gon; Rajasekaran, Nirmal; Lee, Dukhee; Kim, Nam Ah; Jung, Hun Soon; Hong, Sungyoul; Shin, Young Kee; Kang, Eunah; Jeong, Seong Hoon

2017-09-20

Nanodiamonds have been discovered as a new exogenous material source in biomedical applications. As a new potent form of nanodiamond (ND), polyamidoamine-decorated nanodiamonds (PAMAM-NDs) were prepared for E7 or E6 oncoprotein-suppressing siRNA gene delivery for high risk human papillomavirus-induced cervical cancer, such as types 16 and 18. It is critical to understand the physicochemical properties of siRNA complexes immobilized on cationic solid ND surfaces in the aspect of biomolecular structural and conformational changes, as the new inert carbon material can be extended into the application of a gene delivery vector. A spectral study of siRNA/PAMAM-ND complexes using differential scanning calorimetry and circular dichroism spectroscopy proved that the hydrogen bonding and electrostatic interactions between siRNA and PAMAM-NDs decreased endothermic heat capacity. Moreover, siRNA/PAMAM-ND complexes showed low cell cytotoxicity and significant suppressing effects for forward target E6 and E7 oncogenic genes, proving functional and therapeutic efficacy. The cellular uptake of siRNA/PAMAM-ND complexes at 8 h was visualized by macropinocytes and direct endosomal escape of the siRNA/PAMAM-ND complexes. It is presumed that PAMAM-NDs provided a buffering cushion to adjust the pH and hard mechanical stress to escape endosomes. siRNA/PAMAM-ND complexes provide a potential organic/inorganic hybrid material source for gene delivery carriers.

WIPI1 Coordinates Melanogenic Gene Transcription and Melanosome Formation via TORC1 Inhibition*

PubMed Central

Ho, Hsiang; Kapadia, Rubina; Al-Tahan, Sejad; Ahmad, Safoora; Ganesan, Anand K.

2011-01-01

Recent studies implicate a role for WD repeat domain, phosphoinositide-interacting 1 (WIPI1) in the biogenesis of melanosomes, cell type-specific lysosome-related organelles. In this study, we determined that WIPI1, an ATG18 homologue that is shown to localize to both autophagosomes and early endosomes, inhibited mammalian target of rapamycin (MTOR) signaling, leading to increased transcription of melanogenic enzymes and the formation of mature melanosomes. WIPI1 suppressed the target of rapamycin complex 1 (TORC1) activity, resulting in glycogen synthase kinase 3β inhibition, β-Catenin stabilization, and increased transcription of microphthalmia transcription factor and its target genes. WIPI1-depleted cells accumulated stage I melanosomes but lacked stage III-IV melanosomes. Inhibition of TORC1 by rapamycin treatment resulted in the accumulation of stage IV melanosomes but not autophagosomes, whereas starvation resulted in the formation of autophagosomes but not melanin accumulation. Taken together, our studies define a distinct role for WIPI1 and TORC1 signaling in controlling the transcription of melanogenic enzymes and melanosome maturation, a process that is distinct from starvation-induced autophagy. PMID:21317285

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.

PubMed

Wang, Ming; Zuris, John A; Meng, Fantao; Rees, Holly; Sun, Shuo; Deng, Pu; Han, Yong; Gao, Xue; Pouli, Dimitra; Wu, Qi; Georgakoudi, Irene; Liu, David R; Xu, Qiaobing

2016-03-15

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL.

PubMed

Bartuzi, Paulina; Billadeau, Daniel D; Favier, Robert; Rong, Shunxing; Dekker, Daphne; Fedoseienko, Alina; Fieten, Hille; Wijers, Melinde; Levels, Johannes H; Huijkman, Nicolette; Kloosterhuis, Niels; van der Molen, Henk; Brufau, Gemma; Groen, Albert K; Elliott, Alison M; Kuivenhoven, Jan Albert; Plecko, Barbara; Grangl, Gernot; McGaughran, Julie; Horton, Jay D; Burstein, Ezra; Hofker, Marten H; van de Sluis, Bart

2016-03-11

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking.

The structure and function of presynaptic endosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jähne, Sebastian, E-mail: sebastian.jaehne1@stud.uni-goettingen.de; International Max Planck Research School for Neurosciences, 37077 Göttingen; Rizzoli, Silvio O.

The function of endosomes and of endosome-like structures in the presynaptic compartment is still controversial. This is in part due to the absence of a consensus on definitions and markers for these compartments. Synaptic endosomes are sometimes seen as stable organelles, permanently present in the synapse. Alternatively, they are seen as short-lived intermediates in synaptic vesicle recycling, arising from the endocytosis of large vesicles from the plasma membrane, or from homotypic fusion of small vesicles. In addition, the potential function of the endosome is largely unknown in the synapse. Some groups have proposed that the endosome is involved in themore » sorting of synaptic vesicle proteins, albeit others have produced data that deny this possibility. In this review, we present the existing evidence for synaptic endosomes, we discuss their potential functions, and we highlight frequent technical pitfalls in the analysis of this elusive compartment. We also sketch a roadmap to definitely determine the role of synaptic endosomes for the synaptic vesicle cycle. Finally, we propose a common definition of synaptic endosome-like structures.« less

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

PubMed Central

Upadhyay, Srijana; Xu, Xinping

2016-01-01

ABSTRACT Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. PMID:27879337

Discovery of a vezatin-like protein for dynein-mediated early endosome transport

PubMed Central

Yao, Xuanli; Arst, Herbert N.; Wang, Xiangfeng; Xiang, Xin

2015-01-01

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo. PMID:26378255

Syndapin/SDPN-1 is required for endocytic recycling and endosomal actin association in the Caenorhabditis elegans intestine

PubMed Central

Gleason, Adenrele M.; Nguyen, Ken C. Q.; Hall, David H.; Grant, Barth D.

2016-01-01

Syndapin/pascin-family F-BAR domain proteins bind directly to membrane lipids and are associated with actin dynamics at the plasma membrane. Previous reports also implicated mammalian syndapin 2 in endosome function during receptor recycling, but precise analysis of a putative recycling function for syndapin in mammalian systems is difficult because of its effects on the earlier step of endocytic uptake and potential redundancy among the three separate genes that encode mammalian syndapin isoforms. Here we analyze the endocytic transport function of the only Caenorhabditis elegans syndapin, SDPN-1. We find that SDPN-1 is a resident protein of the early and basolateral recycling endosomes in the C. elegans intestinal epithelium, and sdpn-1 deletion mutants display phenotypes indicating a block in basolateral recycling transport. sdpn-1 mutants accumulate abnormal endosomes positive for early endosome and recycling endosome markers that are normally separate, and such endosomes accumulate high levels of basolateral recycling cargo. Furthermore, we observed strong colocalization of endosomal SDPN-1 with the F-actin biosensor Lifeact and found that loss of SDPN-1 greatly reduced Lifeact accumulation on early endosomes. Taken together, our results provide strong evidence for an in vivo function of syndapin in endocytic recycling and suggest that syndapin promotes transport via endosomal fission. PMID:27630264

A role for the ESCRT system in cell division in archaea.

PubMed

Samson, Rachel Y; Obita, Takayuki; Freund, Stefan M; Williams, Roger L; Bell, Stephen D

2008-12-12

Archaea are prokaryotic organisms that lack endomembrane structures. However, a number of hyperthermophilic members of the Kingdom Crenarchaea, including members of the Sulfolobus genus, encode homologs of the eukaryotic endosomal sorting system components Vps4 and ESCRT-III (endosomal sorting complex required for transport-III). We found that Sulfolobus ESCRT-III and Vps4 homologs underwent regulation of their expression during the cell cycle. The proteins interacted and we established the structural basis of this interaction. Furthermore, these proteins specifically localized to the mid-cell during cell division. Overexpression of a catalytically inactive mutant Vps4 in Sulfolobus resulted in the accumulation of enlarged cells, indicative of failed cell division. Thus, the archaeal ESCRT system plays a key role in cell division.

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

PubMed Central

Lucas, María; Gaspar, Andrew H.; Pallara, Chiara; Rojas, Adriana Lucely; Fernández-Recio, Juan; Machner, Matthias P.; Hierro, Aitor

2014-01-01

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization. PMID:25114243

Checkpoints for vesicular traffic?

PubMed

Fiset, A; Faure, R

2001-01-01

During interphase the transport of material between different intracellular organelles requires accurate regulation of fusiogenic domains. Recent studies on hepatic endosomes indicated that compartmentalized Cdk2-cyclin E complexes act by braking fusion events. These Cdk2 complexes integrate tyrosine phosphorylation and dephosphorylation inputs, resulting in the control of the number of rounds of fusion at discrete domains. This leads to changes in the intracellular location of internalized receptors and ultimately their biological response.

AP1S3 Mutations Are Associated with Pustular Psoriasis and Impaired Toll-like Receptor 3 Trafficking

PubMed Central

Setta-Kaffetzi, Niovi; Simpson, Michael A.; Navarini, Alexander A.; Patel, Varsha M.; Lu, Hui-Chun; Allen, Michael H.; Duckworth, Michael; Bachelez, Hervé; Burden, A. David; Choon, Siew-Eng; Griffiths, Christopher E.M.; Kirby, Brian; Kolios, Antonios; Seyger, Marieke M.B.; Prins, Christa; Smahi, Asma; Trembath, Richard C.; Fraternali, Franca; Smith, Catherine H.; Barker, Jonathan N.; Capon, Francesca

2014-01-01

Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis. PMID:24791904

Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish

PubMed Central

Clark, Brian S.; Winter, Mark; Cohen, Andrew R.; Link, Brian A.

2011-01-01

The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions. PMID:21976318

Fusion of Enveloped Viruses in Endosomes

PubMed Central

White, Judith M.; Whittaker, Gary R.

2016-01-01

Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH, and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion triggering mechanisms. A key take home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors, and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion. PMID:26935856

A Novel Type III Endosome Transmembrane Protein, TEMP

PubMed Central

Aturaliya, Rajith N.; Kerr, Markus C.; Teasdale, Rohan D.

2012-01-01

As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP’s plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport. PMID:24710541

An albumin nanocomplex-based endosomal pH-activatable on/off probe system.

PubMed

Lee, Changkyu; Lee, Seunghyun; Thao, Le Quang; Hwang, Ha Shin; Kim, Jong Oh; Lee, Eun Seong; Oh, Kyung Taek; Shin, Beom Soo; Choi, Han-Gon; Youn, Yu Seok

2016-08-01

Albumin has gained considerable interest as a material for fabricating nanoparticulate systems due to its biomedical advantages, such as biocompatibility and chemical functionality. Here, we report a new pH-sensitive albumin nanocomplex prototype with a zinc-imidazole coordination bond. Albumin was conjugated with 1-(3-aminopropyl)imidazole and mPEG10kDa-NHS, and the resulting albumin conjugate (PBI) was then modified with either Cy5.5 or BHQ-3. The newly formed albumin nanocomplex (C/BQ-PBI Zn NCs: ∼116nm) system was facilely self-assembled around pH 7.4 in the presence of Zn(2+), but it quickly disassembled in an acidic environment (∼pH 5.0). Based on this pH-sensitivity, C/BQ-PBI Zn NCs emitted strong near-infrared fluorescence and released Zn(2+), turning "off" at pH ∼7.4 (e.g., plasma) and "on" at pH ∼5.0 (e.g., endo/lysosomes in tumor cells) on account of fluorescence resonance energy transfer. C/BQ-PBI Zn NCs displayed significant cytotoxicity due to an increase in cellular Zn(2+) in response to endosomal pH (∼5.0) in breast cancer MCF-7 cells and lung adenocarcinoma A549 cells. Particularly, confocal laser scanning microscopic images showed a strong fluorescence signal caused by the disassembly of C/BQ-PBI Zn NCs in the endosomal region of MCF-7 cells. Based on these results, we believe that this albumin nanocomplex is an attractive biocompatible tumor targeting probe carrier for the theranostic purpose. Copyright © 2016 Elsevier B.V. All rights reserved.

Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin.

PubMed

Feinstein, Timothy N; Yui, Naofumi; Webber, Matthew J; Wehbi, Vanessa L; Stevenson, Hilary P; King, J Darwin; Hallows, Kenneth R; Brown, Dennis; Bouley, Richard; Vilardaga, Jean-Pierre

2013-09-27

The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na(+) transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel "noncanonical" regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex.

Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin

PubMed Central

Chia, Pei Zhi Cheryl; Gasnereau, Isabelle; Lieu, Zi Zhao; Gleeson, Paul A.

2011-01-01

The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail. PMID:21693586

The COMMD Family Regulates Plasma LDL Levels and Attenuates Atherosclerosis Through Stabilizing the CCC Complex in Endosomal LDLR Trafficking.

PubMed

Fedoseienko, Alina; Wijers, Melinde; Wolters, Justina C; Dekker, Daphne; Smit, Marieke; Huijkman, Nicolette; Kloosterhuis, Niels; Klug, Helene; Schepers, Aloys; Willems van Dijk, Ko; Levels, Johannes H M; Billadeau, Daniel D; Hofker, Marten H; van Deursen, Jan; Westerterp, Marit; Burstein, Ezra; Kuivenhoven, Jan Albert; van de Sluis, Bart

2018-06-08

COMMD (copper metabolism MURR1 domain)-containing proteins are a part of the CCC (COMMD-CCDC22 [coiled-coil domain containing 22]-CCDC93 [coiled-coil domain containing 93]) complex facilitating endosomal trafficking of cell surface receptors. Hepatic COMMD1 inactivation decreases CCDC22 and CCDC93 protein levels, impairs the recycling of the LDLR (low-density lipoprotein receptor), and increases plasma low-density lipoprotein cholesterol levels in mice. However, whether any of the other COMMD members function similarly as COMMD1 and whether perturbation in the CCC complex promotes atherogenesis remain unclear. The main aim of this study is to unravel the contribution of evolutionarily conserved COMMD proteins to plasma lipoprotein levels and atherogenesis. Using liver-specific Commd1 , Commd6 , or Commd9 knockout mice, we investigated the relation between the COMMD proteins in the regulation of plasma cholesterol levels. Combining biochemical and quantitative targeted proteomic approaches, we found that hepatic COMMD1, COMMD6, or COMMD9 deficiency resulted in massive reduction in the protein levels of all 10 COMMDs. This decrease in COMMD protein levels coincided with destabilizing of the core (CCDC22, CCDC93, and chromosome 16 open reading frame 62 [C16orf62]) of the CCC complex, reduced cell surface levels of LDLR and LRP1 (LDLR-related protein 1), followed by increased plasma low-density lipoprotein cholesterol levels. To assess the direct contribution of the CCC core in the regulation of plasma cholesterol levels, Ccdc22 was deleted in mouse livers via CRISPR/Cas9-mediated somatic gene editing. CCDC22 deficiency also destabilized the complete CCC complex and resulted in elevated plasma low-density lipoprotein cholesterol levels. Finally, we found that hepatic disruption of the CCC complex exacerbates dyslipidemia and atherosclerosis in ApoE3*Leiden mice. Collectively, these findings demonstrate a strong interrelationship between COMMD proteins and the core of the CCC complex in endosomal LDLR trafficking. Hepatic disruption of either of these CCC components causes hypercholesterolemia and exacerbates atherosclerosis. Our results indicate that not only COMMD1 but all other COMMDs and CCC components may be potential targets for modulating plasma lipid levels in humans. © 2018 American Heart Association, Inc.

Stochastic acidification, activation of hemagglutinin and escape of influenza viruses from an endosome

NASA Astrophysics Data System (ADS)

Lagache, Thibault; Sieben, Christian; Meyer, Tim; Herrmann, Andreas; Holcman, David

2017-06-01

Influenza viruses enter the cell inside an endosome. During the endosomal journey, acidification triggers a conformational change of the virus spike protein hemagglutinin (HA) that results in escape of the viral genome from the endosome into the cytoplasm. It is still unclear how the interplay between acidification and HA conformation changes affects the kinetics of the viral endosomal escape. We develop here a stochastic model to estimate the change of conformation of HAs inside the endosome nanodomain. Using a Markov process, we model the arrival of protons to HA binding sites and compute the kinetics of their accumulation. We compute the Mean First Passage Time (MFPT) of the number of HA bound sites to a threshold, which is used to estimate the HA activation rate for a given pH concentration. The present analysis reveals that HA proton binding sites possess a high chemical barrier, ensuring a stability of the spike protein at sub-acidic pH. We predict that activating more than 3 adjacent HAs is necessary to trigger endosomal fusion and this configuration prevents premature release of viruses from early endosomes

APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

PubMed Central

Kalaidzidis, Inna; Miaczynska, Marta; Brewińska-Olchowik, Marta; Hupalowska, Anna; Ferguson, Charles; Parton, Robert G.; Kalaidzidis, Yannis

2015-01-01

Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway. PMID:26459602

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

PubMed

Sriram, V; Krishnan, K S; Mayor, Satyajit

2003-05-12

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Shedding PEG Palisade by Temporal Photostimulation and Intracellular Reducing Milieu for Facilitated Intracellular Trafficking and DNA Release.

PubMed

Wang, Tieyan; Chen, Qixian; Lu, Hongguang; Li, Wei; Li, Zaifen; Ma, Jianbiao; Gao, Hui

2016-08-17

The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired us to develop an intracellularly sheddable PEG palisade for synthetic delivery systems. Here, we attempted to conjugate PEG to polyethylenimine (PEI) through tandem linkages of disulfide-bridge susceptible to cytoplasmic reduction and an azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosomes upon photoirradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellularly charged species, ultimately contributing to efficient gene expression.

GPCR Signaling and Trafficking: The Long and Short of It

PubMed Central

Pavlos, Nathan J.; Friedman, Peter A.

2016-01-01

Emerging findings disclose unexpected components of G protein-coupled receptor (GPCR) signaling and cell biology. Select GPCRs exhibit classical signaling that is restricted to cell membranes and newly described persistent signaling that depends on internalization of the GPCR bound to β-arrestins. Termination of non-canonical endosomal signaling requires intraluminal acidification and sophisticated protein trafficking machineries. Recent studies reveal the structural determinants of the trafficking chaperones. This review summarizes advances in GPCR signaling and trafficking with a focus on the parathyroid hormone receptor as prototype, and the actin-SNX27-retromer tubule complex, an endosomal sorting hub responsible for recycling and preservation of cell surface receptors. The findings are integrated into a model of PTHR trafficking with implications for signal transduction, bone growth, and mineral-ion metabolism. PMID:27889227

Rab2 promotes autophagic and endocytic lysosomal degradation

PubMed Central

Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Scita, Giorgio

2017-01-01

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. PMID:28483915

A phorbol ester-binding protein is required downstream of Rab5 in endosome fusion.

PubMed

Aballay, A; Barbieri, M A; Colombo, M I; Arenas, G N; Stahl, P D; Mayorga, L S

1998-12-28

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.

RAB-7 Antagonizes LET-23 EGFR Signaling during Vulva Development in Caenorhabditis elegans

PubMed Central

Skorobogata, Olga; Rocheleau, Christian E.

2012-01-01

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(−) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans. PMID:22558469

RAB-7 antagonizes LET-23 EGFR signaling during vulva development in Caenorhabditis elegans.

PubMed

Skorobogata, Olga; Rocheleau, Christian E

2012-01-01

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.

Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.

2009-09-11

G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesizemore » that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.« less

Changes in membrane lipids drive increased endocytosis following Fas ligation.

PubMed

Degli Esposti, Mauro; Matarrese, Paola; Tinari, Antonella; Longo, Agostina; Recalchi, Serena; Khosravi-Far, Roya; Malorni, Walter; Misasi, Roberta; Garofalo, Tina; Sorice, Maurizio

2017-05-01

Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles.

The Arf6 GTPase-activating proteins ARAP2 and ACAP1 define distinct endosomal compartments that regulate integrin α5β1 traffic.

PubMed

Chen, Pei-Wen; Luo, Ruibai; Jian, Xiaoying; Randazzo, Paul A

2014-10-31

Arf6 and the Arf6 GTPase-activating protein (GAP) ACAP1 are established regulators of integrin traffic important to cell adhesion and migration. However, the function of Arf6 with ACAP1 cannot explain the range of Arf6 effects on integrin-based structures. We propose that Arf6 has different functions determined, in part, by the associated Arf GAP. We tested this idea by comparing the Arf6 GAPs ARAP2 and ACAP1. We found that ARAP2 and ACAP1 had opposing effects on apparent integrin β1 internalization. ARAP2 knockdown slowed, whereas ACAP1 knockdown accelerated, integrin β1 internalization. Integrin β1 association with adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif (APPL)-positive endosomes and EEA1-positive endosomes was affected by ARAP2 knockdown and depended on ARAP2 GAP activity. ARAP2 formed a complex with APPL1 and colocalized with Arf6 and APPL in a compartment distinct from the Arf6/ACAP1 tubular recycling endosome. In addition, although ACAP1 and ARAP2 each colocalized with Arf6, they did not colocalize with each other and had opposing effects on focal adhesions (FAs). ARAP2 overexpression promoted large FAs, but ACAP1 overexpression reduced FAs. Taken together, the data support a model in which Arf6 has at least two sites of opposing action defined by distinct Arf6 GAPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Calnuc Function in Endosomal Sorting of Lysosomal Receptors.

PubMed

Larkin, Heidi; Costantino, Santiago; Seaman, Matthew N J; Lavoie, Christine

2016-04-01

Calnuc is a ubiquitous Ca(2+)-binding protein present on the trans-Golgi network (TGN) and endosomes. However, the precise role of Calnuc in these organelles is poorly characterized. We previously highlighted the role of Calnuc in the transport of LRP9, a new member of a low-density lipoprotein (LDL) receptor subfamily that cycles between the TGN and endosomes. The objective of this study was to explore the role of Calnuc in the endocytic sorting of mannose-6-phosphate receptor (MPR) and Sortilin, two well-characterized lysosomal receptors that transit between the TGN and endosomes. Using biochemical and microscopy assays, we showed that Calnuc depletion [by small interfering RNA (siRNA)] causes the misdelivery to and degradation in lysosomes of cationic-independent mannose-6-phosphate receptor (CI-MPR) and Sortilin due to a defect in the endosomal recruitment of retromers, which are key components of the endosome-to-Golgi retrieval machinery. Indeed, we demonstrated that Calnuc depletion impairs the activation and membrane association of Rab7, a small G protein required for the endosomal recruitment of retromers. Overall, our data indicate a novel role for Calnuc in the endosome-to-TGN retrograde transport of lysosomal receptors through the regulation of Rab7 activity and the recruitment of retromers to endosomes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

PubMed

Cifuentes-Muñoz, Nicolás; Sun, Weina; Ray, Greeshma; Schmitt, Phuong Tieu; Webb, Stacy; Gibson, Kathleen; Dutch, Rebecca Ellis; Schmitt, Anthony P

2017-07-15

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites. IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly. Copyright © 2017 American Society for Microbiology.

PIKfyve mediates the motility of late endosomes and lysosomes in neuronal dendrites.

PubMed

Tsuruta, Fuminori; Dolmetsch, Ricardo E

2015-09-25

The endosome/lysosome system in the nervous system is critically important for a variety of neuronal functions such as neurite outgrowth, retrograde transport, and synaptic plasticity. In neurons, the endosome/lysosome system is crucial for the activity-dependent internalization of membrane proteins and contributes to the regulation of lipid level on the plasma membrane. Although homeostasis of membrane dynamics plays important roles in the properties of central nervous systems, it has not been elucidated how endosome/lysosome system is regulated. Here, we report that phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) mediates the motility of late endosomes and lysosomes in neuronal dendrites. Endosomes and lysosomes are highly motile in resting neurons, however knockdown of PIKfyve led to a significant reduction in late endosomes and lysosomes motility. We also found that vesicle acidification is crucial for their motility and PIKfyve is associated with this process indirectly. These data suggest that PIKfyve mediates vesicle motility through the regulation of vesicle integrity in neurons. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes

PubMed Central

Villaseñor, Roberto; Nonaka, Hidenori; Del Conte-Zerial, Perla; Kalaidzidis, Yannis; Zerial, Marino

2015-01-01

An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response. DOI: http://dx.doi.org/10.7554/eLife.06156.001 PMID:25650738

Enhanced Endosomal Escape by Light-Fueled Liquid-Metal Transformer.

PubMed

Lu, Yue; Lin, Yiliang; Chen, Zhaowei; Hu, Quanyin; Liu, Yang; Yu, Shuangjiang; Gao, Wei; Dickey, Michael D; Gu, Zhen

2017-04-12

Effective endosomal escape remains as the "holy grail" for endocytosis-based intracellular drug delivery. To date, most of the endosomal escape strategies rely on small molecules, cationic polymers, or pore-forming proteins, which are often limited by the systemic toxicity and lack of specificity. We describe here a light-fueled liquid-metal transformer for effective endosomal escape-facilitated cargo delivery via a chemical-mechanical process. The nanoscale transformer can be prepared by a simple approach of sonicating a low-toxicity liquid-metal. When coated with graphene quantum dots (GQDs), the resulting nanospheres demonstrate the ability to absorb and convert photoenergy to drive the simultaneous phase separation and morphological transformation of the inner liquid-metal core. The morphological transformation from nanospheres to hollow nanorods with a remarkable change of aspect ratio can physically disrupt the endosomal membrane to promote endosomal escape of payloads. This metal-based nanotransformer equipped with GQDs provides a new strategy for facilitating effective endosomal escape to achieve spatiotemporally controlled drug delivery with enhanced efficacy.

Spatiotemporal Dynamics of Adenovirus Membrane Rupture and Endosomal Escape

PubMed Central

Maier, Oana; Marvin, Shauna A.; Wodrich, Harald; Campbell, Edward M.

2012-01-01

A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live- and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1+) early endosomes or LAMP-1+ late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3+ endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3+ endosomes, pVI appears to remain associated with the gal3+ ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events. PMID:22855481

Pathogenic cascades in lysosomal disease-Why so complex?

PubMed

Walkley, S U

2009-04-01

Lysosomal disease represents a large group of more than 50 clinically recognized conditions resulting from inborn errors of metabolism affecting the organelle known as the lysosome. The lysosome is an integral part of the larger endosomal/lysosomal system, and is closely allied with the ubiquitin-proteosomal and autophagosomal systems, which together comprise essential cell machinery for substrate degradation and recycling, homeostatic control, and signalling. More than two-thirds of lysosomal diseases affect the brain, with neurons appearing particularly vulnerable to lysosomal compromise and showing diverse consequences ranging from specific axonal and dendritic abnormalities to neuron death. While failure of lysosomal function characteristically leads to lysosomal storage, new studies argue that lysosomal diseases may also be appropriately viewed as 'states of deficiency' rather than simply overabundance (storage). Interference with signalling events and salvage processing normally controlled by the endosomal/lysosomal system may represent key mechanisms accounting for the inherent complexity of lysosomal disorders. Analysis of lysosomal disease pathogenesis provides a unique window through which to observe the importance of the greater lysosomal system for normal cell health.

GRAF1 forms a complex with MICAL-L1 and EHD1 to cooperate in tubular recycling endosome vesiculation

PubMed Central

Cai, Bishuang; Xie, Shuwei; Caplan, Steve; Naslavsky, Naava

2014-01-01

The biogenesis of tubular recycling endosomes (TREs) and their subsequent vesiculation after cargo-sorting has occurred, is essential for receptor and lipid recycling to the plasma membrane. Although recent studies have implicated the C-terminal Eps15 Homology Domain (EHD) protein, EHD1, as a key regulator of TRE vesiculation, additional proteins involved in this process have been largely uncharacterized. In the present study, we identify the GTPase Regulator Associated with Focal adhesion kinase-1 (GRAF1) protein in a complex with EHD1 and the TRE hub protein, Molecules Interacting with CasL-Like1 (MICAL-L1). Over-expression of GRAF1 caused vesiculation of MICAL-L1-containing TRE, whereas GRAF1-depletion led to impaired TRE vesiculation and delayed receptor recycling. Moreover, co-addition of purified EHD1 and GRAF1 in a semi-permeabilized cell vesiculation assay produced synergistic TRE vesiculation. Overall, based on our data, we suggest that in addition to its roles in clathrin-independent endocytosis, GRAF1 synergizes with EHD1 to support TRE vesiculation. PMID:25364729

Soluble Glucan Is Internalized and Trafficked to the Golgi Apparatus in Macrophages via a Clathrin-Mediated, Lipid Raft-Regulated Mechanism

PubMed Central

Goldman, Matthew P.; Kalbfleisch, John H.; Williams, David L.

2012-01-01

Glucans are natural product carbohydrates that stimulate immunity. Glucans are internalized by the pattern recognition receptor, Dectin-1. Glucans were thought to be trafficked to phagolysosomes, but this is unproven. We examined the internalization and trafficking of soluble glucans in macrophages. Incubation of macrophages with glucan resulted in internalization of Dectin-1 and glucan. Inhibition of clathrin blocked internalization of the Dectin-1/glucan complex. Lipid raft depletion resulted in decreased Dectin levels and glucan uptake. Once internalized, glucans colocalized with early endosomes at 0 to 15 min, with the Golgi apparatus at 15 min to 24 h, and with Dectin-1 immediately (0 h) and again later (15 min-24 h). Glucans did not colocalize with lysosomes at any time interval examined. We conclude that the internalization of Dectin-1/glucan complexes in macrophages is mediated by clathrin and negatively regulated by lipid rafts and/or caveolin-1. Upon internalization, soluble glucans are trafficked via endosomes to the Golgi apparatus, not lysosomes. PMID:22700434

CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

PubMed Central

Bartuzi, Paulina; Billadeau, Daniel D.; Favier, Robert; Rong, Shunxing; Dekker, Daphne; Fedoseienko, Alina; Fieten, Hille; Wijers, Melinde; Levels, Johannes H.; Huijkman, Nicolette; Kloosterhuis, Niels; van der Molen, Henk; Brufau, Gemma; Groen, Albert K.; Elliott, Alison M.; Kuivenhoven, Jan Albert; Plecko, Barbara; Grangl, Gernot; McGaughran, Julie; Horton, Jay D.; Burstein, Ezra; Hofker, Marten H.; van de Sluis, Bart

2016-01-01

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking. PMID:26965651

Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly

PubMed Central

Shen, Qing-Tao; Schuh, Amber L.; Zheng, Yuqing; Quinney, Kyle; Wang, Lei; Hanna, Michael; Mitchell, Julie C.; Otegui, Marisa S.; Ahlquist, Paul; Cui, Qiang

2014-01-01

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts. PMID:25202029

Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry.

PubMed

Qiu, Shirley; Leung, Anders; Bo, Yuxia; Kozak, Robert A; Anand, Sai Priya; Warkentin, Corina; Salambanga, Fabiola D R; Cui, Jennifer; Kobinger, Gary; Kobasa, Darwyn; Côté, Marceline

2018-01-01

For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P 2 ), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P 2 -positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P 2 production in cells to promote efficient delivery to NPC1. Copyright © 2017 Elsevier Inc. All rights reserved.

Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R.

PubMed

Filippakis, Harilaos; Alesi, Nicola; Ogorek, Barbara; Nijmeh, Julie; Khabibullin, Damir; Gutierrez, Catherine; Valvezan, Alexander J; Cunningham, James; Priolo, Carmen; Henske, Elizabeth P

2017-06-13

Tuberous sclerosis complex (TSC) is a multisystem disease associated with hyperactive mTORC1. The impact of TSC1/2 deficiency on lysosome-mediated processes is not fully understood. We report here that inhibition of lysosomal function using chloroquine (CQ) upregulates cholesterol homeostasis genes in TSC2-deficient cells. This TSC2-dependent transcriptional signature is associated with increased accumulation and intracellular levels of both total cholesterol and cholesterol esters. Unexpectedly, engaging this CQ-induced cholesterol uptake pathway together with inhibition of de novo cholesterol synthesis allows survival of TSC2-deficient, but not TSC2-expressing cells. The underlying mechanism of TSC2-deficient cell survival is dependent on exogenous cholesterol uptake via LDL-R, and endosomal trafficking mediated by Vps34. Simultaneous inhibition of lysosomal and endosomal trafficking inhibits uptake of esterified cholesterol and cell growth in TSC2-deficient, but not TSC2-expressing cells, highlighting the TSC-dependent lysosome-mediated regulation of cholesterol homeostasis and pointing toward the translational potential of these pathways for the therapy of TSC.

AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking.

PubMed

Setta-Kaffetzi, Niovi; Simpson, Michael A; Navarini, Alexander A; Patel, Varsha M; Lu, Hui-Chun; Allen, Michael H; Duckworth, Michael; Bachelez, Hervé; Burden, A David; Choon, Siew-Eng; Griffiths, Christopher E M; Kirby, Brian; Kolios, Antonios; Seyger, Marieke M B; Prins, Christa; Smahi, Asma; Trembath, Richard C; Fraternali, Franca; Smith, Catherine H; Barker, Jonathan N; Capon, Francesca

2014-05-01

Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

PubMed

Mizuno, Tadahaya; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

2015-06-01

By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRβ inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRβ interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRβ and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system. © 2015 American Heart Association, Inc.

Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis.

PubMed

Marsh, M; Schmid, S; Kern, H; Harms, E; Male, P; Mellman, I; Helenius, A

1987-04-01

Endosomes are prelysosomal organelles that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organelles. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postnuclear supernatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP-dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 micrograms protein) sufficient for biochemical, immunological, and functional analysis.

GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

PubMed Central

Toh, Wei Hong; Chia, Pei Zhi Cheryl; Hossain, Mohammed Iqbal; Gleeson, Paul A.

2018-01-01

The diversion of the membrane-bound β-site amyloid precursor protein–(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production. PMID:29142073

Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin*

PubMed Central

Feinstein, Timothy N.; Yui, Naofumi; Webber, Matthew J.; Wehbi, Vanessa L.; Stevenson, Hilary P.; King, J. Darwin; Hallows, Kenneth R.; Brown, Dennis; Bouley, Richard; Vilardaga, Jean-Pierre

2013-01-01

The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na+ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex. PMID:23935101

Modifications of the endosomal compartment in peripheral blood mononuclear cells and fibroblasts from Alzheimer's disease patients

PubMed Central

Corlier, F; Rivals, I; Lagarde, J; Hamelin, L; Corne, H; Dauphinot, L; Ando, K; Cossec, J-C; Fontaine, G; Dorothée, G; Malaplate-Armand, C; Olivier, J-L; Dubois, B; Bottlaender, M; Duyckaerts, C; Sarazin, M; Potier, M-C; Alnajjar-Carpentier, Dr Amer; Logak, Dr Michel; Leder, Dr Sara; Marchal, Dr Dominique; Pitti-Ferandi, Dr Hélène; Brugeilles, Dr Hélene; Roualdes, Dr Brigitte; Michon, Dr Agnes

2015-01-01

Identification of blood-based biomarkers of Alzheimer's disease (AD) remains a challenge. Neuropathological studies have identified enlarged endosomes in post-mortem brains as the earliest cellular change associated to AD. Here the presence of enlarged endosomes was investigated in peripheral blood mononuclear cells from 48 biologically defined AD patients (25 with mild cognitive impairment and 23 with dementia (AD-D)), and 23 age-matched healthy controls using immunocytochemistry and confocal microscopy. The volume and number of endosomes were not significantly different between AD and controls. However, the percentage of cells containing enlarged endosomes was significantly higher in the AD-D group as compared with controls. Furthermore, endosomal volumes significantly correlated to [C11]PiB cortical index measured by positron emission tomography in the AD group, independently of the APOE genotype, but not to the levels of amyloid-beta, tau and phosphorylated tau measured in the cerebrospinal fluid. Importantly, we confirmed the presence of enlarged endosomes in fibroblasts from six unrelated AD-D patients as compared with five cognitively normal controls. This study is the first, to our knowledge, to report morphological alterations of the endosomal compartment in peripheral cells from AD patients correlated to amyloid load that will now be evaluated as a possible biomarker. PMID:26151923

Pathogenic mechanisms in lysosomal disease: a reappraisal of the role of the lysosome.

PubMed

Walkley, Steven U

2007-04-01

The view that lysosomes simply represent end organelles in the serial degradation of polymeric molecules derived from the cell surface and its interior has led to major misconceptions about the nature of lysosomal storage diseases and the pathogenic cascades that characterize them. Accordingly, lysosomal storage bodies are often considered 'inert', inducing cell dysfunction and death primarily through mechanical overcrowding of normal organelles or by other non-specific means leading to generalized cytotoxicity. However, modern studies of lysosomes and their component proteins provide evidence to support a far greater role for these organelles in cell metabolism. In intimate association with endosomal, autophagosomal and related vesicular systems, the greater lysosomal system can be conceptualized as a vital recycling centre that serves as a central metabolic coordinator, influencing literally every aspect of the cell, from signal transduction to regulation of gene expression. This broader view of the role of lysosomes in cells not only provides insight into how single gene defects impacting on lysosomal function can result in the plethora of complex cellular transformations characteristic of these diseases, but also suggests new and innovative therapies that may hold considerable promise for ameliorating disease progression.

Variable-angle epifluorescence microscopy characterizes protein dynamics in the vicinity of plasma membrane in plant cells.

PubMed

Chen, Tong; Ji, Dongchao; Tian, Shiping

2018-03-14

The assembly of protein complexes and compositional lipid patterning act together to endow cells with the plasticity required to maintain compositional heterogeneity with respect to individual proteins. Hence, the applications for imaging protein localization and dynamics require high accuracy, particularly at high spatio-temporal level. We provided experimental data for the applications of Variable-Angle Epifluorescence Microscopy (VAEM) in dissecting protein dynamics in plant cells. The VAEM-based co-localization analysis took penetration depth and incident angle into consideration. Besides direct overlap of dual-color fluorescence signals, the co-localization analysis was carried out quantitatively in combination with the methodology for calculating puncta distance and protein proximity index. Besides, simultaneous VAEM tracking of cytoskeletal dynamics provided more insights into coordinated responses of actin filaments and microtubules. Moreover, lateral motility of membrane proteins was analyzed by calculating diffusion coefficients and kymograph analysis, which represented an alternative method for examining protein motility. The present study presented experimental evidence on illustrating the use of VAEM in tracking and dissecting protein dynamics, dissecting endosomal dynamics, cell structure assembly along with membrane microdomain and protein motility in intact plant cells.

PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells

NASA Astrophysics Data System (ADS)

Kim, Sang-Woo; Lee, Yeon Kyung; Lee, Jong Yeon; Hong, Jeong Hee; Khang, Dongwoo

2017-11-01

Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

Comparison of the cellular transport mechanism of cationic, star-shaped polymers and liposomes in HaCat cells.

PubMed

Luo, Heng-Cong; Li, Na; Yan, Li; Mai, Kai-Jin; Sun, Kan; Wang, Wei; Lao, Guo-Juan; Yang, Chuan; Zhang, Li-Ming; Ren, Meng

2017-01-01

Several biological barriers must be overcome to achieve efficient nonviral gene delivery. These barriers include target cell uptake, lysosomal degradation, and dissociation from the carrier. In this study, we compared the differences in the uptake mechanism of cationic, star-shaped polymer/MMP-9siRNA complexes (β-CD-(D3)7/MMP-9siRNA complexes: polyplexes) and commercial liposome/MMP-9siRNA complexes (Lipofectamine ® 2000/MMP-9siRNA complexes: liposomes). The uptake pathway and transfection efficiency of the polyplexes and liposomes were determined by fluorescence microscopy, flow cytometry, and reverse transcriptase-polymerase chain reaction. The occurrence of intracellular processing was assessed by confocal laser scanning microscopy. Endosomal acidification inhibitors were used to explore the endosomal escape mechanisms of the polyplexes and lysosomes. We concluded that the polyplexes were internalized by non-caveolae- and non-clathrin-mediated pathways, with no lysosomal trafficking, thereby inducing successful transfection, while the majority of liposomes were internalized by clathrin-dependent endocytosis (CDE), caveolae-mediated endocytosis, and macropinocytosis, and only CDE induced successful transfection. Liposomes might escape more quickly than polyplexes, and the digestion effect of acidic organelles on liposomes was faint compared to the polyplexes, although both complexes escaped from endolysosomes via the proton sponge mechanism. This may be the key aspect that leads to the lower transfection efficiency of the β-CD-(D3)7/MMP-9siRNA complexes. The present study may offer some insights for the rational design of novel delivery systems with increased transfection efficiency but decreased toxicity.

Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy*

PubMed Central

Morozova, Kateryna; Clement, Cristina C.; Kaushik, Susmita; Stiller, Barbara; Arias, Esperanza; Ahmad, Atta; Rauch, Jennifer N.; Chatterjee, Victor; Melis, Chiara; Scharf, Brian; Gestwicki, Jason E.; Cuervo, Ana-Maria; Zuiderweg, Erik R. P.; Santambrogio, Laura

2016-01-01

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 μm. This interaction is specific and involves a total of 4–5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes. PMID:27405763

Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy.

PubMed

Morozova, Kateryna; Clement, Cristina C; Kaushik, Susmita; Stiller, Barbara; Arias, Esperanza; Ahmad, Atta; Rauch, Jennifer N; Chatterjee, Victor; Melis, Chiara; Scharf, Brian; Gestwicki, Jason E; Cuervo, Ana-Maria; Zuiderweg, Erik R P; Santambrogio, Laura

2016-08-26

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 μm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The structure of the yeast plasma membrane SNARE complex reveals destabilizing water-filled cavities.

PubMed

Strop, Pavel; Kaiser, Stephen E; Vrljic, Marija; Brunger, Axel T

2008-01-11

SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7A resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

Differential Requirements in Endocytic Trafficking for Penetration of Dengue Virus

PubMed Central

Acosta, Eliana G.; Castilla, Viviana; Damonte, Elsa B.

2012-01-01

The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection. PMID:22970315

The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis.

PubMed

Spitzer, Christoph; Li, Faqiang; Buono, Rafael; Roschzttardtz, Hannetz; Chung, Taijoon; Zhang, Min; Osteryoung, Katherine W; Vierstra, Richard D; Otegui, Marisa S

2015-02-01

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents. © 2015 American Society of Plant Biologists. All rights reserved.

Silica nanogelling of environment-responsive PEGylated polyplexes for enhanced stability and intracellular delivery of siRNA.

PubMed

Gouda, Noha; Miyata, Kanjiro; Christie, R James; Suma, Tomoya; Kishimura, Akihiro; Fukushima, Shigeto; Nomoto, Takahiro; Liu, Xueying; Nishiyama, Nobuhiro; Kataoka, Kazunori

2013-01-01

In this study, poly(ethylene glycol) (PEG)-block-polycation/siRNA complexes (PEGylated polyplexes) were wrapped with a hydrated silica, termed "silica nanogelling", in order to enhance their stability and functionality. Silica nanogelling was achieved by polycondensation of soluble silicates onto the surface of PEGylated polyplexes comprising a disulfide cross-linked core. Formation of silica nanogel layer on the PEGylated cross-linked polyplexes was confirmed by particle size increase, surface charge reduction, and elemental analysis of transmission electron micrographs. Silica nanogelling substantially improved polyplex stability against counter polyanion-induced dissociation under non-reductive condition, without compromising the reductive environment-responsive siRNA release triggered by disulfide cleavage. Silica nanogelling significantly enhanced the sequence-specific gene silencing activity of the polyplexes in HeLa cells without associated cytotoxicity, probably due lower endosomal entrapment (or lysosomal degradation) of delivered siRNA. The lower endosomal entrapment of the silica nanogel system could be explained by an accelerated endosomal escape triggered by deprotonated silanol groups in the silica (the proton sponge hypothesis) and/or a modulated intracellular trafficking, possibly via macropinocytosis, as evidenced by the cellular uptake inhibition assay. Henceforth, silica nanogelling of PEGylated siRNA polyplexes is a promising strategy for preparation of stable and functional siRNA delivery vehicles. Copyright © 2012 Elsevier Ltd. All rights reserved.

Integrated experimental and model-based analysis reveals the spatial aspects of EGFR activation dynamics

PubMed Central

Shankaran, Harish; Zhang, Yi; Chrisler, William B.; Ewald, Jonathan A.; Wiley, H. Steven; Resat, Haluk

2012-01-01

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models to determine the contributions of receptor dimerization and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: 1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, 2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and 3) receptor phosphorylation kinetics at the cell surface and early endosomes are comparable. We validated the last finding by measuring the EGFR dephosphorylation rates at various times following ligand addition both in whole cells and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. This study demonstrates that an iterative cycle of experiments and modeling can be used to gain mechanistic insight regarding complex cell signaling networks. PMID:22952062

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

PubMed Central

Nayak, Ramesh C.; Keshava, Shiva; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

2013-01-01

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa. PMID:23555015

GIV/Girdin activates Gαi and inhibits Gαs via the same motif

PubMed Central

Gupta, Vijay; Bhandari, Deepali; Leyme, Anthony; Aznar, Nicolas; Midde, Krishna K.; Lo, I-Chung; Ear, Jason; Niesman, Ingrid; López-Sánchez, Inmaculada; Blanco-Canosa, Juan Bautista; von Zastrow, Mark; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta

2016-01-01

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK–ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV–Gαi complexes and activates GIV’s GEF function; then a second phosphomodification terminates GIV’s GEF function, triggers the assembly of GIV–Gαs complexes, and activates GIV’s GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK–ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses. PMID:27621449

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology.

PubMed

Skjørringe, Tina; Burkhart, Annette; Johnsen, Kasper Bendix; Moos, Torben

2015-01-01

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology

PubMed Central

Skjørringe, Tina; Burkhart, Annette; Johnsen, Kasper Bendix; Moos, Torben

2015-01-01

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail. PMID:26106291

Overexpression of CHMP7 from rapeseed and Arabidopsis causes dwarfism and premature senescence in Arabidopsis.

PubMed

Yang, Hongli; Liu, Jing; Lin, Jiulu; Deng, Linbin; Fan, Shihang; Guo, Yan; Sun, Fengming; Hua, Wei

2016-10-01

Endosomal sorting complexes required for transport (ESCRT) are well known in mammalians and yeast and plays an essential role in the formation of multi-vesicular bodies. Accumulating evidence has shown that ESCRT proteins contribute to proper plant development. CHMP7 (charged multi-vesicular body protein 7) is an ESCRT-III-related protein and functions in the endosomal sorting pathway in humans. However, its function in plants has not been explored in detail. In this study, we isolate the putative homolog of CHMP7 from rapeseed, BnCHMP7, which contains eight exons and encodes a protein consisting of 423 amino acid residues. Compared with the wild-type, overexpression of BnCHMP7 in Arabidopsis disturbs plant growth and decreases seed yield. Moreover, the transgenic plants also display early leaf senescence and hypersensitivity to dark treatment due to defects in autophagic degradation. Further study showed that BnCHMP7 is highly expressed in leaves and that YFP-BnCHMP7 is predominantly localized in endosome. Compared with human CHMP7, we found that BnCHMP7 not only interacts with ESCRT-III subunits SNF7.2 (CHMP4B), but also with VPS2.2 and CHMP1B. As expected, microarray analysis revealed that the expression of ESCRT transport genes is significantly affected. Additionally, the expression of some genes that are involved in senescence, protein synthesis and protein degradation is also altered in BnCHMP7-overexpressing plants. Taken together, BnCHMP7 encodes an endosome-localized protein, which causes dwarfism and leaf senescence as an ESCRT-III-related component. Copyright © 2016 Elsevier GmbH. All rights reserved.

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

PubMed

Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

2001-09-18

It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

A novel requirement for C. elegans Alix/ALX-1 in RME-1 mediated membrane transport

PubMed Central

Shi, Anbing; Pant, Saumya; Balklava, Zita; Chen, Carlos Chih-Hsiung; Figueroa, Vanesa; Grant, Barth D.

2007-01-01

Summary Background Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition Alix is associated with the actin cytoskeleton and may regulate cytoskeletal dynamics. Results Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane called RME-1. Analysis of alx-1 mutants indicates that ALX-1 is required for endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by analysis of rme-1 mutants. Expression of truncated human Alix in HeLa cells disrupts recycling of MHCI, a known Ehd1/RME-1 dependent transport step, suggesting phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears dispensable for ALX-1 function in MVEs/late endosomes. Conclusions This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1. PMID:17997305

Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

PubMed Central

2015-01-01

To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In conclusion, biomechanical interactions with membrane lipids are involved in cellular uptake and endosomal escape of NPs. Biophysical interaction studies could help us better understand the role of membrane lipids in cellular uptake and intracellular trafficking of NPs. PMID:24911361

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating

PubMed Central

Kim, Deok-Song; Kim, Ji-Yun; Park, Jun-Gyu; Alfajaro, Mia Madel; Baek, Yeong-Bin; Cho, Eun-Hyo; Kwon, Joseph; Choi, Jong-Soon; Kang, Mun-Il; Park, Sang-Ik; Cho, Kyoung-Oh

2018-01-01

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections. PMID:29352319

Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging.

PubMed

Padilla-Parra, Sergi; Marin, Mariana; Kondo, Naoyuki; Melikyan, Gregory B

2014-06-16

The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

PubMed Central

Topalidou, Irini; Cattin-Ortolá, Jérôme; MacCoss, Michael J.

2016-01-01

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment. PMID:27191843

The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit Vps25.

PubMed

Emerman, Amy B; Blower, Michael

2018-06-14

RNA-binding proteins (RBPs) are critical regulators of gene expression. Recent studies have uncovered hundreds of mRNA-binding proteins that do not contain annotated RNA-binding domains and have well-established roles in other cellular processes. Investigation of these nonconventional RBPs is critical for revealing novel RNA-binding domains and may disclose connections between RNA regulation and other aspects of cell biology. Endosomal sorting complex required for transport II (ESCRT-II) is a nonconventional RNA-binding complex that has a canonical role in multivesicular body formation. ESCRT-II previously has been identified as an RNA-binding complex in Drosophila oocytes, but whether its RNA-binding properties extend beyond Drosophila is unknown. In this study, we found that the RNA-binding properties of ESCRT-II are conserved in Xenopus eggs, where ESCRT-II interacted with hundreds of mRNAs. Using a UV-crosslinking approach, we demonstrated that ESCRT-II binds directly to RNA through its subunit Vps25. UV-crosslinking and immunoprecipitation (CLIP)-Seq revealed that Vps25 specifically recognizes a polypurine (i.e. GA-rich) motif in RNA. Using purified components, we could reconstitute the selective Vps25-mediated binding of the polypurine motif in vitro. Our results provide insight into the mechanism by which ESCRT-II selectively binds to mRNAs and also suggest an unexpected link between endosome biology and RNA regulation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Cationic Shell-crosslinked Knedel-like (cSCK) Nanoparticles for Highly Efficient PNA Delivery

PubMed Central

Fang, Huafeng; Zhang, Ke; Shen, Gang; Wooley, Karen L.; Taylor, John-Stephen A.

2009-01-01

Peptide nucleic acids have a number of features that make them an ideal platform for the development of in vitro biological probes and tools. Unfortunately, their inability to pass through membranes has limited their in vivo application as diagnostic and therapeutic agents. Herein, we describe the development of cationic shell-crosslinked knedel-like (cSCK) nanoparticles as highly efficient vehicles for the delivery of PNAs into cells, either through electrostatic complexation with a PNA•ODN hybrid, or through a bioreductively cleavable disulfide linkage to a PNA. These delivery systems are better than the standard lipofectamine/ODN-mediated method and much better than the Arg9-mediated method for PNA delivery in HeLa cells, showing lower toxicity and higher bioactivity. The cSCKs were also found to facilitate both endocytosis and endosomal release of the PNAs, while themselves remaining trapped in the endosomes. PMID:19231840

PIKfyve regulates melanosome biogenesis

PubMed Central

Jahid, Sohail; Sasaki, Junko; Sasaki, Takehiko; Boissy, Raymond E.; Ganesan, Anand K.

2018-01-01

PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes. PMID:29584722

Recent developments in nucleic acid delivery with polyethylenimines.

PubMed

Neuberg, Patrick; Kichler, Antoine

2014-01-01

Polyethylenimines (PEIs) have proven to be highly efficient and versatile agents for nucleic acid delivery in vitro and in vivo. Despite the low biodegradability of these polymers, they have been used in several clinical trials and the results suggest that the nucleic acid/PEI complexes have a good safety profile. The high transfection efficiency of PEIs probably relies on the fact that these polymers possess a stock of amines that can undergo protonation during the acidification of endosomes. This buffering capacity likely enhances endosomal escape of the polyplexes through the "proton sponge" effect. PEIs have also attracted great interest because the presence of many amino groups allow for easy chemical modifications or conjugation of targeting moieties and hydrophilic polymers. In the present chapter, we summarize and discuss the mechanism of PEI-mediated transfection, as well as the recent developments in PEI-mediated DNA, antisense oligonucleotide, and siRNA delivery.

Clostridium botulinum C2 toxin--new insights into the cellular up-take of the actin-ADP-ribosylating toxin.

PubMed

Aktories, Klaus; Barth, Holger

2004-04-01

Clostridium botulinum C2 toxin is a member of the family of binary actin-ADP-ribosylating toxins. It consists of the enzyme component C2I, and the separated binding/translocation component C2II. Proteolytically activated C2II forms heptamers and binds to a carbohydrate cell surface receptor. After attachment of C2I, the toxin complex is endocytosed to reach early endosomes. At low pH of endosomes, C2II-heptamers insert into the membrane, form pores and deliver C2I into the cytosol. Here, C2I ADP-ribosylates actin at Arg177 to block actin polymerization and to induce depolymerization of actin filaments. The mini-review describes main properties of C2 toxin and discusses new findings on the involvement of chaperones in the up-take process of the toxin.

Enhancing endosomal escape for nanoparticle mediated siRNA delivery

NASA Astrophysics Data System (ADS)

Ma, Da

2014-05-01

Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

The two-pore channel TPC1 is required for efficient protein processing through early and recycling endosomes.

PubMed

Castonguay, Jan; Orth, Joachim H C; Müller, Thomas; Sleman, Faten; Grimm, Christian; Wahl-Schott, Christian; Biel, Martin; Mallmann, Robert Theodor; Bildl, Wolfgang; Schulte, Uwe; Klugbauer, Norbert

2017-08-30

Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and pathogenicity of Ebola viruses. Here, we investigate the cellular function of TPC1 using protein toxins as model substrates for distinct endosomal processing routes. Toxin uptake and activation through early endosomes but not processing through other compartments were reduced in TPC1 knockout cells. Detailed co-localization studies with subcellular markers confirmed predominant localization of TPC1 to early and recycling endosomes. Proteomic analysis of native TPC1 channels finally identified direct interaction with a distinct set of syntaxins involved in fusion of intracellular vesicles. Together, our results demonstrate a general role of TPC1 for uptake and processing of proteins in early and recycling endosomes, likely by providing high local Ca 2+ concentrations required for SNARE-mediated vesicle fusion.

Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes.

PubMed

Salas-Cortes, Laura; Ye, Fei; Tenza, Danièle; Wilhelm, Claire; Theos, Alexander; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

2005-10-15

Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B-chain.

PubMed

Authier, François; Djavaheri-Mergny, Mojgan; Lorin, Séverine; Frénoy, Jean-Pierre; Desbuquois, Bernard

2016-12-01

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome-associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5-90 min) and was later (15-90 min) partially translocated to cytosol as A- and B-chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell-free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time-dependent translocation of ricin across the endosomal membrane occurred in cell-free endosomes. Endosome-located thioredoxin reductase-1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase-9 and caspase-3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B-chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B-chain with mitochondria is involved in ricin-induced apoptosis. © 2016 John Wiley & Sons Ltd.

The Parkinson’s Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway

PubMed Central

Imai, Yuzuru; Kobayashi, Yoshito; Inoshita, Tsuyoshi; Meng, Hongrui; Arano, Taku; Uemura, Kengo; Asano, Takeshi; Yoshimi, Kenji; Zhang, Chang-Liang; Matsumoto, Gen; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Kiyonari, Hiroshi; Shioi, Go; Nukina, Nobuyuki; Hattori, Nobutaka; Takahashi, Ryosuke

2015-01-01

Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2. PMID:26355680

Targeted transplantation of mitochondria to hepatocytes

PubMed Central

Gupta, Nidhi; Wu, Catherine H; Wu, George Y

2016-01-01

Background Mitochondrial defects in hepatocytes can result in liver dysfunction and death. Hepatocytes have cell-surface asialoglycoprotein receptors (AsGRs) which internalize AsGs within endosomes. The aim of this study was to determine whether mitochondria could be targeted to hepatocytes by AsGR-mediated endocytosis. Materials and methods An AsG, AsOR, was linked to polylysine to create a conjugate, AsOR-PL, and complexed with healthy and functional mitochondria (defined by normal morphology, cytochrome c assays, and oxygen-consumption rates). Huh7 (AsGR+) and SK Hep1 (AsGR−) cells were treated with a mitochondrial toxin to form Huh7-Mito− and SK Hep1-Mito− cells, lacking detectable mitochondrial DNA. An endosomolytic peptide, LLO, was coupled to AsOR to form AsOR-LLO. A lysosomal inhibitor, amantadine, was used in mitochondria-uptake studies as a control for nonspecific endosomal release. Results Coincubation of complexed mitochondria and AsOR-LLO with Huh7-Mito− cells increased mitochondrial DNA to >9,700-fold over control at 7 days (P<0.001), and increased mitochondrial oxygen-consumption rates to >90% of control by 10 days. Conclusion Rescue of mitochondria-damaged hepatocytes can be achieved by targeted uptake of normal mitochondria through receptor-mediated endocytosis. PMID:27942238

Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

PubMed

Zheng, Hao; Tang, Cui; Yin, Chunhua

2015-06-01

Present study aimed at exploring advantages/disadvantages of amino acid modified trimethylated chitosan in conquering multiple gene delivery obstacles and thus providing comprehensive understandings for improved transfection efficiency. Arginine, cysteine, and histidine modified trimethyl chitosan were synthesized and employed to self-assemble with plasmid DNA (pDNA) to form nanocomplexes, namely TRNC, TCNC, and THNC, respectively. They were assessed by structural stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. Besides, sodium tripolyphosphate (TPP) was added into TRNC to compromise certain disadvantageous attributes for pDNA delivery. Optimal endosomal escape ability failed to bring in satisfactory transfection efficiency of THNC due to drawbacks in structural stability, cellular uptake, pDNA liberation, and nuclear distribution. TCNC evoked the most potent gene expression owing to multiple advantages including sufficient stability, preferable uptake, efficient pDNA release, and high nucleic accumulation. Undesirable stability and insufficient pDNA release adversely affected TRNC-mediated gene transfer. However, incorporation of TPP could improve such disadvantages and consequently resulted in enhanced transfection efficiencies. Coordination of multiple contributing effects to conquer all delivery obstacles was necessitated for improved transfection efficiency, which would provide insights into rational design of gene delivery vehicles.

Endosomal NOX2 oxidase exacerbates virus pathogenicity and is a target for antiviral therapy.

PubMed

To, Eunice E; Vlahos, Ross; Luong, Raymond; Halls, Michelle L; Reading, Patrick C; King, Paul T; Chan, Christopher; Drummond, Grant R; Sobey, Christopher G; Broughton, Brad R S; Starkey, Malcolm R; van der Sluis, Renee; Lewin, Sharon R; Bozinovski, Steven; O'Neill, Luke A J; Quach, Tim; Porter, Christopher J H; Brooks, Doug A; O'Leary, John J; Selemidis, Stavros

2017-07-12

The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.Production of reactive oxygen species is an ancient antimicrobial mechanism, but its role in antiviral defense in mammals is unclear. Here, To et al. show that virus infection activates endosomal NOX2 oxidase and restricts TLR7 signaling, and that an endosomal NOX2 inhibitor decreases viral pathogenicity.

First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

PubMed

Canfrán-Duque, Alberto; Barrio, Luis C; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A; Busto, Rebeca

2016-03-18

First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling

PubMed Central

Diering, Graham H.; Numata, Yuka; Fan, Steven; Church, John; Numata, Masayuki

2013-01-01

To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na+/H+ exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase–Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation. PMID:24006492

Vps26B-retromer negatively regulates plasma membrane resensitization of PAR-2.

PubMed

Bugarcic, Andrea; Vetter, Irina; Chalmers, Silke; Kinna, Genevieve; Collins, Brett M; Teasdale, Rohan D

2015-11-01

Retromer is a trimeric complex composed of Vps26, Vps29, and Vps35 and has been shown to be involved in trafficking and sorting of transmembrane proteins within the endosome. The Vps26 paralog, Vps26B, defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. Although endosomally associated, Vps26B-retromer does not bind the established retromer transmembrane cargo protein, cation-independent mannose 6-phosphate receptor (CI-M6PR), indicating it has a distinct role to retromer containing the Vps26A paralog. In the present study we use the previously established Vps26B-expressing HEK293 cell model to address the role of Vps26B-retromer in trafficking of the protease activated G-protein coupled receptor PAR-2 to the plasma membrane. In these cells there is no apparent defect in the initial activation of the receptor, as evidenced by release of intracellular calcium, ERK1/2 signaling and endocytosis of activated receptor PAR-2 into degradative organelles. However, we observe a significant delay in plasma membrane repopulation of the protease activated G protein-coupled receptor PAR-2 following stimulation, resulting in a defect in PAR-2 activation after resensitization. Here we propose that PAR-2 plasma membrane repopulation is regulated by Vps26B-retromer, describing a potential novel role for this complex. © 2015 International Federation for Cell Biology.

Versatile polyion complex micelles for peptide and siRNA vectorization to engineer tolerogenic dendritic cells.

PubMed

Mebarek, Naila; Vicente, Rita; Aubert-Pouëssel, Anne; Quentin, Julie; Mausset-Bonnefont, Anne-Laure; Devoisselle, Jean-Marie; Jorgensen, Christian; Bégu, Sylvie; Louis-Plence, Pascale

2015-05-01

Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly(methacrylic acid) and poly(ethylene oxide) blocks and able to entrap therapeutic molecules, which did not induce DC maturation. In the current study, the intrinsic destabilizing membrane properties of the polymers were used to optimize endosomal escape property of the micelles in order to propose various strategies to restore tolerance. On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favor the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favor the cytosolic release of micelle-entrapped small interfering RNAs, dampening the DCs immunogenicity. Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila

PubMed Central

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P.

2017-01-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. PMID:28381425

Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains*

PubMed Central

Guo, Emily Z.; Xu, Zhaohui

2015-01-01

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. PMID:25657007

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

PubMed Central

Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

2015-01-01

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

MHC class II presentation of gp100 epitopes in melanoma cells requires the function of conventional endosomes, and is influenced by melanosomes1

PubMed Central

Robila, Valentina; Ostankovitch, Marina; Altrich-VanLith, Michelle L.; Theos, Alexander C.; Drover, Sheila; Marks, Michael S.; Restifo, Nicholas; Engelhard, Victor H.

2009-01-01

Many human solid tumors express MHC II molecules, and proteins normally localized to melanosomes give rise to MHC II restricted epitopes in melanoma. However, the pathways by which this occurs have not been defined. We analyzed the processing of one such epitope, gp10044-59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1hi/MHC II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044-59 presentation. By depletion of the AP2 adaptor protein using siRNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044-59 epitope production. Gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC II molecules. Gp10044-59 presentation is dramatically reduced, and processing occurs entirely in early endosomes / stage I melanosomes. This suggests that melanosomes are inefficient antigen processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition. PMID:19017974

Intracellular mediators of transforming growth factor beta superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo.

PubMed

Rajagopal, Ramya; Ishii, Shunsuke; Beebe, David C

2007-06-25

Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Proteins that are downstream of the transforming growth factor-beta superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFbeta superfamily for their normal development. Phosphorylated Smad1 (pSmad1), pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA) and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-beta superfamily to endosomes is important for the regulation of growth factor signaling.

Infection of XC Cells by MLVs and Ebola Virus Is Endosome-Dependent but Acidification-Independent

PubMed Central

Kamiyama, Haruka; Kakoki, Katsura; Yoshii, Hiroaki; Iwao, Masatomo; Igawa, Tsukasa; Sakai, Hideki; Hayashi, Hideki; Matsuyama, Toshifumi; Yamamoto, Naoki; Kubo, Yoshinao

2011-01-01

Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells. PMID:22022555

Molecular Mechanisms Controlling GLUT4 Intracellular Retention

PubMed Central

Blot, Vincent

2008-01-01

In basal adipocytes, glucose transporter 4 (GLUT4) is sequestered intracellularly by an insulin-reversible retention mechanism. Here, we analyze the roles of three GLUT4 trafficking motifs (FQQI, TELEY, and LL), providing molecular links between insulin signaling, cellular trafficking machinery, and the motifs in the specialized trafficking of GLUT4. Our results support a GLUT4 retention model that involves two linked intracellular cycles: one between endosomes and a retention compartment, and the other between endosomes and specialized GLUT4 transport vesicles. Targeting of GLUT4 to the former is dependent on the FQQI motif and its targeting to the latter is dependent on the TELEY motif. These two motifs act independently in retention, with the TELEY-dependent step being under the control of signaling downstream of the AS160 rab GTPase activating protein. Segregation of GLUT4 from endosomes, although positively correlated with the degree of basal retention, does not completely account for GLUT4 retention or insulin-responsiveness. Mutation of the LL motif slows return to basal intracellular retention after insulin withdrawal. Knockdown of clathrin adaptin protein complex-1 (AP-1) causes a delay in the return to intracellular retention after insulin withdrawal. The effects of mutating the LL motif and knockdown of AP-1 were not additive, establishing that AP-1 regulation of GLUT4 trafficking requires the LL motif. PMID:18550797

The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

PubMed

Zehner, Matthias; Marschall, Andrea L; Bos, Erik; Schloetel, Jan-Gero; Kreer, Christoph; Fehrenschild, Dagmar; Limmer, Andreas; Ossendorp, Ferry; Lang, Thorsten; Koster, Abraham J; Dübel, Stefan; Burgdorf, Sven

2015-05-19

The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER. Copyright © 2015 Elsevier Inc. All rights reserved.

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Mouse Polyomavirus Enters Early Endosomes, Requires Their Acidic pH for Productive Infection, and Meets Transferrin Cargo in Rab11-Positive Endosomes

PubMed Central

Liebl, David; Difato, Francesco; Horníková, Lenka; Mannová, Petra; Štokrová, Jitka; Forstová, Jitka

2006-01-01

Mouse polyomavirus (PyV) virions enter cells by internalization into smooth monopinocytic vesicles, which fuse under the cell membrane with larger endosomes. Caveolin-1 was detected on monopinocytic vesicles carrying PyV particles in mouse fibroblasts and epithelial cells (33). Here, we show that PyV can be efficiently internalized by Jurkat cells, which do not express caveolin-1 and lack caveolae, and that overexpression of a caveolin-1 dominant-negative mutant in mouse epithelial cells does not prevent their productive infection. Strong colocalization of VP1 with early endosome antigen 1 (EEA1) and of EEA1 with caveolin-1 in mouse fibroblasts and epithelial cells suggests that the monopinocytic vesicles carrying the virus (and vesicles containing caveolin-1) fuse with EEA1-positive early endosomes. In contrast to SV40, PyV infection is dependent on the acidic pH of endosomes. Bafilomycin A1 abolished PyV infection, and an increase in endosomal pH by NH4Cl markedly reduced its efficiency when drugs were applied during virion transport towards the cell nucleus. The block of acidification resulted in the retention of a fraction of virions in early endosomes. To monitor further trafficking of PyV, we used fluorescent resonance energy transfer (FRET) to determine mutual localization of PyV VP1 with transferrin and Rab11 GTPase at a 2- to 10-nm resolution. Positive FRET between PyV VP1 and transferrin cargo and between PyV VP1 and Rab11 suggests that during later times postinfection (1.5 to 3 h), the virus meets up with transferrin in the Rab11-positive recycling endosome. These results point to a convergence of the virus and the cargo internalized by different pathways in common transitional compartments. PMID:16611921

Melanosomes – dark organelles enlighten endosomal membrane transport

PubMed Central

Raposo, Graça; Marks, Michael S.

2009-01-01

Melanosomes are tissue-specific “lysosome-related” organelles of pigment cells in which melanins are synthesized and stored. Analyses of the trafficking and fate of melanosomal components are beginning to reveal how melanosomes are formed through novel pathways from early endosomal intermediates. These studies unveil generalized structural and functional modifications of the endosomal system in specialized cells, and provide unexpected insights into the biogenesis of multivesicular bodies and how compartmentalization regulates protein refolding. Moreover, genetic disorders that affect the biogenesis of melanosomes and other lysosome-related organelles have shed light into the molecular machinery that controls specialized endosomal sorting events. PMID:17878918

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

PubMed Central

Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian

2015-01-01

ABSTRACT The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that prime the virus extracellularly for receptor switching, internalization, and possibly uncoating. This step was also important for HPV6 and HPV18, which may suggest that it is conserved among the papillomaviruses. This study advances the understanding of how HPV16 initially infects cells, strengthens the notion that wounding facilitates infection of epidermal tissue, and may help the development of antiviral measures. PMID:25926655

First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

PubMed Central

Canfrán-Duque, Alberto; Barrio, Luis C.; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A.; Busto, Rebeca

2016-01-01

First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

Cellular vacuolation induced by Clostridium perfringens epsilon-toxin.

PubMed

Nagahama, Masahiro; Itohayashi, Yukari; Hara, Hideki; Higashihara, Masahiro; Fukatani, Yusuke; Takagishi, Teruhisa; Oda, Masataka; Kobayashi, Keiko; Nakagawa, Ichiro; Sakurai, Jun

2011-09-01

The epsilon-toxin of Clostridium perfringens forms a heptamer in the membranes of Madin-Darby canine kidney cells, leading to cell death. Here, we report that it caused the vacuolation of Madin-Darby canine kidney cells. The toxin induced vacuolation in a dose-dependent and time-dependent manner. The monomer of the toxin formed oligomers on lipid rafts in membranes of the cells. Methyl-β-cyclodextrin and poly(ethylene glycol) 4000 inhibited the vacuolation. Epsilon-toxin was internalized into the cells. Confocal microscopy revealed that the internalized toxin was transported from early endosomes (early endosome antigen 1 staining) to late endosomes and lysosomes (lysosomal-associated membrane protein 2 staining) and then distributed to the membranes of vacuoles. Furthermore, the vacuolation was inhibited by bafilomycin A1, a V-type ATPase inhibitor, and colchicine and nocodazole, microtubule-depolymerizing agents. The early endosomal marker green fluorescent protein-Rab5 and early endosome antigen 1 did not localize to vacuolar membranes. In contrast, the vacuolar membranes were specifically stained by the late endosomal and lysosomal marker green fluorescent protein-Rab7 and lysosomal-associated membrane protein 2. The vacuoles in the toxin-treated cells were stained with LysoTracker Red DND-99, a marker for late endosomes and lysosomes. A dominant negative mutant of Rab7 prevented the vacuolization, whereas a mutant form of Rab5 was less effective. These results demonstrate, for the first time, that: (a) oligomers of epsilon-toxin formed in lipid rafts are endocytosed; and (b) the vacuoles originating from late endosomes and lysosomes are formed by an oligomer of epsilon-toxin. © 2011 The Authors Journal compilation © 2011 FEBS.

Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release.

PubMed

Steere, Ashley N; Miller, Brendan F; Roberts, Samantha E; Byrne, Shaina L; Chasteen, N Dennis; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

2012-01-17

Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport*♦

PubMed Central

Skalicky, Jack J.; Arii, Jun; Wenzel, Dawn M.; Stubblefield, William-May B.; Katsuyama, Angela; Uter, Nathan T.; Bajorek, Monika; Myszka, David G.; Sundquist, Wesley I.

2012-01-01

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate. PMID:23105106

The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

PubMed Central

Michard, Céline; Sperandio, Daniel; Baïlo, Nathalie; Pizarro-Cerdá, Javier; LeClaire, Lawrence; Chadeau-Argaud, Elise; Pombo-Grégoire, Isabel; Hervet, Eva; Vianney, Anne; Gilbert, Christophe; Faure, Mathias; Cossart, Pascale

2015-01-01

ABSTRACT Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses. PMID:25944859

Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages

NASA Technical Reports Server (NTRS)

Lah, T. T.; Hawley, M.; Rock, K. L.; Goldberg, A. L.

1995-01-01

Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of β-arrestin.

PubMed

Smith, Thomas H; Li, Julia G; Dores, Michael R; Trejo, JoAnn

2017-08-18

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate β-arrestin-mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls β-arrestin-dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for β-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of β-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes β-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for β-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Thermodynamic Comparison of the Catalytic Domain of AMSH and AMSH-LP: Nearly Identical Fold but Different Stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davies, Christopher W.; Paul, Lake N.; Kim, Myung-Il

2012-02-07

AMSH plays a critical role in the ESCRT (endosomal sorting complexes required for transport) machinery, which facilitates the down-regulation and degradation of cell-surface receptors. It displays a high level of specificity toward cleavage of Lys63-linked polyubiquitin chains, the structural basis of which has been understood recently through the crystal structure of a highly related, but ESCRT-independent, protein AMSH-LP (AMSH-like protein). We have determined the X-ray structure of two constructs representing the catalytic domain of AMSH: AMSH244, the JAMM (JAB1/MPN/MOV34)-domain-containing polypeptide segment from residues 244 to 424, and AMSH219{sup E280A}, an active-site mutant, Glu280 to Ala, of the segment from 219more » to 424. In addition to confirming the expected zinc coordination in the protein, the structures reveal that the catalytic domains of AMSH and AMSH-LP are nearly identical; however, guanidine-hydrochloride-induced unfolding studies show that the catalytic domain of AMSH is thermodynamically less stable than that of AMSH-LP, indicating that the former is perhaps structurally more plastic. Much to our surprise, in the AMSH219{sup E280A} structure, the catalytic zinc was still held in place, by the compensatory effect of an aspartate from a nearby loop moving into a position where it could coordinate with the zinc, once again suggesting the plasticity of AMSH. Additionally, a model of AMSH244 bound to Lys63-linked diubiquitin reveals a type of interface for the distal ubiquitin significantly different from that seen in AMSH-LP. Altogether, we believe that our data provide important insight into the structural difference between the two proteins that may translate into the difference in their biological function.« less

pH-Controlled Two-Step Uncoating of Influenza Virus

PubMed Central

Li, Sai; Sieben, Christian; Ludwig, Kai; Höfer, Chris T.; Chiantia, Salvatore; Herrmann, Andreas; Eghiaian, Frederic; Schaap, Iwan A.T.

2014-01-01

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5–6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection. PMID:24703306

SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors.

PubMed

Mari, Muriel; Bujny, Miriam V; Zeuschner, Dagmar; Geerts, Willie J C; Griffith, Janice; Petersen, Claus M; Cullen, Pete J; Klumperman, Judith; Geuze, Hans J

2008-03-01

Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno-electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three-dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.

Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

PubMed Central

Chen, C S; Martin, O C; Pagano, R E

1997-01-01

N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from receptor-mediated endocytosis. We conclude that the spectral properties of C5-DMB-SM can be used to distinguish unique populations of early endosomes from one another and to record dynamic changes in their number and distribution within living cells. Images FIGURE 1 FIGURE 3 FIGURE 4 FIGURE 6 FIGURE 8 FIGURE 9 PMID:8994591

VPS35 in Dopamine Neurons Is Required for Endosome-to-Golgi Retrieval of Lamp2a, a Receptor of Chaperone-Mediated Autophagy That Is Critical for α-Synuclein Degradation and Prevention of Pathogenesis of Parkinson's Disease

PubMed Central

Tang, Fu-Lei; Erion, Joanna R.; Tian, Yun; Liu, Wei; Yin, Dong-Min; Ye, Jian; Tang, Baisha; Mei, Lin

2015-01-01

Vacuolar protein sorting-35 (VPS35) is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with autosomal dominant PD. However, it remains poorly understood if and how VPS35 deficiency or mutation contributes to PD pathogenesis. Here we provide evidence that links VPS35 deficiency to PD-like neuropathology. VPS35 was expressed in mouse dopamine (DA) neurons in substantia nigra pars compacta (SNpc) and STR (striatum)—regions that are PD vulnerable. VPS35-deficient mice exhibited PD-relevant deficits including accumulation of α-synuclein in SNpc-DA neurons, loss of DA transmitter and DA neurons in SNpc and STR, and impairment of locomotor behavior. Further mechanical studies showed that VPS35-deficient DA neurons or DA neurons expressing PD-linked VPS35 mutant (D620N) had impaired endosome-to-Golgi retrieval of lysosome-associated membrane glycoprotein 2a (Lamp2a) and accelerated Lamp2a degradation. Expression of Lamp2a in VPS35-deficient DA neurons reduced α-synuclein, supporting the view for Lamp2a as a receptor of chaperone-mediated autophagy to be critical for α-synuclein degradation. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis. SIGNIFICANCE STATEMENT VPS35 is a key component of the retromer complex that is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with PD. However, if and how VPS35 deficiency or mutation contributes to PD pathogenesis remains unclear. We demonstrated that VPS35 deficiency or mutation (D620N) in mice leads to α-synuclein accumulation and aggregation in the substantia nigra, accompanied with DA neurodegeneration. VPS35-deficient DA neurons exhibit impaired endosome-to-Golgi retrieval of Lamp2a, which may contribute to the reduced α-synuclein degradation through chaperone-mediated autophagy. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis, and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis. PMID:26203154

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes.

PubMed

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian; Kielian, Margaret

2016-07-15

The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

PubMed Central

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian

2016-01-01

ABSTRACT The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment. PMID:27122589

Modulation of Endosomal Escape of IRQ-PEGylated Nano-carrier

NASA Astrophysics Data System (ADS)

Mudhakir, Diky; Akita, Hidetaka; Harashima, Hideyoshi

2011-12-01

The novel IRQ peptide is one of cell penetrating peptides (CPPs) that has ability to induce endosomal escape. It has been demonstrated that IRQ ligand had ability to facilitate an escape of liposomes encapsulating siRNA from the endosomes presumably by fusion-independent mechanism [1,2]. In the present study, we attempted to modulate the intracellular trafficking of IRQ-modified nano-carrier in term of escaping process by changing the lipid composition. The peptide was attached to the terminal end of maleimide group of polyethylene glycol-modified liposomes (IRQ-PEG-Lip). The liposomes were composed of DOTAP, DOPE and cholesterol and it was labeled by water soluble sulpho-rhodamine B (Sr-B). The escape of PEG-coated liposomes was then observed by confocal laser scanning microscope after the endosomes were stained with Lysosensor. The results exhibited that IRQ-PEG-Lip was escaped from endosomal compartment after 1 h transfection when 40% of DOPE was incorporated into the nanostructure comparing to that of PEG-Lip. These results are consistent with the previous results that the IRQ facilitates endosomal escape via independent-mechanism. However, IRQ-PEG-Lip were then completely co-localized in the acidic compartment when density of DOPE was reduced approximately 20%. These results indicated that the utilizing of DOPE is important for the escape process even in the presence of hydrophilic PEG polymer. In conclusion, the regulation of endosomal escape ability of the PEGylated-IRQ nano-carrier was induced by fusion-independent manner as well as fusogenic lipid.

Ulk1 Governs Nerve Growth Factor/TrkA Signaling by Mediating Rab5 GTPase Activation in Porcine Hemagglutinating Encephalomyelitis Virus-Induced Neurodegenerative Disorders.

PubMed

Li, Zi; Zhao, Kui; Lv, Xiaoling; Lan, Yungang; Hu, Shiyu; Shi, Junchao; Guan, Jiyu; Yang, Yawen; Lu, Huijun; He, Hongbin; Gao, Feng; He, Wenqi

2018-06-06

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus and causes neurological dysfunction in the central nervous system (CNS), but the neuropathological mechanism of PHEV remains poorly understood. We report that Unc51-like kinase 1 (Ulk1/Unc51.1) is a pivotal regulator of PHEV-induced neurological disorders and functions to selectively control the initiation of NGF/TrkA endosome trafficking. We first identified the function of Ulk1 by histopathologic evaluation in PHEV-infected mouse model where neuronal loss was accompanied by the suppression of Ulk1 expression. Morphogenesis assessments in the primary cortical neurons revealed that overexpression or mutations of Ulk1 modulated neurite outgrowth, collateral sprouting, and endosomal transport. Likewise, Ulk1 expression was decreased following PHEV infection, suggesting that there was a correlation between the neurodegeneration and functional Ulk1 deficiency. We then showed that Ulk1 forms a multiprotein complex with TrkA and the early endosome marker Rab5 and that Ulk1 defects lead to either blocking of NGF/TrkA endocytosis or premature degradation of pTrkA via constitutive activation of the Rab5 GTPase. Further investigation determined that the ectopic expression of Rab5 mutants induces aberrant endosomal accumulation of activated pTrkA, proving that targeting of Ulk1-TrkA-NGF signaling to the retrograde transport route in the neurodegenerative process that underlies PHEV infection is dependent on Rab5 GTPase activity. Therefore, we described a long-distance signaling mechanism of PHEV-driven deficits in neurons and suggested that such Ulk1 repression may result in limited NGF/TrkA retrograde signaling within activated Rab5 endosomes, explaining the progressive failure of neurite outgrowth and survival. IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV) is neurotropic coronavirus and targets neurons in the nervous system for proliferation, frequently leaving behind grievous neurodegeneration. Structural plasticity disorders occur in the axons, dendrites, and dendritic spines of PHEV-infected neurons, and dysfunction of this neural process may contribute to neurologic pathologies, but the mechanisms remain undetermined. Further understanding of the neurological manifestations underlying PHEV infection in the CNS may provide insights into both neurodevelopmental and neurodegenerative diseases that may be necessarily conducive to targeted approaches for treatment. The significance of our research is in identifying an Ulk1-related neurodegenerative mechanism, focusing on the regulatory functions of Ulk1 in the transport of long-distance trophic signaling endosomes, thereby explaining the progressive failure of neurite outgrowth and survival associated with PHEV aggression. This is the first report to define a mechanistic link between alterations in signaling from endocytic pathways and the neuropathogenesis of PHEV-induced CNS disease. Copyright © 2018 American Society for Microbiology.

Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum.

PubMed

Cianciola, Nicholas L; Chung, Stacey; Manor, Danny; Carlin, Cathleen R

2017-03-15

Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDα via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDα. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich "lipid rafts." Collectively, these data show that RIDα utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes. IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune-mediated pathology have been a particularly rich source of information regarding intracellular protein trafficking. Our studies with the early region 3-encoded RIDα protein also provided fundamental new information regarding mechanisms of nonvesicular lipid transport and the flow of molecular information at membrane contacts between different organelles. We describe a new pathway that delivers cholesterol from endosomes to the endoplasmic reticulum, where it is esterified and stored in lipid droplets. Although lipid droplets are attracting renewed interest from the standpoint of normal physiology and human diseases, including those resulting from viral infections, experimental model systems for evaluating how and why they accumulate are still limited. Our studies also revealed an intriguing relationship between lipid droplets and innate immunity that may represent a new paradigm for viruses utilizing these organelles. Copyright © 2017 American Society for Microbiology.

Endosomal sorting complexes required for ESCRTing cells toward death during neurogenesis, neurodevelopment and neurodegeneration.

PubMed

Kaul, Zenia; Chakrabarti, Oishee

2018-03-25

The endosomal sorting complexes required for transport (ESCRT) proteins help in the recognition, sorting and degradation of ubiquitinated cargoes from the cell surface, long-lived proteins or aggregates, and aged organelles present in the cytosol. These proteins take part in the endo-lysosomal system of degradation. The ESCRT proteins also play an integral role in cytokinesis, viral budding and mRNA transport. Many neurodegenerative diseases are caused by toxic accumulation of cargo in the cell, which causes stress and ultimately leads to neuronal death. This accumulation of cargo occurs because of defects in the endo-lysosomal degradative pathway-loss of function of ESCRTs has been implicated in this mechanism. ESCRTs also take part in many survival processes, lack of which can culminate in neuronal cell death. While the role played by the ESCRT proteins in maintaining healthy neurons is known, their role in neurodegenerative diseases is still poorly understood. In this review, we highlight the importance of ESCRTs in maintaining healthy neurons and then suggest how perturbations in many of the survival mechanisms governed by these proteins could eventually lead to cell death; quite often these correlations are not so obviously laid out. Extensive neuronal death eventually culminates in neurodegeneration. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Identification of cellular compartments involved in processing of cathepsin E in primary cultures of rat microglia.

PubMed

Sastradipura, D F; Nakanishi, H; Tsukuba, T; Nishishita, K; Sakai, H; Kato, Y; Gotow, T; Uchiyama, Y; Yamamoto, K

1998-05-01

Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic compartments.

Integrated Experimental and Model-based Analysis Reveals the Spatial Aspects of EGFR Activation Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shankaran, Harish; Zhang, Yi; Chrisler, William B.

2012-10-02

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models for deconvolving the contributions of receptor dimerizationmore » and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: 1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, 2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and 3) receptor dephosphorylation rates at the cell surface and early endosomes are comparable. We validated the last finding by measuring EGFR dephosphorylation rates at various times following ligand addition both in whole cells, and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. Further, the mathematical model described here can be extended to determine receptor dimer abundances in cells co-expressing various levels of ErbB receptors. This study demonstrates that an iterative cycle of experiments and modeling can be used to gain mechanistic insight regarding complex cell signaling networks.« less

Distinct mechanisms of recognizing endosomal sorting complex required for transport III (ESCRT-III) protein IST1 by different microtubule interacting and trafficking (MIT) domains.

PubMed

Guo, Emily Z; Xu, Zhaohui

2015-03-27

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

DOE PAGES

Guo, Emily Z.; Xu, Zhaohui

2015-02-05

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). In this paper, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed thatmore » IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. Finally, these observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode.« less

Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Schmidlin, Fabien; Steinhoff, Martin; Bunnett, Nigel W

2004-07-16

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Co-factors Required for TLR7- and TLR9- dependent Innate Immune Responses

PubMed Central

Chiang, Chih-yuan; Engel, Alex; Opaluch, Amanda M.; Ramos, Irene; Maestre, Ana M.; Secundino, Ismael; De Jesus, Paul D.; Nguyen, Quy T.; Welch, Genevieve; Bonamy, Ghislain M.C.; Miraglia, Loren J.; Orth, Anthony P.; Nizet, Victor; Fernandez-Sesma, Ana; Zhou, Yingyao; Barton, Gregory M.; Chanda, Sumit K.

2012-01-01

SUMMARY Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent pro-inflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis we identify 190 co-factors required for TLR7- and TLR9-directed signaling responses. A set of co-factors were cross-profiled for their activities downstream of several immunoreceptors, and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection. PMID:22423970

Structure and membrane remodeling activity of ESCRT-III helical polymers

PubMed Central

McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; Skowyra, Michael L.; Saunders, Marissa G.; Naismith, Teresa V.; Colf, Leremy A.; Afonine, Pavel; Arthur, Christopher; Sundquist, Wesley I.; Hanson, Phyllis I.; Frost, Adam

2015-01-01

The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 Å resolution cryo-EM reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, CHMP1B and IST1. The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecture, and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively-curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature. PMID:26634441

Recognition-mediated activation of therapeutic gold nanoparticles inside living cells

NASA Astrophysics Data System (ADS)

Kim, Chaekyu; Agasti, Sarit S.; Zhu, Zhengjiang; Isaacs, Lyle; Rotello, Vincent M.

2010-11-01

Supramolecular chemistry provides a versatile tool for the organization of molecular systems into functional structures and the actuation of these assemblies for applications through the reversible association between complementary components. Use of this methodology in living systems, however, represents a significant challenge owing to the chemical complexity of cellular environments and lack of selectivity of conventional supramolecular interactions. Herein, we present a host-guest system featuring diaminohexane-terminated gold nanoparticles (AuNP-NH2) and complementary cucurbit[7]uril (CB[7]). In this system, threading of CB[7] on the particle surface reduces the cytotoxicity of AuNP-NH2 through sequestration of the particle in endosomes. Intracellular triggering of the therapeutic effect of AuNP-NH2 was then achieved through the administration of 1-adamantylamine (ADA), removing CB[7] from the nanoparticle surface, causing the endosomal release and concomitant in situ cytotoxicity of AuNP-NH2. This supramolecular strategy for intracellular activation provides a new tool for potential therapeutic applications.

The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR

PubMed Central

Boncompain, Gaelle; Laketa, Vibor; Poser, Ina; Beck, Martin; Bork, Peer

2016-01-01

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane. PMID:27872256

Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

PubMed Central

Bowen, Aaron B; Bourke, Ashley M; Hiester, Brian G; Hanus, Cyril

2017-01-01

Neurons face the challenge of regulating the abundance, distribution and repertoire of integral membrane proteins within their immense, architecturally complex dendritic arbors. While the endoplasmic reticulum (ER) supports dendritic translation, most dendrites lack the Golgi apparatus (GA), an essential organelle for conventional secretory trafficking. Thus, whether secretory cargo is locally trafficked in dendrites through a non-canonical pathway remains a fundamental question. Here we define the dendritic trafficking itinerary for key synaptic molecules in rat cortical neurons. Following ER exit, the AMPA-type glutamate receptor GluA1 and neuroligin 1 undergo spatially restricted entry into the dendritic secretory pathway and accumulate in recycling endosomes (REs) located in dendrites and spines before reaching the plasma membrane. Surprisingly, GluA1 surface delivery occurred even when GA function was disrupted. Thus, in addition to their canonical role in protein recycling, REs also mediate forward secretory trafficking in neuronal dendrites and spines through a specialized GA-independent trafficking network. PMID:28875935

Axonal autophagosomes recruit dynein for retrograde transport through fusion with late endosomes

PubMed Central

Cheng, Xiu-Tang; Zhou, Bing; Lin, Mei-Yao; Cai, Qian

2015-01-01

Efficient degradation of autophagic vacuoles (AVs) via lysosomes is an important cellular homeostatic process. This is particularly challenging for neurons because mature acidic lysosomes are relatively enriched in the soma. Although dynein-driven retrograde transport of AVs was suggested, a fundamental question remains how autophagosomes generated at distal axons acquire dynein motors for retrograde transport toward the soma. In this paper, we demonstrate that late endosome (LE)–loaded dynein–snapin complexes drive AV retrograde transport in axons upon fusion of autophagosomes with LEs into amphisomes. Blocking the fusion with syntaxin17 knockdown reduced recruitment of dynein motors to AVs, thus immobilizing them in axons. Deficiency in dynein–snapin coupling impaired AV transport, resulting in AV accumulation in neurites and synaptic terminals. Altogether, our study provides the first evidence that autophagosomes recruit dynein through fusion with LEs and reveals a new motor–adaptor sharing mechanism by which neurons may remove distal AVs engulfing aggregated proteins and dysfunctional organelles for efficient degradation in the soma. PMID:25940348

The reconstructed ancestral subunit a functions as both V-ATPase isoforms Vph1p and Stv1p in Saccharomyces cerevisiae

PubMed Central

Finnigan, Gregory C.; Hanson-Smith, Victor; Houser, Benjamin D.; Park, Hae J.; Stevens, Tom H.

2011-01-01

The vacuolar-type, proton-translocating ATPase (V-ATPase) is a multisubunit enzyme responsible for organelle acidification in eukaryotic cells. Many organisms have evolved V-ATPase subunit isoforms that allow for increased specialization of this critical enzyme. Differential targeting of the V-ATPase to specific subcellular organelles occurs in eukaryotes from humans to budding yeast. In Saccharomyces cerevisiae, the two subunit a isoforms are the only difference between the two V-ATPase populations. Incorporation of Vph1p or Stv1p into the V-ATPase dictates the localization of the V-ATPase to the vacuole or late Golgi/endosome, respectively. A duplication event within fungi gave rise to two subunit a genes. We used ancestral gene reconstruction to generate the most recent common ancestor of Vph1p and Stv1p (Anc.a) and tested its function in yeast. Anc.a localized to both the Golgi/endosomal network and vacuolar membrane and acidified these compartments as part of a hybrid V-ATPase complex. Trafficking of Anc.a did not require retrograde transport from the late endosome to the Golgi that has evolved for retrieval of the Stv1p isoform. Rather, Anc.a localized to both structures through slowed anterograde transport en route to the vacuole. Our results suggest an evolutionary model that describes the differential localization of the two yeast V-ATPase isoforms. PMID:21737673

HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2

PubMed Central

Lubben, Nienke B.; Sahlender, Daniela A.; Motley, Alison M.; Lehner, Paul J.; Benaroch, Philippe

2007-01-01

Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2–depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment. PMID:17581864

Pro-metastatic NEDD9 regulates individual cell migration via caveolin-1-dependent trafficking of integrins

PubMed Central

Kozyulina, Polina Y.; Loskutov, Yuriy V.; Kozyreva, Varvara K.; Rajulapati, Anuradha; Ice, Ryan J.; Jones, Brandon. C.; Pugacheva, Elena N.

2014-01-01

The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of pro-metastatic protein, NEDD9, in breast cancer (BC) cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and co-localizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand/integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Re-expression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9 depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. PMID:25319010

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

PubMed

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans

PubMed Central

Grussendorf, Kelly A.; Trezza, Christopher J.; Salem, Alexander T.; Al-Hashimi, Hikmat; Mattingly, Brendan C.; Kampmeyer, Drew E.; Khan, Liakot A.; Hall, David H.; Göbel, Verena; Ackley, Brian D.; Buechner, Matthew

2016-01-01

Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans. In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn’s disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling. PMID:27334269

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

PubMed Central

Nour, Adel M.; Li, Yue; Wolenski, Joseph; Modis, Yorgo

2013-01-01

Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion. PMID:24039574

Trans-infection but not infection from within endosomal compartments after cell-to-cell HIV-1 transfer to CD4+ T cells.

PubMed

Permanyer, Marc; Ballana, Ester; Badia, Roger; Pauls, Eduardo; Clotet, Bonaventura; Esté, José A

2012-09-14

Cellular contacts between HIV-1-infected donor cells and uninfected primary CD4(+) T lymphocytes lead to virus transfer into endosomes. Recent evidence suggests that HIV particles may fuse with endosomal membranes to initiate a productive infection. To explore the role of endocytosis in the entry and replication of HIV, we evaluated the infectivity of transferred HIV particles in a cell-to-cell culture model of virus transmission. Endocytosed virus led to productive infection of cells, except when cells were cultured in the presence of the anti-gp120 mAb IgGb12, an agent that blocks virus attachment to CD4, suggesting that endocytosed virus was recycled to the outer cell surface. Confocal microscopy confirmed the colocalization of internalized virus antigen and the endosomal marker dynamin. Additionally, virus transfer, fusion, or productive infection was not blocked by dynasore, dynamin-dependent endosome-scission inhibitor, at subtoxic concentrations, suggesting that the early capture of virus into intracellular compartments did not depend on endosomal maturation. Our results suggest that endocytosis is not a mechanism of infection of primary CD4 T cells, but may serve as a reservoir capable of inducing trans-infection of cells after the release of HIV particles to the extracellular environment.

pH-Controlled two-step uncoating of influenza virus.

PubMed

Li, Sai; Sieben, Christian; Ludwig, Kai; Höfer, Chris T; Chiantia, Salvatore; Herrmann, Andreas; Eghiaian, Frederic; Schaap, Iwan A T

2014-04-01

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5-6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion

PubMed Central

Chin, Christopher R.; Savidis, George; Brass, Abraham L.; Melikyan, Gregory B.

2014-01-01

Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes. PMID:24699674

Rab11a and its binding partners regulate the recycling of the β1-adrenergic receptor

PubMed Central

Gardner, Lidia A.; Hajjhussein, Hassan; Frederick, Katherine C.; Bahouth, Suleiman W.

2010-01-01

β1-adrenergic receptors (β1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the β1-AR (S312A) is internalized but does not recycle. We determined that WT β1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by >70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT β1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT β1-AR were colocalized by >70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT β1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the β1-AR. Next, we determined the effect of each of the rab11-intercating proteins on trafficking of the WT β1-AR. The recycling of the β1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role recycling of the human β1-AR. PMID:20727405

The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease.

PubMed

Prasad, Hari; Rao, Rajini

2015-02-27

Early intervention may be key to safe and effective therapies in patients with Alzheimer disease. Endosomal dysfunction is an early step in neurodegeneration. Endosomes are a major site of production of Aβ peptide from the processing of amyloid precursor protein (APP) by clipping enzymes (β- and γ-secretases). The β-secretase enzyme BACE1 requires acidic lumen pH for optimum function, and acid pH promotes Aβ aggregation. The Na(+)/H(+) exchanger NHE6 provides a leak pathway for protons, limiting luminal acidification by proton pumps. Like APP, NHE6 expression was induced upon differentiation of SH-SY5Y neuroblastoma cells and localized to an endosomal compartment. Therefore, we investigated whether NHE6 expression altered APP localization and processing in a stably transfected cell culture model of human APP expression. We show that co-expression with NHE6 or treatment with the Na(+)/H(+) ionophore monensin shifted APP away from the trans-Golgi network into early and recycling endosomes in HEK293 cells. NHE6 alkalinized the endosomal lumen, similar to monensin, and significantly attenuated APP processing and Aβ secretion. In contrast, Aβ production was elevated upon NHE6 knockdown. We show that NHE6 transcript and protein levels are lowered in Alzheimer brains relative to control. These findings, taken together with emerging genetic evidence linking endosomal Na(+)/H(+) exchangers with Alzheimer disease, suggest that proton leak pathways may regulate Aβ generation and contribute to disease etiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Role for Dynamin in Late Endosome Dynamics and Trafficking of the Cation-independent Mannose 6-Phosphate Receptor

PubMed Central

Nicoziani, Paolo; Vilhardt, Frederik; Llorente, Alicia; Hilout, Leila; Courtoy, Pierre J.; Sandvig, Kirsten; van Deurs, Bo

2000-01-01

It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)–containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules. PMID:10679008

The Na+/H+ Exchanger NHE6 Modulates Endosomal pH to Control Processing of Amyloid Precursor Protein in a Cell Culture Model of Alzheimer Disease*

PubMed Central

Prasad, Hari; Rao, Rajini

2015-01-01

Early intervention may be key to safe and effective therapies in patients with Alzheimer disease. Endosomal dysfunction is an early step in neurodegeneration. Endosomes are a major site of production of Aβ peptide from the processing of amyloid precursor protein (APP) by clipping enzymes (β- and γ-secretases). The β-secretase enzyme BACE1 requires acidic lumen pH for optimum function, and acid pH promotes Aβ aggregation. The Na+/H+ exchanger NHE6 provides a leak pathway for protons, limiting luminal acidification by proton pumps. Like APP, NHE6 expression was induced upon differentiation of SH-SY5Y neuroblastoma cells and localized to an endosomal compartment. Therefore, we investigated whether NHE6 expression altered APP localization and processing in a stably transfected cell culture model of human APP expression. We show that co-expression with NHE6 or treatment with the Na+/H+ ionophore monensin shifted APP away from the trans-Golgi network into early and recycling endosomes in HEK293 cells. NHE6 alkalinized the endosomal lumen, similar to monensin, and significantly attenuated APP processing and Aβ secretion. In contrast, Aβ production was elevated upon NHE6 knockdown. We show that NHE6 transcript and protein levels are lowered in Alzheimer brains relative to control. These findings, taken together with emerging genetic evidence linking endosomal Na+/H+ exchangers with Alzheimer disease, suggest that proton leak pathways may regulate Aβ generation and contribute to disease etiology. PMID:25561733

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

PubMed

Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

ESCRT-I function is required for Tyrp1 transport from early endosomes to the melanosome limiting membrane

PubMed Central

Truschel, Steven T.; Simoes, Sabrina; Setty, Subba Rao Gangi; Harper, Dawn C.; Tenza, Danièle; Thomas, Penelope C.; Herman, Kathryn E.; Sackett, Sara D.; Cowan, David C.; Theos, Alexander C.; Raposo, Graça; Marks, Michael S.

2009-01-01

Melanosomes are lysosome-related organelles that coexist with lysosomes within melanocytes. The pathways by which melanosomal proteins are diverted from endocytic organelles toward melanosomes are incompletely defined. In melanocytes from mouse models of Hermansky-Pudlak syndrome (HPS) that lack BLOC-1, melanosomal proteins such as Tyrp1 accumulate in early endosomes. Whether this accumulation represents an anomalous pathway or an arrested normal intermediate in melanosome protein trafficking is not clear. Here we show that early endosomes are requisite intermediates in the trafficking of Tyrp1 from the Golgi to late stage melanosomes in normal melanocytic cells. Kinetic analyses show that very little newly synthesized Tyrp1 traverses the cell surface and that internalized Tyrp1 is inefficiently sorted to melanosomes. Nevertheless, nearly all Tyrp1 traverses early endosomes since it becomes trapped within enlarged, modified endosomes upon overexpression of Hrs. Although Tyrp1 localization is not affected by Hrs depletion, depletion of the ESCRT-I component, Tsg101, or inhibition of ESCRT function by dominant negative approaches results in a dramatic redistribution of Tyrp1 to aberrant endosomal membranes that are largely distinct from those harboring traditional ESCRT-dependent, ubiquitylated cargoes such as MART-1. The lysosomal protein content of some of these membranes and the lack of Tyrp1 recycling to the plasma membrane in Tsg101-depleted cells suggests that ESCRT-I functions downstream of BLOC-1. Our data delineate a novel pathway for Tyrp1 trafficking and illustrate a requirement for ESCRT-I function in controlling protein sorting from vacuolar endosomes to the limiting membrane of a lysosome-related organelle. PMID:19624486

Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin.

PubMed

Chichger, Havovi; Braza, Julie; Duong, Huetran; Boni, Geraldine; Harrington, Elizabeth O

2016-06-01

Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

Quantum dot bioconjugates: uptake into cells and induction of changes in normal cellular transport

NASA Astrophysics Data System (ADS)

Iversen, Tore-Geir; Frerker, Nadine; Sandvig, Kirsten

2009-02-01

Can quantum dots (QDs) act as relevant intracellular probes to investigate routing of ligands in live cells? To answer this question we studied intracellular trafficking of QDs that were coupled to the plant toxin ricin, Shiga toxin or the ligand transferrin (Tf) by confocal fluorescence microscopy in three different cell lines. The Tf:QDs were internalized but instead of being recycled they accumulated within endosomes in all cell lines. However, for the HEp-2 and SW480 cells a higher fraction colocalized with a lysosomal marker as compared with HeLa cells. The Shiga:QD bioconjugate was internalized slowly and with poor efficiency in the HEp-2 and SW480 cells as compared with HeLa cells, and was not routed to the Golgi apparatus in any of the cell lines. The internalized ricin:QD bioconjugates localized to the same endosomes as ricin itself, but could in contrast to ricin not be visualized in the Golgi apparatus. Importantly, we find that the endosomal accumulation of either ricin:QDs or transferrin:QDs affects endosome-to-Golgi transport of both ricin and Shiga toxin: Transport of ricin was reduced whereas transport of Shiga toxin was increased. In conclusion, the data from different cells reveal that in general these ligand-coupled QD nanoparticles are arrested within endosomes, and somehow perturb the normal endosomal sorting in cells.

Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling CompartmentD⃞

PubMed Central

Strick, David J.; Elferink, Lisa A.

2005-01-01

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways. PMID:16195351

Investigation of endosome and lysosome biology by ultra pH-sensitive nanoprobes.

PubMed

Wang, Chensu; Zhao, Tian; Li, Yang; Huang, Gang; White, Michael A; Gao, Jinming

2017-04-01

Endosomes and lysosomes play a critical role in various aspects of cell physiology such as nutrient sensing, receptor recycling, protein/lipid catabolism, and cell death. In drug delivery, endosomal release of therapeutic payloads from nanocarriers is also important in achieving efficient delivery of drugs to reach their intracellular targets. Recently, we invented a library of ultra pH-sensitive (UPS) nanoprobes with exquisite fluorescence response to subtle pH changes. The UPS nanoprobes also displayed strong pH-specific buffer effect over small molecular bases with broad pH responses (e.g., chloroquine and NH 4 Cl). Tunable pH transitions from 7.4 to 4.0 of UPS nanoprobes cover the entire physiological pH of endocytic organelles (e.g., early and late endosomes) and lysosomes. These unique physico-chemical properties of UPS nanoprobes allowed a 'detection and perturbation' strategy for the investigation of luminal pH in cell signaling and metabolism, which introduces a nanotechnology-enabled paradigm for the biological studies of endosomes and lysosomes. Published by Elsevier B.V.

Endosomal system genetics and autism spectrum disorders: A literature review.

PubMed

Patak, Jameson; Zhang-James, Yanli; Faraone, Stephen V

2016-06-01

Autism spectrum disorders (ASDs) are a group of debilitating neurodevelopmental disorders thought to have genetic etiology, due to their high heritability. The endosomal system has become increasingly implicated in ASD pathophysiology. In an attempt to summarize the association between endosomal system genes and ASDs we performed a systematic review of the literature. We searched PubMed for relevant articles. Simons Foundation Autism Research Initiative (SFARI) gene database was used to exclude articles regarding genes with less than minimal evidence for association with ASDs. Our search retained 55 articles reviewed in two categories: genes that regulate and genes that are regulated by the endosomal system. Our review shows that the endosomal system is a novel pathway implicated in ASDs as well as other neuropsychiatric disorders. It plays a central role in aspects of cellular physiology on which neurons and glial cells are particularly reliant, due to their unique metabolic and functional demands. The system shows potential for biomarkers and pharmacological intervention and thus more research into this pathway is warranted. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interferon-induced Sus scrofa Mx1 blocks endocytic traffic of incoming influenza A virus particles.

PubMed

Palm, Mélanie; Garigliany, Mutien-Marie; Cornet, François; Desmecht, Daniel

2010-01-01

The interferon-induced Mx proteins of vertebrates are dynamin-like GTPases, some isoforms of which can additionally inhibit the life cycle of certain RNA viruses. Here we show that the porcine Mx1 protein (poMx1) inhibits replication of influenza A virus and we attempt to identify the step at which the viral life cycle is blocked. In infected cells expressing poMx1, the level of transcripts encoding the viral nucleoprotein is significantly lower than normal, even when secondary transcription is prevented by exposure to cycloheximide. This reveals that a pretranscriptional block participates to the anti-influenza activity. Binding and internalization of incoming virus particles are normal in the presence of poMx1 but centripetal traffic to the late endosomes is interrupted. Surprisingly but decisively, poMx1 significantly alters binding of early endosome autoantigen 1 to early endosomes and/or early endosome size and spatial distribution. This is compatible with impairment of traffic of the endocytic vesicles to the late endosomes. INRA, EDP Sciences, 2010.

OCRL controls trafficking through early endosomes via PtdIns4,5P2-dependent regulation of endosomal actin

PubMed Central

Vicinanza, Mariella; Di Campli, Antonella; Polishchuk, Elena; Santoro, Michele; Di Tullio, Giuseppe; Godi, Anna; Levtchenko, Elena; De Leo, Maria Giovanna; Polishchuk, Roman; Sandoval, Lisette; Marzolo, Maria-Paz; De Matteis, Maria Antonietta

2011-01-01

Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P2 in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P2 and F-actin at the EEs is essential for exporting cargoes that transit this compartment. PMID:21971085

Sphingosine Kinase 1 Cooperates with Autophagy to Maintain Endocytic Membrane Trafficking.

PubMed

Young, Megan M; Takahashi, Yoshinori; Fox, Todd E; Yun, Jong K; Kester, Mark; Wang, Hong-Gang

2016-11-01

Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, Sphk1 also appears to facilitate endosomal fusion independent of its catalytic activity, given that catalytically inactive Sphk1 G82D is recruited to endocytic membranes by sphingosine or sphingosine-based Sphk1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceramide synthase, lysosomal biogenesis, and the restoration of autophagic flux. Collectively, these studies uncover intersecting roles for Sphk1, sphingosine, and autophagic machinery in endocytic membrane trafficking. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Crucial role of neuron-enriched endosomal protein of 21 kDa in sorting between degradation and recycling of internalized G-protein-coupled receptors.

PubMed

Debaigt, Colin; Hirling, Harald; Steiner, Pascal; Vincent, Jean-Pierre; Mazella, Jean

2004-08-20

Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway.

Bacterial uracil modulates Drosophila DUOX-dependent gut immunity via Hedgehog-induced signaling endosomes.

PubMed

Lee, Kyung-Ah; Kim, Boram; Bhin, Jinhyuk; Kim, Do Hun; You, Hyejin; Kim, Eun-Kyoung; Kim, Sung-Hee; Ryu, Ji-Hwan; Hwang, Daehee; Lee, Won-Jae

2015-02-11

Genetic studies in Drosophila have demonstrated that generation of microbicidal reactive oxygen species (ROS) through the NADPH dual oxidase (DUOX) is a first line of defense in the gut epithelia. Bacterial uracil acts as DUOX-activating ligand through poorly understood mechanisms. Here, we show that the Hedgehog (Hh) signaling pathway modulates uracil-induced DUOX activation. Uracil-induced Hh signaling is required for intestinal expression of the calcium-dependent cell adhesion molecule Cadherin 99C (Cad99C) and subsequent Cad99C-dependent formation of endosomes. These endosomes play essential roles in uracil-induced ROS production by acting as signaling platforms for PLCβ/PKC/Ca2+-dependent DUOX activation. Animals with impaired Hh signaling exhibit abolished Cad99C-dependent endosome formation and reduced DUOX activity, resulting in high mortality during enteric infection. Importantly, endosome formation, DUOX activation, and normal host survival are restored by genetic reintroduction of Cad99C into enterocytes, demonstrating the important role for Hh signaling in host resistance to enteric infection. Copyright © 2015 Elsevier Inc. All rights reserved.

A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5

DOE PAGES

Vild, Cody J.; Li, Yan; Guo, Emily Z.; ...

2015-01-30

Disassembly of the endosomal sorting complex required for transport (ESCRT) machinery from biological membranes is a critical final step in cellular processes that require the ESCRT function. This reaction is catalyzed by VPS4, an AAA-ATPase whose activity is tightly regulated by a host of proteins, including LIP5 and the ESCRT-III proteins. In this paper, we present structural and functional analyses of molecular interactions between human VPS4, LIP5, and the ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly inhibits the stimulation. Both of these observations are distinct frommore » what was previously described for homologous yeast proteins. The crystal structure of LIP5NTD in complex with the MIT (microtubule-interacting and transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, was determined at 1 Å resolution. It reveals an ESCRT-III binding induced moderate conformational change in LIP5NTD, which results from insertion of a conserved CHMP5 tyrosine residue (Tyr 182) at the core of LIP5NTD structure. Finally, mutation of Tyr 182 partially relieves the inhibition displayed by CHMP5. Together, these results suggest a novel mechanism of VPS4 regulation in metazoans, where CHMP5 functions as a negative allosteric switch to control LIP5-mediated stimulation of VPS4.« less

The p25 Subunit of the Dynactin Complex is Required for Dynein-Early Endosome Interaction

DTIC Science & Technology

2011-01-01

Recently, spectrin mutations in Drosophila melanogaster have been shown to also cause defective axonal transport and neu- ronal degeneration...A and study showed that in Arp1-RNAi–treated Drosophila S2 cells, although dynactin cannot be fully assembled and vesicle transport is defective...Muresan et al., 2001; Schroer 2004). However, this idea has been under debate recently, mainly because results from a study in Drosophila S2 cells

Sibling rivalry: competition between MHC class II family members inhibits immunity.

PubMed

Denzin, Lisa K; Cresswell, Peter

2013-01-01

Peptide loading of major histocompatibility complex (MHC) class II molecules in the endosomes and lysosomes of antigen-presenting cells is catalyzed by human leukocyte antigen-DM (HLA-DM) and modulated by HLA-DO. In a structural study in this issue, Guce et al. show that HLA-DO is an MHC class II mimic and functions as a competitive and essentially irreversible inhibitor of HLA-DM activity, thereby inhibiting MHC class II antigen presentation.

[INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

PubMed

Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

2015-01-01

To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only phospho-ERK2 could be detected after 60 min of endocytosis. In both cases, MAP-kinase activation dynamics was significantly different from the control. Our results suggest involvement of EGF-stimulated MAP-kinase pathway in cytoskeleton regulation. At the same time, they demonstrate that the two studied and widely used inhibitors are not equivalent with respect to not only the effect on MAP-kinase activity but also to such interdependent processes such as changes in cytoskeleton organization and signaling receptor' endocytosis.

Lamp1 Increases the Efficiency of Lassa Virus Infection by Promoting Fusion in Less Acidic Endosomal Compartments.

PubMed

Hulseberg, Christine E; Fénéant, Lucie; Szymańska, Katarzyna M; White, Judith M

2018-01-02

Lassa virus (LASV) is an arenavirus whose entry into host cells is mediated by a glycoprotein complex (GPC) comprised of a receptor binding subunit, GP1, a fusogenic transmembrane subunit, GP2, and a stable signal peptide. After receptor-mediated internalization, arenaviruses converge in the endocytic pathway, where they are thought to undergo low-pH-triggered, GPC-mediated fusion with a late endosome membrane. A unique feature of LASV entry is a pH-dependent switch from a primary cell surface receptor (α-dystroglycan) to an endosomal receptor, lysosomal-associated membrane protein (Lamp1). Despite evidence that the interaction between LASV GP1 and Lamp1 is critical, the function of Lamp1 in promoting LASV infection remains poorly characterized. Here we used wild-type (WT) and Lamp1 knockout (KO) cells to show that Lamp1 increases the efficiency of, but is not absolutely required for, LASV entry and infection. We then used cell-cell and pseudovirus-cell surface fusion assays to demonstrate that LASV GPC-mediated fusion occurs at a significantly higher pH when Lamp1 is present compared to when Lamp1 is missing. Correspondingly, we found that LASV entry occurs through less acidic endosomes in WT (Lamp1-positive) versus Lamp1 KO cells. We propose that, by elevating the pH threshold for fusion, Lamp1 allows LASV particles to exit the endocytic pathway before they encounter an increasingly acidic and harsh proteolytic environment, which could inactivate a significant percentage of incoming viruses. In this manner Lamp1 increases the overall efficiency of LASV entry and infection. IMPORTANCE Lassa virus is the most clinically important member of the Arenaviridae , a family that includes six additional biosafety level 4 (BSL4) hemorrhagic fever viruses. The lack of specific antiviral therapies for Lassa fever drives an urgent need to identify druggable targets, and interventions that block infection at the entry stage are particularly attractive. Lassa virus is only the second virus known to employ an intracellular receptor, the first being Ebola virus. Here we show that interaction with its intracellular receptor, Lamp1, enhances and upwardly shifts the pH dependence of fusion and consistently permits Lassa virus entry into cells through less acidic endosomes. We propose that in this manner, Lamp1 increases the overall efficiency of Lassa virus infection. Copyright © 2018 Hulseberg et al.

The zebrafish mutant vps18 as a model for vesicle-traffic related hypopigmentation diseases.

PubMed

Maldonado, Ernesto; Hernandez, Fabiola; Lozano, Carlos; Castro, Marta E; Navarro, Rosa E

2006-08-01

Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky-Pudlak, Chediak-Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18(hi2499A), which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18(hi2499A) the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18(hi2499A) larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18(hi2499A) can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

PubMed Central

Zhou, Sharon; Davidson, Cristin; McGlynn, Robert; Stephney, Gloria; Dobrenis, Kostantin; Vanier, Marie T.; Walkley, Steven U.

2011-01-01

Niemann-Pick disease type C (NPC) is a severe neurovisceral lysosomal storage disorder caused by defects in NPC1 or NPC2 proteins. Although numerous studies support the primacy of cholesterol storage, neurons of double-mutant mice lacking both NPC1 and an enzyme required for synthesis of all complex gangliosides (β1,4GalNAc transferase) have been reported to exhibit dramatically reduced cholesterol sequestration. Here we show that NPC2-deficient mice lacking this enzyme also exhibit reduced cholesterol, but that genetically restricting synthesis to only a-series gangliosides fully restores neuronal cholesterol storage to typical disease levels. Examining the subcellular locations of sequestered compounds in neurons lacking NPC1 or NPC2 by confocal microscopy revealed that cholesterol and the two principal storage gangliosides (GM2 and GM3) were not consistently co-localized within the same intracellular vesicles. To determine whether the lack of GM2 and GM3 co-localization was due to differences in synthetic versus degradative pathway expression, we generated mice lacking both NPC1 and lysosomal β-galactosidase, and therefore unable to generate GM2 and GM3 in lysosomes. Double mutants lacked both gangliosides, indicating that each is the product of endosomal/lysosomal processing. Unexpectedly, GM1 accumulation in double mutants increased compared to single mutants consistent with a direct role for NPC1 in ganglioside salvage. These studies provide further evidence that NPC1 and NPC2 proteins participate in endosomal/lysosomal processing of both sphingolipids and cholesterol. PMID:21708114

Multivesicular Bodies in Neurons: Distribution, Protein Content, and Trafficking Functions

PubMed Central

VON BARTHELD, CHRISTOPHER S.; ALTICK, AMY L.

2011-01-01

Summary Multivesicular bodies (MVBs) are intracellular endosomal organelles characterized by multiple internal vesicles that are enclosed within a single outer membrane. MVBs were initially regarded as purely prelysosomal structures along the degradative endosomal pathway of internalized proteins. MVBs are now known to be involved in numerous endocytic and trafficking functions, including protein sorting, recycling, transport, storage, and release. This review of neuronal MVBs summarizes their research history, morphology, distribution, accumulation of cargo and constitutive proteins, transport, and theories of functions of MVBs in neurons and glia. Due to their complex morphologies, neurons have expanded trafficking and signaling needs, beyond those of “geometrically simpler” cells, but it is not known whether neuronal MVBs perform additional transport and signaling functions. This review examines the concept of compartment-specific MVB functions in endosomal protein trafficking and signaling within synapses, axons, dendrites and cell bodies. We critically evaluate reports of the accumulation of neuronal MVBs based on evidence of stress-induced MVB formation. Furthermore, we discuss potential functions of neuronal and glial MVBs in development, in dystrophic neuritic syndromes, injury, disease, and aging. MVBs may play a role in Alzheimer’s, Huntington’s, and Niemann-Pick diseases, some types of frontotemporal dementia, prion and virus trafficking, as well as in adaptive responses of neurons to trauma and toxin or drug exposure. Functions of MVBs in neurons have been much neglected, and major gaps in knowledge currently exist. Developing truly MVB-specific markers would help to elucidate the roles of neuronal MVBs in intra- and intercellular signaling of normal and diseased neurons. PMID:21216273

The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

PubMed Central

Sandri, Chiara; Caccavari, Francesca; Valdembri, Donatella; Camillo, Chiara; Veltel, Stefan; Santambrogio, Martina; Lanzetti, Letizia; Bussolino, Federico; Ivaska, Johanna; Serini, Guido

2012-01-01

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1. PMID:22825554

TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

PubMed Central

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

A silencing-mediated enhancement of osteogenic differentiation by supramolecular ternary siRNA polyplexes comprising biocleavable cationic polyrotaxanes and anionic fusogenic peptides.

PubMed

Inada, Takasuke; Tamura, Atsushi; Terauchi, Masahiko; Yamaguchi, Satoshi; Yui, Nobuhiko

2018-01-30

Gene silencing of noggin by small interfering RNA (siRNA) is a promising approach for the treatment of bone defects, because noggin deactivates bone morphogenetic protein-2 (BMP-2) and suppresses osteogenic differentiation. Here, we demonstrated the silencing of the noggin gene by siRNA polyplexes composed of noggin-targeted siRNA and biocleavable cationic polyrotaxanes (DMAE-SS-PRX). To improve the endosomal escape efficiencies of the DMAE-SS-PRX/siRNA polyplexes, anionic and fusogenic GALA peptides were integrated onto the DMAE-SS-PRX/siRNA polyplexes via simple electrostatic interactions. The formation of ternary complexes was confirmed by gel electrophoresis, dynamic light scattering, and zeta-potential measurements. Although the association of GALA peptides with the DMAE-SS-PRX/siRNA polyplexes did not remarkably affect the cellular uptake efficiency of siRNA, the endosomal escape efficiency was remarkably increased for GALA/DMAE-SS-PRX/siRNA ternary polyplexes because of the endosomal and lysosomal membrane destabilization by GALA peptides. Consequently, GALA/DMAE-SS-PRX/siRNA ternary polyplexes showed significantly higher gene silencing efficiency against noggin and enhanced the BMP-2-mediated osteogenic differentiation efficiency. Therefore, we concluded that GALA/DMAE-SS-PRX/siRNA ternary polyplexes can be effective siRNA carriers for suppressing the expression of specific endogenous genes. Consequently, we believe that a more practical approach in vivo will be the combined use of BMP-2 and GALA/DMAE-SS-PRX/siRNA ternary polyplexes, because it will improve the efficacy of bone regeneration therapy.

ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis

PubMed Central

Konopacki, Filip A.; Dwivedy, Asha; Bellon, Anaïs; Blower, Michael D.

2016-01-01

Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo. These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS. PMID:27248654

GLUT4 Retention in Adipocytes Requires Two Intracellular Insulin-regulated Transport Steps

PubMed Central

Zeigerer, Anja; Lampson, Michael A.; Karylowski, Ola; Sabatini, David D.; Adesnik, Milton; Ren, Mindong; McGraw, Timothy E.

2002-01-01

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level. PMID:12134080

GLUT4 retention in adipocytes requires two intracellular insulin-regulated transport steps.

PubMed

Zeigerer, Anja; Lampson, Michael A; Karylowski, Ola; Sabatini, David D; Adesnik, Milton; Ren, Mindong; McGraw, Timothy E

2002-07-01

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level.

Evidence for a role of SNX16 in regulating traffic between the early and later endosomal compartments.

PubMed

Hanson, Brendon J; Hong, Wanjin

2003-09-05

Sorting nexins (SNXs) are a growing family of proteins characterized by the presence of a PX domain. The PX domain mediates membrane association by interaction with phosphoinositides. The SNXs are generally believed to participate in membrane trafficking, but information regarding the function of individual proteins is limited. In this report, we describe the major characteristics of one member, SNX16. SNX16 is a novel 343-amino acid protein consisting of a central PX domain followed by a potential coiled-coil domain and a C-terminal region. Like other sorting nexins, SNX16 associates with the membrane via the PX domain which interacts with the phospholipid phosphatidylinositol 3-phosphate. We show via biochemical and cellular studies that SNX16 is distributed in both early and late endosome/lysosome structures. The coiled-coil domain is necessary for localization to the later endosomal structures, as mutant SNX16 lacking this domain was found only in early endosomes. Trafficking of internalized epidermal growth factor was also delayed by this SNX16 mutant, as these cells showed a delay in the segregation of epidermal growth factor in the early endosome for its delivery to later compartments. In addition, the coiled-coil domain is shown here to be important for homo-oligomerization of SNX16. Taken together, these results suggest that SNX16 is a sorting nexin that may function in the trafficking of proteins between the early and late endosomal compartments.

Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation

PubMed Central

Jopling, Helen M.; Odell, Adam F.; Pellet-Many, Caroline; Latham, Antony M.; Frankel, Paul; Sivaprasadarao, Asipu; Walker, John H.; Zachary, Ian C.; Ponnambalam, Sreenivasan

2014-01-01

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis. PMID:24785348

Anterograde Trafficking of KCa3.1 in Polarized Epithelia Is Rab1- and Rab8-Dependent and Recycling Endosome-Independent

PubMed Central

Bertuccio, Claudia A.; Lee, Shih-Liang; Wu, Guangyu; Butterworth, Michael B.; Hamilton, Kirk L.; Devor, Daniel C.

2014-01-01

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells. PMID:24632741

Modular cell-internalizing aptamer nanostructure enables targeted delivery of large functional RNAs in cancer cell lines.

PubMed

Porciani, David; Cardwell, Leah N; Tawiah, Kwaku D; Alam, Khalid K; Lange, Margaret J; Daniels, Mark A; Burke, Donald H

2018-06-11

Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19-21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50-80 kDa, 175-250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

PubMed

Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

2009-03-23

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Polymeric micelles based on poly(methacrylic acid) block-containing copolymers with different membrane destabilizing properties for cellular drug delivery.

PubMed

Mebarek, Naila; Aubert-Pouëssel, Anne; Gérardin, Corine; Vicente, Rita; Devoisselle, Jean-Marie; Bégu, Sylvie

2013-10-01

Poly(methacrylic acid)-b-poly(ethylene oxide) are double hydrophilic block copolymers, which are able to form micelles by complexation with a counter-polycation, such as poly-l-lysine. A study was carried out on the ability of the copolymers to interact with model membranes as a function of their molecular weights and as a function of pH. Different behaviors were observed: high molecular weight copolymers respect the membrane integrity, whereas low molecular weight copolymers with a well-chosen asymmetry degree can induce a membrane alteration. Hence by choosing the appropriate molecular weight, micelles with distinct membrane interaction behaviors can be obtained leading to different intracellular traffics with or without endosomal escape, making them interesting tools for cell engineering. Especially micelles constituted of low molecular weight copolymers could exhibit the endosomal escape property, which opens vast therapeutic applications. Moreover micelles possess a homogeneous nanometric size and show variable properties of disassembly at acidic pH, of stability in physiological conditions, and finally of cyto-tolerance. Copyright © 2013 Elsevier B.V. All rights reserved.

Structure and membrane remodeling activity of ESCRT-III helical polymers

DOE PAGES

McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; ...

2015-12-18

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecturemore » and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. In conclusion, our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.« less

Decreased function of survival motor neuron protein impairs endocytic pathways.

PubMed

Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S; O'Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C Q; Cook, Steven J; Poulogiannis, George; Atwood, Walter J; Hall, David H; Hart, Anne C

2016-07-26

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.

Decreased function of survival motor neuron protein impairs endocytic pathways

PubMed Central

Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S.; O’Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C. Q.; Cook, Steven J.; Poulogiannis, George; Atwood, Walter J.; Hall, David H.; Hart, Anne C.

2016-01-01

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death. PMID:27402754

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

PubMed Central

Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C.; Nelson, Christian D. S.; Atwood, Walter J.; DiMaio, Daniel

2013-01-01

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed. PMID:23569269

Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

PubMed

Mallet, W G; Maxfield, F R

1999-07-26

Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J. Cell Biol.142:923-936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments.

PubMed

Stoops, Emily H; Hull, Michael; Caplan, Michael J

2016-12-01

Polarized epithelial cells sort newly synthesized and recycling plasma membrane proteins into distinct trafficking pathways directed to either the apical or basolateral membrane domains. While the trans-Golgi network is a well-established site of protein sorting, increasing evidence indicates a key role for endosomes in the initial trafficking of newly synthesized proteins. Both basolateral and apical proteins have been shown to traverse endosomes en route to the plasma membrane. In particular, apical proteins traffic through either subapical early or recycling endosomes. Here we use the SNAP tag system to analyze the trafficking of the apical protein gp135, also known as podocalyxin. We show that newly synthesized gp135 traverses the apical recycling endosome, but not the apical early endosomes (AEEs). In contrast, post-endocytic gp135 is delivered to the AEE before recycling back to the apical membrane. The pathways pursued by the newly synthesized and recycling gp135 populations do not detectably intersect, demonstrating that the biosynthetic and post-endocytic pools of this protein are subjected to distinct sorting processes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Role for Peptides in Overcoming Endosomal Entrapment in siRNA Delivery – A Focus on Melittin

PubMed Central

Hou, Kirk K.; Pan, Hua; Schlesinger, Paul H.; Wickline, Samuel A.

2015-01-01

siRNA has the possibility to revolutionize medicine by enabling highly specific and efficient silencing of proteins involved in disease pathogenesis. Despite nearly 20 years of research dedicated to translating siRNA from a research tool into a clinically relevant therapeutic, minimal success has been had to date. Access to RNA interference machinery located in the cytoplasm is often overlooked, but must be considered when designing the next generation of siRNA delivery strategies. Peptide transduction domains (PTD) have demonstrated moderate siRNA transfection, which is primarily limited by endosomal entrapment. Strategies aimed at overcoming endosomal entrapment associated with peptide vectors are reviewed here, including osmotic methods, lipid conjugation, and fusogenic peptides. As an alternative to traditional PTD, the hemolytic peptide melittin exhibits the native capacity for endosomal disruption but causes cytotoxicity. However, appropriate packaging and protection of melittin with activation and release in the endosomal compartment has allowed melittin-based strategies to demonstrate both in vitro and in vivo safety and efficacy. These data suggest that melittin's membrane disruptive properties can enable safe and effective endosomolysis, building a case for melittin as a key component in a new generation of siRNA therapeutics. PMID:26025036

Stargazin-related protein γ7 is associated with signalling endosomes in superior cervical ganglion neurons and modulates neurite outgrowth

PubMed Central

Waithe, Dominic; Ferron, Laurent; Dolphin, Annette C.

2011-01-01

The role(s) of the newly discovered stargazin-like γ-subunit proteins remains unclear; although they are now widely accepted to be transmembrane AMPA receptor regulatory proteins (TARPs), rather than Ca2+ channel subunits, it is possible that they have more general roles in trafficking within neurons. We previously found that γ7 subunit is associated with vesicles when it is expressed in neurons and other cells. Here, we show that γ7 is present mainly in retrogradely transported organelles in sympathetic neurons, where it colocalises with TrkA–YFP, and with the early endosome marker EEA1, suggesting that γ7 localises to signalling endosomes. It was not found to colocalise with markers of the endoplasmic reticulum, mitochondria, lysosomes or late endosomes. Furthermore, knockdown of endogenous γ7 by short hairpin RNA transfection into sympathetic neurons reduced neurite outgrowth. The same was true in the PC12 neuronal cell line, where neurite outgrowth was restored by overexpression of human γ7. These findings open the possibility that γ7 has an essential trafficking role in relation to neurite outgrowth as a component of endosomes involved in neurite extension and growth cone remodelling. PMID:21610096

Evaluation of cellular uptake and gene transfer efficiency of pegylated poly-L-lysine compacted DNA: implications for cancer gene therapy.

PubMed

Walsh, M; Tangney, M; O'Neill, M J; Larkin, J O; Soden, D M; McKenna, S L; Darcy, R; O'Sullivan, G C; O'Driscoll, C M

2006-01-01

Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the efficiency of gene delivery.

An AGEF-1/Arf GTPase/AP-1 Ensemble Antagonizes LET-23 EGFR Basolateral Localization and Signaling during C. elegans Vulva Induction

PubMed Central

Skorobogata, Olga; Escobar-Restrepo, Juan M.; Rocheleau, Christian E.

2014-01-01

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling. PMID:25329472

An AGEF-1/Arf GTPase/AP-1 ensemble antagonizes LET-23 EGFR basolateral localization and signaling during C. elegans vulva induction.

PubMed

Skorobogata, Olga; Escobar-Restrepo, Juan M; Rocheleau, Christian E

2014-10-01

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

PubMed Central

Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

2015-01-01

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous). PMID:25799564

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures.

PubMed

Riquelme, Meritxell; Aguirre, Jesús; Bartnicki-García, Salomon; Braus, Gerhard H; Feldbrügge, Michael; Fleig, Ursula; Hansberg, Wilhelm; Herrera-Estrella, Alfredo; Kämper, Jörg; Kück, Ulrich; Mouriño-Pérez, Rosa R; Takeshita, Norio; Fischer, Reinhard

2018-06-01

Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established. Copyright © 2018 American Society for Microbiology.

IFITM3 Restricts Human Metapneumovirus Infection.

PubMed

McMichael, Temet M; Zhang, Yu; Kenney, Adam D; Zhang, Lizhi; Zani, Ashley; Lu, Mijia; Chemudupati, Mahesh; Li, Jianrong; Yount, Jacob S

2018-06-15

Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein Toll-like receptor 7 (TLR7), and that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells.

Biogenesis and Function of Multivesicular Bodies

PubMed Central

Piper, Robert C.; Katzmann, David J.

2010-01-01

The two major cellular sites for membrane protein degradation are the proteasome and the lysosome. Ubiquitin attachment is a sorting signal for both degradation routes. For lysosomal degradation, ubiquitination triggers the sorting of cargo proteins into the lumen of late endosomal multivesicular bodies (MVBs)/endosomes. MVB formation occurs when a portion of the limiting membrane of an endosome invaginates and buds into its own lumen. Intralumenal vesicles are degraded when MVBs fuse to lysosomes. The proper delivery of proteins to the MVB interior relies on specific ubiquitination of cargo, recognition and sorting of ubiquitinated cargo to endosomal subdomains, and the formation and scission of cargo-filled intralumenal vesicles. Over the past five years, a number of proteins that may directly participate in these aspects of MVB function and biogenesis have been identified. However, major questions remain as to exactly what these proteins do at the molecular level and how they may accomplish these tasks. PMID:17506697

Challenges in carrier-mediated intracellular delivery: moving beyond endosomal barriers.

PubMed

Stewart, Martin P; Lorenz, Anna; Dahlman, James; Sahay, Gaurav

2016-05-01

The deployment of molecular to microscale carriers for intracellular delivery has tremendous potential for biology and medicine, especially for in vivo therapies. The field remains limited, however, by a poor understanding of how carriers gain access to the cell interior. In this review, we provide an overview of the different types of carriers, their speculated modes of entry, putative pathways of vesicular transport, and sites of endosomal escape. We compare this alongside pertinent examples from the cell biology of how viruses, bacteria, and their effectors enter cells and escape endosomal confinement. We anticipate insights into the mechanisms of cellular entry and endosomal escape will benefit future research efforts on effective carrier-mediated intracellular delivery. WIREs Nanomed Nanobiotechnol 2016, 8:465-478. doi: 10.1002/wnan.1377 For further resources related to this article, please visit the WIREs website. © 2015 Wiley Periodicals, Inc.

A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells.

PubMed

Hertzano, Ronna; Shalit, Ella; Rzadzinska, Agnieszka K; Dror, Amiel A; Song, Lin; Ron, Uri; Tan, Joshua T; Shitrit, Alina Starovolsky; Fuchs, Helmut; Hasson, Tama; Ben-Tal, Nir; Sweeney, H Lee; de Angelis, Martin Hrabe; Steel, Karen P; Avraham, Karen B

2008-10-03

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or 'gating' in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI-impaired hair cells, and ultimately leading to deafness.

A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila.

PubMed

Sevrioukov, E A; He, J P; Moghrabi, N; Sunio, A; Krämer, H

1999-10-01

Deep orange and carnation are two of the classic eye color genes in Drosophila. Here, we demonstrate that Deep orange is part of a protein complex that localizes to endosomal compartments. A second component of this complex is Carnation, a homolog of Sec1p-like regulators of membrane fusion. Because complete loss of deep orange function is lethal, the role of this complex in intracellular trafficking was analyzed in deep orange mutant clones. Retinal cells devoid of deep orange function completely lacked pigmentation and exhibited exaggerated multivesicular structures. Furthermore, a defect in endocytic trafficking was visualized in developing photoreceptor cells. These results provide direct evidence that eye color mutations of the granule group also disrupt vesicular trafficking to lysosomes.

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2.

PubMed

Selyunin, Andrey S; Mukhopadhyay, Somshuvra

2015-12-01

Shiga toxin-producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A-subunits block protein synthesis, while the B-subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B-subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B-subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome-to-Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface-exposed loop in STx2B (β4-β5 loop) is required for its endosome-to-Golgi trafficking. We previously demonstrated that residues in the corresponding β4-β5 loop of STx1B are required for interaction with GPP130, the STx1B-specific endosomal receptor, and for endosome-to-Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

PubMed

Isono, Erika; Kalinowska, Kamila

2017-12-01

To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

PubMed Central

Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves

2016-01-01

ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. PMID:27803188

PalC, One of Two Bro1 Domain Proteins in the Fungal pH Signalling Pathway, Localizes to Cortical Structures and Binds Vps32

PubMed Central

Galindo, Antonio; Hervás-Aguilar, América; Rodríguez-Galán, Olga; Vincent, Olivier; Arst, Herbert N; Tilburn, Joan; Peñalva, Miguel A

2007-01-01

PalC, distantly related to Saccharomyces cerevisiaeperipheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulanspH signalling proteins, contains a Bro1 domain. Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active. PalC recruitment to these structures is dependent on the seven transmembrane domain (7-TMD) receptor and likely pH sensor PalH. PalC is a two-hybrid interactor of the ESCRT-III Vps20/Vps32 subcomplex and binds Vps32 directly. This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence. In contrast, Arg442Δ impairs Vps32 binding and prevents PalC-GFP recruitment to cortical structures. pH signalling involves a plasma membrane complex including the 7-TMD receptor PalH and the arrestin-like PalF and an endosomal membrane complex involving the PalB protease, the transcription factor PacC and the Vps32 binding, Bro1-domain-containing protein PalA. PalC, which localizes to cortical structures and can additionally bind a component of ESCRT-III, has the features required to bridge these two entities. A likely S. cerevisiaeorthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes. PMID:17696968

BLOC-2, AP-3, and AP-1 Proteins Function in Concert with Rab38 and Rab32 Proteins to Mediate Protein Trafficking to Lysosome-related Organelles*

PubMed Central

Bultema, Jarred J.; Ambrosio, Andrea L.; Burek, Carolyn L.; Di Pietro, Santiago M.

2012-01-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis. PMID:22511774

BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.

PubMed

Bultema, Jarred J; Ambrosio, Andrea L; Burek, Carolyn L; Di Pietro, Santiago M

2012-06-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

PubMed

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P

2017-06-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1 -knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. © 2017 Kaur et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Tumor-targeted pH/redox dual-sensitive unimolecular nanoparticles for efficient siRNA delivery.

PubMed

Chen, Guojun; Wang, Yuyuan; Xie, Ruosen; Gong, Shaoqin

2017-08-10

A unique pH/redox dual-sensitive cationic unimolecular nanoparticle (NP) enabling excellent endosomal/lysosomal escape and efficient siRNA decomplexation inside the target cells was developed for tumor-targeted delivery of siRNA. siRNA was complexed into the cationic core of the unimolecular NP through electrostatic interactions. The cationic core used for complexing siRNA contained reducible disulfide bonds that underwent intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH) inside the cells, thereby facilitating the decomplexation of siRNA from the unimolecular NPs. The cationic polymers were conjugated onto the hyperbranched core (H40) via a pH-sensitive bond, which further facilitated the decomplexation of siRNA from the NPs. In vitro studies on the siRNA release behaviors showed that dual stimuli (pH=5.3, 10mM GSH) induced the quickest release of siRNA from the NPs. In addition, the imidazole groups attached to the cationic polymer segments enhanced the endosomal/lysosomal escape of NPs via the proton sponge effect. Intracellular tracking studies revealed that siRNA delivered by unimolecular NPs was efficiently released to the cytosol. Moreover, the GE11 peptide, an anti-EGFR peptide, enhanced the cellular uptake of NPs in MDA-MB-468, an EFGR-overexpressing triple negative breast cancer (TNBC) cell line. The GE11-conjugated, GFP-siRNA-complexed NPs exhibited excellent GFP gene silencing efficiency in GFP-MDA-MB-468 TNBC cells without any significant cytotoxicity. Therefore, these studies suggest that this smart unimolecular NP could be a promising nanoplatform for targeted siRNA delivery to EFGR-overexpressing cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

PubMed

Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

2015-03-13

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

PubMed Central

Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

2015-01-01

The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier. PMID:26123532

Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

NASA Astrophysics Data System (ADS)

Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

2015-06-01

The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

PubMed Central

Zacchi, Paola; Stenmark, Harald; Parton, Robert G.; Orioli, Donata; Lim, Filip; Giner, Angelika; Mellman, Ira; Zerial, Marino; Murphy, Carol

1998-01-01

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells. PMID:9490718

IQGAP1 promotes CXCR4 chemokine receptor function and trafficking via EEA-1+ endosomes

PubMed Central

Bamidele, Adebowale O.; Kremer, Kimberly N.; Hirsova, Petra; Clift, Ian C.; Gores, Gregory J.; Billadeau, Daniel D.

2015-01-01

IQ motif–containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell–derived factor-1 (SDF-1; also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1+ endosomes, and decreased efficiency of CXCR4 recycling. SDF-1–induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to α-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1+ endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1+ endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types. PMID:26195666

A proteomic approach to identify endosomal cargoes controlling cancer invasiveness

PubMed Central

Diaz-Vera, Jesica; Palmer, Sarah; Hernandez-Fernaud, Juan Ramon; Dornier, Emmanuel; Mitchell, Louise E.; Macpherson, Iain; Edwards, Joanne; Zanivan, Sara

2017-01-01

ABSTRACT We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype. PMID:28062852

Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction

PubMed Central

Gray, Lindsay; Tsurudome, Kazuya; El-Mounzer, Wassim; Elazzouzi, Fatima; Baim, Christopher; Calderon, Mario R.; Kauwe, Grant

2018-01-01

Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons. PMID:29373576

The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

NASA Astrophysics Data System (ADS)

Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

2014-07-01

Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

Spatial separation of the processing and MHC class I loading compartments for cross-presentation of the tumor-associated antigen HER2/neu by human dendritic cells

PubMed Central

Baleeiro, Renato B; Rietscher, René; Diedrich, Andrea; Czaplewska, Justyna A; Lehr, Claus-Michael; Scherließ, Regina; Hanefeld, Andrea; Gottschaldt, Michael; Walden, Peter

2015-01-01

Cross-presentation is the process by which professional antigen presenting cells (APCs) (B cells, dendritic cells (DCs) and macrophages) present endocytosed antigens (Ags) via MHC-I to CD8+ T cells. This process is crucial for induction of adaptive immune responses against tumors and infected cells. The pathways and cellular compartments involved in cross-presentation are unresolved and controversial. Among the cells with cross-presenting capacity, DCs are the most efficient, which was proposed to depend on prevention of endosomal acidification to block degradation of the epitopes. Contrary to this view, we show in this report that some cargoes induce strong endosomal acidification following uptake by human DCs, while others not. Moreover, processing of the tumor-associated antigen HER2/neu delivered in nanoparticles (NP) for cross-presentation of the epitope HER2/neu369–377 on HLA-A2 depended on endosomal acidification and cathepsin activity as well as proteasomes, and newly synthesized HLA class I. However, the HLA-A*0201/HER2/neu369–377 complexes were not found in the endoplasmic reticulum (ER) nor in endolysosomes but in hitherto not described vesicles. The data thus indicate spatial separation of antigen processing and loading of MHC-I for cross-presentation: antigen processing occurs in the uptake compartment and the cytosol whereas MHC-I loading with peptide takes place in a distinct subcellular compartment. The findings further elucidate the cellular pathways involved in the cross-presentation of a full-length, clinically relevant tumor-associated antigen by human DCs, and the impact of the vaccine formulation on antigen processing and CD8+ T cell induction. PMID:26985398

Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1-Dependent Trafficking of Integrins.

PubMed

Kozyulina, Polina Y; Loskutov, Yuriy V; Kozyreva, Varvara K; Rajulapati, Anuradha; Ice, Ryan J; Jones, Brandon C; Pugacheva, Elena N

2015-03-01

The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of the prometastatic protein, NEDD9, in breast cancer cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and colocalizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand-integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Reexpression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9-depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. This study provides valuable new insight into the potential therapeutic benefit of NEDD9 depletion to reduce dissemination of tumor cells and discovers a new regulatory role of NEDD9 in promoting migration through modulation of CAV1-dependent trafficking of integrins. ©2014 American Association for Cancer Research.

Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells.

PubMed

Barth, Holger; Stiles, Bradley G

2008-01-01

Binary bacterial toxins are unique AB-type toxins, composed of two non-linked proteins that act as a binding/translocation component and an enzyme component. All known actin-ADP-ribosylating toxins from clostridia possess this binary structure. This toxin family is comprised of the prototypical Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile CDT, and Clostridium spiroforme toxin. Once in the cytosol of host cells, these toxins transfer an ADP-ribose moiety from nicotinamide-adenosine-dinucleotide onto G-actin that then leads to depolymerization of actin filaments. In recent years much progress has been made towards understanding the cellular uptake mechanism of binary actin-ADP-ribosylating toxins, and in particular that of C2 toxin. Both components act in a precisely concerted manner to intoxicate eukaryotic cells. The binding/translocation (B-) component forms a complex with the enzyme (A-) component and mediates toxin binding to a cell-surface receptor. Following receptor-mediated endocytosis, the enzyme component escapes from acidic endosomes into the cytosol. Acidification of endosomes triggers pore formation by the binding/translocation component in endosomal membranes and the enzyme component subsequently translocates through the pore. This step requires a host cell chaperone, Hsp90. Due to their unique structure, binary toxins are naturally "tailor made" for transporting foreign proteins into the cytosol of host cells. Several highly specific and cell-permeable recombinant fusion proteins have been designed and successfully used in experimental cell research. This review will focus on the recent progress in studying binary actin ADP-ribosylating toxins as highly effective virulence factors and innovative tools for cell physiology as well as pharmacology.

Aldolase directly interacts with ARNO and modulates cell morphology and acidic vesicle distribution

PubMed Central

Merkulova, Maria; Hurtado-Lorenzo, Andrés; Hosokawa, Hiroyuki; Zhuang, Zhenjie; Brown, Dennis; Ausiello, Dennis A.

2011-01-01

Previously, we demonstrated that the vacuolar-type H+-ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1–a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: KD = 2.84 × 10−10 M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms. PMID:21307348

Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

PubMed

Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

2013-05-01

P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient. © 2013 John Wiley & Sons A/S.

The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity*

PubMed Central

Clement, Cristina C.; Becerra, Aniuska; Yin, Liusong; Zolla, Valerio; Huang, Liling; Merlin, Simone; Follenzi, Antonia; Shaffer, Scott A.; Stern, Lawrence J.; Santambrogio, Laura

2016-01-01

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of “self-recognition” as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis. PMID:26740625

Structural Fine-Tuning of MIT-Interacting Motif 2 (MIM2) and Allosteric Regulation of ESCRT-III by Vps4 in Yeast.

PubMed

Kojima, Rieko; Obita, Takayuki; Onoue, Kousuke; Mizuguchi, Mineyuki

2016-06-05

The endosomal sorting complex required for transport (ESCRT) facilitates roles in membrane remodeling, such as multivesicular body biogenesis, enveloped virus budding and cell division. In yeast, Vps4 plays a crucial role in intraluminal vesicle formation by disassembling ESCRT proteins. Vps4 is recruited by ESCRT-III proteins to the endosomal membrane through the interaction between the microtubule interacting and trafficking (MIT) domain of Vps4 and the C-terminal MIT-interacting motif (MIM) of ESCRT-III proteins. Here, we have determined the crystal structure of Vps4-MIT in a complex with Vps20, a member of ESCRT-III, and revealed that Vps20 adopts a unique MIM2 conformation. Based on structural comparisons with other known MIM2s, we have refined the consensus sequence of MIM2. We have shown that another ESCRT-III protein, Ist1, binds to Vps4-MIT via its C-terminal MIM1 with higher affinity than Vps2, but lacks MIM2 by surface plasmon resonance. Surprisingly, the Ist1 MIM1 competed with the MIM2 of Vfa1, a regulator of Vps4, for binding to Vps4-MIT, even though these MIMs bind in non-overlapping sites on the MIT. These findings provide insight into the allosteric recognition of MIMs of ESCRT-III by Vps4 and also the regulation of ESCRT machinery at the last step of membrane remodeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

KinG Is a Plant-Specific Kinesin That Regulates Both Intra- and Intercellular Movement of SHORT-ROOT.

PubMed

Spiegelman, Ziv; Lee, Chin-Mei; Gallagher, Kimberly L

2018-01-01

Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR. © 2018 American Society of Plant Biologists. All Rights Reserved.

Biocompatible mannosylated endosomal-escape nanoparticles enhance selective delivery of short nucleotide sequences to tumor associated macrophages

NASA Astrophysics Data System (ADS)

Ortega, Ryan A.; Barham, Whitney J.; Kumar, Bharat; Tikhomirov, Oleg; McFadden, Ian D.; Yull, Fiona E.; Giorgio, Todd D.

2014-12-01

Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create a pro-tumor niche. Manipulation of the TAM phenotype is a novel, potential therapeutic approach to engage anti-cancer immunity. siRNA is a molecular tool for knockdown of specific mRNAs that is tunable in both strength and duration. The use of siRNA to reprogram TAMs to adopt an immunogenic, anti-tumor phenotype is an attractive alternative to ablation of this cell population. One current difficulty with this approach is that TAMs are difficult to specifically target and transfect. We report here successful utilization of novel mannosylated polymer nanoparticles (MnNP) that are capable of escaping the endosomal compartment to deliver siRNA to TAMs in vitro and in vivo. Transfection with MnNP-siRNA complexes did not significantly decrease TAM cell membrane integrity in culture, nor did it create adverse kidney or liver function in mice, even at repeated doses of 5 mg kg-1. Furthermore, MnNP effectively delivers labeled nucleotides to TAMs in mice with primary mammary tumors. We also confirmed TAM targeting in the solid tumors disseminated throughout the peritoneum of ovarian tumor bearing mice following injection of fluorescently labeled MnNP-nucleotide complexes into the peritoneum. Finally, we show enhanced uptake of MnNP in lung metastasis associated macrophages compared to untargeted particles when using an intubation delivery method. In summary, we have shown that MnNP specifically and effectively deliver siRNA to TAMs in vivo.

A new liposome-based gene delivery system targeting lung epithelial cells using endothelin antagonist.

PubMed

Allon, Nahum; Saxena, Ashima; Chambers, Carolyn; Doctor, Bhupendra P

2012-06-10

We formulated a new gene delivery system based on targeted liposomes. The efficacy of the delivery system was demonstrated in in vitro and in vivo models. The targeting moiety consists of a high-affinity 7-amino-acid peptide, covalently and evenly conjugated to the liposome surface. The targeting peptide acts as an endothelin antagonist, and accelerates liposome binding and internalization. It is devoid of other biological activity. Liposomes with high phosphatidyl serine (PS) were specially formulated to help their fusion with the endosomal membrane at low pH and enable release of the liposome payload into the cytoplasm. A DNA payload, pre-compressed by protamine, was encapsulated into the liposomes, which directed the plasmid into the cell's nucleus. Upon exposure to epithelial cells, binding of the liposomes occurred within 5-10 min, followed by facilitated internalization of the complex. Endosomal escape was complete within 30 min, followed by DNA accumulation in the nucleus 2h post-transfection. A549 lung epithelial cells transfected with plasmid encoding for GFP encapsulated in targeted liposomes expressed significantly more protein than those transfected with plasmid complexed with Lipofectamine. The intra-tracheal instillation of plasmid encoding for GFP encapsulated in targeted liposomes into rat lungs resulted in the expression of GFP in bronchioles and alveoli within 5 days. These results suggest that this delivery system has great potential in targeting genes to lungs. Copyright © 2011 Elsevier B.V. All rights reserved.

Human heat shock protein 70 enhances tumor antigen presentation through complex formation and intracellular antigen delivery without innate immune signaling.

PubMed

Bendz, Henriette; Ruhland, Sibylle C; Pandya, Maya J; Hainzl, Otmar; Riegelsberger, Stefan; Braüchle, Christoph; Mayer, Matthias P; Buchner, Johannes; Issels, Rolf D; Noessner, Elfriede

2007-10-26

Heat shock proteins (HSPs) have shown promise for the optimization of protein-based vaccines because they can transfer exogenous antigens to dendritic cells and at the same time induce their maturation. Great care must be exercised in interpretating HSP-driven studies, as by-products linked to the recombinant generation of these proteins have been shown to mediate immunological effects. We generated highly purified human recombinant Hsp70 and demonstrated that it strongly enhances the cross-presentation of exogenous antigens resulting in better antigen-specific T cell stimulation. Augmentation of T cell stimulation was a direct function of the degree of complex formation between Hsp70 and peptides and correlated with improved antigen delivery to endosomal compartments. The Hsp70 activity was independent of TAP proteins and was not inhibited by exotoxin A or endosomal acidification. Consequently, Hsp70 enhanced cross-presentation of various antigenic sequences, even when they required different post-uptake processing and trafficking, as exemplified by the tumor antigens tyrosinase and Melan-A/MART-1. Furthermore, Hsp70 enhanced cross-presentation by different antigen-presenting cells (APCs), including dendritic cells and B cells. Importantly, enhanced cross-presentation and antigen-specific T cell activation were observed in the absence of innate signals transmitted by Hsp70. As Hsp70 supports the cross-presentation of different antigens and APCs and is inert to APC function, it may show efficacy in various settings of immune modulation, including induction of antigen-specific immunity or tolerance.

Structural analysis of the coordination of dinitrogen to transition metal complexes.

PubMed

Peigné, Benjamin; Aullón, Gabriel

2015-06-01

Transition-metal complexes show a wide variety of coordination modes for the nitrogen molecule. A structural database study has been undertaken for dinitrogen complexes, and geometrical parameters around the L(n)M-N2 unit are retrieved from the Cambridge Structural Database. These data were classified in families of compounds, according to metal properties, to determine the degree of lengthening for the dinitrogen bonding. The importance of the nature of the metal center, such as coordination number and electronic configuration, is reported. Our study reveals poor activation by coordination of dinitrogen in mononuclear complexes, always having end-on coordination. However, partial weakening of nitrogen-nitrogen bonding is found for end-on binuclear complexes, whereas side-on complexes can be completely activated.

Cytotoxicity of Cyclometalated Platinum Complexes Based on Tridentate NCN and CNN-coordinating ligands: Remarkable Coordination Dependence

PubMed Central

Vezzu, Dileep A. k.; Lu, Qun; Chen, Yan-Hua; Huo, Shouquan

2014-01-01

A series of cyclometalated platinum complexes with diverse coordination patterns and geometries were screened for their anticancer activity. It was discovered that the NʌCʌN-coordinated platinum complex based on 1,3-di(pyridyl)benzene displayed much higher cytotoxicity against human lung cancer cells NCI-H522, HCC827, and NCI-H1299, and human prostate cancer cell RV1 than cisplatin. In a sharp contrast, the CʌNʌN-coordinated platinum complex based on 6-phenyl-2,2′-bipyridine was ineffective on these cancer cells. This remarkable difference in cytotoxicity displayed by NʌCʌN- and CʌNʌN-coordinated platinum complexes was related to the trans effect of the carbon donor in the cyclometalated platinum complexes, which played a crucial role in facilitating the dissociation of the chloride ligand to create an active binding site. The DNA binding was studied for the NʌCʌN-coordinated platinum complex using electrophoresis and emission titration. The cellular uptake observed by fluorescent microscope showed the complex is largely concentrated in the cytoplasm. The possible pathways for the cell apoptosis was studied by western blot analysis and the activation of PARP via caspase 7 was observed. PMID:24531534

Endothelin-converting enzyme-1 degrades internalized somatostatin-14.

PubMed

Roosterman, Dirk; Kempkes, Cordula; Cottrell, Graeme S; Padilla, Benjamin E; Bunnett, Nigel W; Turck, Christoph W; Steinhoff, Martin

2008-05-01

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

Cellular Auxin Homeostasis under High Temperature Is Regulated through a SORTING NEXIN1–Dependent Endosomal Trafficking Pathway[C][W

PubMed Central

Hanzawa, Taiki; Shibasaki, Kyohei; Numata, Takahiro; Kawamura, Yukio; Gaude, Thierry; Rahman, Abidur

2013-01-01

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature. PMID:24003052

Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

PubMed Central

Hsu, FoSheng; Spannl, Stephanie; Ferguson, Charles; Hyman, Anthony A; Parton, Robert G

2018-01-01

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS. PMID:29469808

dOCRL maintains immune cell quiescence by regulating endosomal traffic

PubMed Central

Del Signore, Steven J.; Biber, Sarah A.; Lehmann, Katherine S.; Heimler, Stephanie R.; Rosenfeld, Benjamin H.; Eskin, Tania L.

2017-01-01

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome. PMID:29028801

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

PubMed Central

Bentley, Marvin; Decker, Helena; Luisi, Julie

2015-01-01

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations. PMID:25624392

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin.

PubMed

Lesteberg, Kelsey; Orange, Jordan; Makedonas, George

2017-10-01

Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein, we aimed to determine how new perforin transits to the synapse if not via lytic granules. We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response.

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

PubMed Central

Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

2018-01-01

Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles

PubMed Central

Wang, Chensu; Wang, Yiguang; Li, Yang; Bodemann, Brian; Zhao, Tian; Ma, Xinpeng; Huang, Gang; Hu, Zeping; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming

2015-01-01

Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells. Deployment in cells allows quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. Together, these biological applications indicate the robustness and adaptability of this nanotechnology-enabled ‘detection and perturbation' strategy. PMID:26437053

Identification of different coordination geometries by XAFS in copper(II) complexes with trimesic acid

NASA Astrophysics Data System (ADS)

Gaur, A.; Klysubun, W.; Soni, Balram; Shrivastava, B. D.; Prasad, J.; Srivastava, K.

2016-10-01

X-ray absorption spectroscopy (XAS) is very useful in revealing the information about geometric and electronic structure of a transition-metal absorber and thus commonly used for determination of metal-ligand coordination. But XAFS analysis becomes difficult if differently coordinated metal centers are present in a system. In the present investigation, existence of distinct coordination geometries around metal centres have been studied by XAFS in a series of trimesic acid Cu(II) complexes. The complexes studied are: Cu3(tma)2(im)6 8H2O (1), Cu3(tma)2(mim)6 17H2O (2), Cu3(tma)2(tmen)3 8.5H2O (3), Cu3(tma) (pmd)3 6H2O (ClO4)3 (4) and Cu3(tma)2 3H2O (5). These complexes have not only Cu metal centres with different coordination but in complexes 1-3, there are multiple coordination geometries present around Cu centres. Using XANES spectra, different coordination geometries present in these complexes have been identified. The variation observed in the pre-edge features and edge features have been correlated with the distortion of the specific coordination environment around Cu centres in the complexes. XANES spectra have been calculated for the distinct metal centres present in the complexes by employing ab-initio calculations. These individual spectra have been used to resolve the spectral contribution of the Cu centres to the particular XANES features exhibited by the experimental spectra of the multinuclear complexes. Also, the variation in the 4p density of states have been calculated for the different Cu centres and then correlated with the features originated from corresponding coordination of Cu. Thus, these spectral features have been successfully utilized to detect the presence of the discrete metal centres in a system. The inferences about the coordination geometry have been supported by EXAFS analysis which has been used to determine the structural parameters for these complexes.

Specific biomolecule corona is associated with ring-shaped organization of silver nanoparticles in cells

NASA Astrophysics Data System (ADS)

Drescher, Daniela; Guttmann, Peter; Büchner, Tina; Werner, Stephan; Laube, Gregor; Hornemann, Andrea; Tarek, Basel; Schneider, Gerd; Kneipp, Janina

2013-09-01

We correlate the localization of silver nanoparticles inside cells with respect to the cellular architecture with the molecular information in the vicinity of the particle surface by combining nanoscale 3D cryo-soft X-ray tomography (cryo-SXT) with surface-enhanced Raman scattering (SERS). The interaction of the silver nanoparticle surface with small molecules and biopolymers was monitored by SERS in vitro over time in living cells. The spectra indicate a stable, time-independent surface composition of silver nanoparticles, despite the changing environment in the endosomal structure. Cryo-SXT reveals a characteristic ring-shaped organization of the silver nanoparticles in endosomes of different cell types. The ring-like structures inside the endosomes suggest a strong association among silver particles and with membrane structures. The comparison of the data with those obtained with gold nanoparticles suggests that the interactions between the nanoparticles and with the endosomal component are influenced by the molecular composition of the corona.We correlate the localization of silver nanoparticles inside cells with respect to the cellular architecture with the molecular information in the vicinity of the particle surface by combining nanoscale 3D cryo-soft X-ray tomography (cryo-SXT) with surface-enhanced Raman scattering (SERS). The interaction of the silver nanoparticle surface with small molecules and biopolymers was monitored by SERS in vitro over time in living cells. The spectra indicate a stable, time-independent surface composition of silver nanoparticles, despite the changing environment in the endosomal structure. Cryo-SXT reveals a characteristic ring-shaped organization of the silver nanoparticles in endosomes of different cell types. The ring-like structures inside the endosomes suggest a strong association among silver particles and with membrane structures. The comparison of the data with those obtained with gold nanoparticles suggests that the interactions between the nanoparticles and with the endosomal component are influenced by the molecular composition of the corona. Electronic supplementary information (ESI) available: Description of additional experiments. Explanation of transmitted intensity and linear absorption coefficient in a cryo-XRT experiment (Fig. S1 and S2). Additional X-ray data (Fig. S3 and Movie S1). Toxicity of silver nanoparticles (Fig. S4). X-ray microscopy and SERS experiments with gold nanoparticles (Fig. S5 and S6). Size, plasmonic properties, and stability of silver and gold nanoparticles (Fig. S7-S9). Distribution of the silver nanoparticles in the cells using SERS mapping (Fig. S10). Tentative band assignments (Table S1). See DOI: 10.1039/c3nr02129g

Cytotoxicity of cyclometalated platinum complexes based on tridentate NCN and CNN-coordinating ligands: remarkable coordination dependence.

PubMed

Vezzu, Dileep A K; Lu, Qun; Chen, Yan-Hua; Huo, Shouquan

2014-05-01

A series of cyclometalated platinum complexes with diverse coordination patterns and geometries were screened for their anticancer activity. It was discovered that the N^C^N-coordinated platinum complex based on 1,3-di(pyridyl)benzene displayed much higher cytotoxicity against human lung cancer cells NCI-H522, HCC827, and NCI-H1299, and human prostate cancer cell RV1 than cisplatin. In a sharp contrast, the C^N^N-coordinated platinum complex based on 6-phenyl-2,2'-bipyridine was ineffective on these cancer cells. This remarkable difference in cytotoxicity displayed by N^C^N- and C^N^N-coordinated platinum complexes was related to the trans effect of the carbon donor in the cyclometalated platinum complexes, which played a crucial role in facilitating the dissociation of the chloride ligand to create an active binding site. The DNA binding was studied for the N^C^N-coordinated platinum complex using electrophoresis and emission titration. The cellular uptake observed by fluorescent microscope showed that the complex is largely concentrated in the cytoplasm. The possible pathways for the cell apoptosis were studied by western blot analysis and the activation of PARP via caspase 7 was observed. Copyright © 2014 Elsevier Inc. All rights reserved.

Linking clinician interaction and coordination to clinical performance in Patient-Aligned Care Teams.

PubMed

Hysong, Sylvia J; Thomas, Candice L; Spitzmüller, Christiane; Amspoker, Amber B; Woodard, LeChauncy; Modi, Varsha; Naik, Aanand D

2016-01-15

Team coordination within clinical care settings is a critical component of effective patient care. Less is known about the extent, effectiveness, and impact of coordination activities among professionals within VA Patient-Aligned Care Teams (PACTs). This study will address these gaps by describing the specific, fundamental tasks and practices involved in PACT coordination, their impact on performance measures, and the role of coordination task complexity. First, we will use a web-based survey of coordination practices among 1600 PACTs in the national VHA. Survey findings will characterize PACT coordination practices and assess their association with clinical performance measures. Functional job analysis, using 6-8 subject matter experts who are 3rd and 4th year residents in VA Primary Care rotations, will be utilized to identify the tasks involved in completing clinical performance measures to standard. From this, expert ratings of coordination complexity will be used to determine the level of coordinative complexity required for each of the clinical performance measures drawn from the VA External Peer Review Program (EPRP). For objective 3, data collected from the first two methods will evaluate the effect of clinical complexity on the relationships between measures of PACT coordination and their ratings on the clinical performance measures. Results from this study will support successful implementation of coordinated team-based work in clinical settings by providing knowledge regarding which aspects of care require the most complex levels of coordination and how specific coordination practices impact clinical performance.

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

PubMed

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2017-04-01

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

BAD-LAMP controls TLR9 trafficking and signalling in human plasmacytoid dendritic cells.

PubMed

Combes, Alexis; Camosseto, Voahirana; N'Guessan, Prudence; Argüello, Rafael J; Mussard, Julie; Caux, Christophe; Bendriss-Vermare, Nathalie; Pierre, Philippe; Gatti, Evelina

2017-10-13

Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1 + late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3 +/ LAMP2 +/ LAMP1 - endolysosome compartment exists in pDCs from which TLR9 activation triggers type I interferon expression. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signalling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours. Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues.TLR9 is highly expressed by plasmacytoid dendritic cells and detects nucleic acids, but to discriminate between host and microbial nucleic acids TLR9 is sorted into different endosomal compartments. Here the authors show that BAD-LAMP limits type 1 interferon responses by sorting TLR9 to late endosomal compartments.

Rab5 and Rab4 Regulate Axon Elongation in the Xenopus Visual System

PubMed Central

Konopacki, Filip A.; Zivraj, Krishna H.; Holt, Christine E.

2014-01-01

The elongation rate of axons is tightly regulated during development. Recycling of the plasma membrane is known to regulate axon extension; however, the specific molecules involved in recycling within the growth cone have not been fully characterized. Here, we investigated whether the small GTPases Rab4 and Rab5 involved in short-loop recycling regulate the extension of Xenopus retinal axons. We report that, in growth cones, Rab5 and Rab4 proteins localize to endosomes, which accumulate markers that are constitutively recycled. Fluorescence recovery after photo-bleaching experiments showed that Rab5 and Rab4 are recruited to endosomes in the growth cone, suggesting that they control recycling locally. Dynamic image analysis revealed that Rab4-positive carriers can bud off from Rab5 endosomes and move to the periphery of the growth cone, suggesting that both Rab5 and Rab4 contribute to recycling within the growth cone. Inhibition of Rab4 function with dominant-negative Rab4 or Rab4 morpholino and constitutive activation of Rab5 decreases the elongation of retinal axons in vitro and in vivo, but, unexpectedly, does not disrupt axon pathfinding. Thus, Rab5- and Rab4-mediated control of endosome trafficking appears to be crucial for axon growth. Collectively, our results suggest that recycling from Rab5-positive endosomes via Rab4 occurs within the growth cone and thereby supports axon elongation. PMID:24403139

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

PubMed Central

Wang, Shiyu; Allen, Nickolas; Vickers, Timothy A; Revenko, Alexey S; Sun, Hong; Liang, Xue-hai; Crooke, Stanley T

2018-01-01

Abstract Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs. PMID:29514240

Processing of Pseudomonas aeruginosa Exotoxin A Is Dispensable for Cell Intoxication▿

PubMed Central

Morlon-Guyot, Juliette; Méré, Jocelyn; Bonhoure, Anne; Beaumelle, Bruno

2009-01-01

Exotoxin A is a major virulence factor of Pseudomonas aeruginosa. This toxin binds to a specific receptor on animal cells, allowing endocytosis of the toxin. Once in endosomes, the exotoxin can be processed by furin to generate a C-terminal toxin fragment that lacks the receptor binding domain and is retrogradely transported to the endoplasmic reticulum for retrotranslocation to the cytosol through the Sec61 channel. The toxin then blocks protein synthesis by ADP ribosylation of elongation factor 2, thereby triggering cell death. A shorter intracellular route has also been described for this toxin. It involves direct translocation of the entire toxin from endosomes to the cytosol and therefore does not rely on furin-mediated cleavage. To examine the implications of endosomal translocation in the intoxication process, we investigated whether the toxin required furin-mediated processing in order to kill cells. We used three different approaches. We first fused to the N terminus of the toxin proteins with different unfolding abilities so that they inhibited or did not inhibit endosomal translocation of the chimera. We then assayed the amount of toxin fragments delivered to the cytosol during cell intoxication. Finally we used furin inhibitors and examined the fate and intracellular localization of the toxin and its receptor. The results showed that exotoxin cytotoxicity results largely from endosomal translocation of the entire toxin. We found that the C-terminal fragment was unstable in the cytosol. PMID:19380469

mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate.

PubMed

Marat, Andrea L; Wallroth, Alexander; Lo, Wen-Ting; Müller, Rainer; Norata, Giuseppe Danilo; Falasca, Marco; Schultz, Carsten; Haucke, Volker

2017-06-02

Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ], synthesized by class II PI3K β (PI3KC2β) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2β hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2β with the Raptor subunit of mTORC1. Local PI(3,4)P 2 synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P 2 production in shutting off mTORC1. Copyright © 2017, American Association for the Advancement of Science.

Neurobeachin and the Kinesin KIF21B Are Critical for Endocytic Recycling of NMDA Receptors and Regulate Social Behavior.

PubMed

Gromova, Kira V; Muhia, Mary; Rothammer, Nicola; Gee, Christine E; Thies, Edda; Schaefer, Irina; Kress, Sabrina; Kilimann, Manfred W; Shevchuk, Olga; Oertner, Thomas G; Kneussel, Matthias

2018-05-29

Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

From mucolipidosis type IV to Ebola: TRPML and two-pore channels at the crossroads of endo-lysosomal trafficking and disease.

PubMed

Grimm, Christian; Butz, Elisabeth; Chen, Cheng-Chang; Wahl-Schott, Christian; Biel, Martin

2017-11-01

What do lysosomal storage disorders such as mucolipidosis type IV have in common with Ebola, cancer cell migration, or LDL-cholesterol trafficking? LDL-cholesterol, certain bacterial toxins and viruses, growth factors, receptors, integrins, macromolecules destined for degradation or secretion are all sorted and transported via the endolysosomal system (ES). There are several pathways known in the ES, e.g. the degradation, the recycling, or the retrograde trafficking pathway. The ES comprises early and late endosomes, lysosomes and recycling endosomes as well as autophagosomes and lysosome related organelles. Contact sites between the ES and the endoplasmic reticulum or the Golgi apparatus may also be considered part of it. Dysfunction of this complex intracellular machinery can cause or contribute to the development of a number of diseases ranging from neurodegenerative, infectious, or metabolic diseases to retinal and pigmentation disorders as well as cancer and autophagy-related diseases. Endolysosomal ion channels such as mucolipins (TRPMLs) and two-pore channels (TPCs) play an important role in intracellular cation/calcium signaling and homeostasis and appear to critically contribute to the proper function of the endolysosomal trafficking network. Copyright © 2017 Elsevier Ltd. All rights reserved.

CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

PubMed Central

Zheng, Ze-Yi; Cheng, Chiang-Min; Fu, Xin-Rong; Chen, Liuh-Yow; Xu, Lizhong; Terrillon, Sonia; Wong, Stephen T.; Bar-Sagi, Dafna; Songyang, Zhou; Chang, Eric C.

2011-01-01

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling. PMID:22231449

Endosomal protein traffic meets nuclear signal transduction head on.

PubMed

Horazdovsky, Bruce

2004-02-01

Rab5 plays a key role in controlling protein traffic through the early stages of the endocytic pathway. Previous studies on the modulators and effectors of Rab5 protein function have tied the regulation of several signal transduction pathways to the movement of protein through endocytic compartments. In the February 6, 2004, issue of Cell, Miaczynska et al. describe a surprising new link between Rab5 function and the nucleus by uncovering two new Rab5 effectors as potential regulators of the nucleosome remodeling and histone deacetylase protein complex NuRD/MeCP1.

Structural and functional organization of the ESCRT-I trafficking complex

PubMed Central

Kostelansky, Michael S.; Sun, Ji; Lee, Sangho; Kim, Jaewon; Ghirlando, Rodolfo; Hierro, Aitor; Emr, Scott D.; Hurley, James H.

2006-01-01

Summary The Endosomal Sorting Complex Required for Transport (ESCRT) complexes are central to receptor downregulation, lysosome biogenesis, and budding of HIV. The yeast ESCRT-I complex contains the Vps23, Vps28, and Vps37 proteins and its assembly is directed by the C-terminal steadiness box of Vps23, the N-terminal half of Vps28, and the C-terminal half of Vps37. The crystal structures of a Vps23:Vps28 core subcomplex and the Vps23:Vps28:Vps37 core were solved at 2.1 and 2.8 Å resolution. Each subunit contains a structurally similar pair of helices that form the core. The N-terminal domain of Vps28 has a hydrophobic binding site on its surface that is conformationally dynamic. The C-terminal domain of Vps28 binds the ESCRT-II complex. The structure shows how ESCRT-I is assembled by a compact core from which the Vps23 UEVdomain, the Vps28 C-domain, and other domains project to bind their partners. PMID:16615894

Armadillo Repeat Containing 8α Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

PubMed Central

Tomaru, Koji; Ueda, Atsuhisa; Suzuki, Takeyuki; Kobayashi, Nobuaki; Yang, Jun; Yamamoto, Masaki; Takeno, Mitsuhiro; Kaneko, Takeshi; Ishigatsubo, Yoshiaki

2010-01-01

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α. PMID:20224683

Electronic structure and reactivity of three-coordinate iron complexes.

PubMed

Holland, Patrick L

2008-08-01

[Reaction: see text]. The identity and oxidation state of the metal in a coordination compound are typically thought to be the most important determinants of its reactivity. However, the coordination number (the number of bonds to the metal) can be equally influential. This Account describes iron complexes with a coordination number of only three, which differ greatly from iron complexes with octahedral (six-coordinate) geometries with respect to their magnetism, electronic structure, preference for ligands, and reactivity. Three-coordinate complexes with a trigonal-planar geometry are accessible using bulky, anionic, bidentate ligands (beta-diketiminates) that steer a monodentate ligand into the plane of their two nitrogen donors. This strategy has led to a variety of three-coordinate iron complexes in which iron is in the +1, +2, and +3 oxidation states. Systematic studies on the electronic structures of these complexes have been useful in interpreting their properties. The iron ions are generally high spin, with singly occupied orbitals available for pi interactions with ligands. Trends in sigma-bonding show that iron(II) complexes favor electronegative ligands (O, N donors) over electropositive ligands (hydride). The combination of electrostatic sigma-bonding and the availability of pi-interactions stabilizes iron(II) fluoride and oxo complexes. The same factors destabilize iron(II) hydride complexes, which are reactive enough to add the hydrogen atom to unsaturated organic molecules and to take part in radical reactions. Iron(I) complexes use strong pi-backbonding to transfer charge from iron into coordinated alkynes and N 2, whereas iron(III) accepts charge from a pi-donating imido ligand. Though the imidoiron(III) complex is stabilized by pi-bonding in the trigonal-planar geometry, addition of pyridine as a fourth donor weakens the pi-bonding, which enables abstraction of H atoms from hydrocarbons. The unusual bonding and reactivity patterns of three-coordinate iron compounds may lead to new catalysts for oxidation and reduction reactions and may be used by nature in transient intermediates of nitrogenase enzymes.

Protein Complexation and pH Dependent Release Using Boronic Acid Containing PEG-Polypeptide Copolymers.

PubMed

Negri, Graciela E; Deming, Timothy J

2017-01-01

New poly(L-lysine)-b-poly(ethylene glycol) copolypeptides have been prepared, where the side-chain amine groups of lysine residues are modified to contain ortho-amine substituted phenylboronic acid, i.e., Wulff-type phenylboronic acid (WBA), groups to improve their pH responsive, carbohydrate binding properties. These block copolymers form nanoscale complexes with glycosylated proteins that are stable at physiological pH, yet dissociate and release the glycoproteins under acidic conditions, similar to those found in endosomal and lysosomal compartments within cells. These results suggest that WBA modified polypeptide copolymers are promising for further development as degradable carriers for intracellular protein delivery. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphatidic acid induces EHD3-containing membrane tubulation and is required for receptor recycling.

PubMed

Henmi, Yuji; Oe, Natsuko; Kono, Nozomu; Taguchi, Tomohiko; Takei, Kohji; Tanabe, Kenji

2016-03-01

EHD3 is localized on the tubular structures of early endosomes, and it regulates their trafficking pathway. However, the regulatory mechanism of EHD3-containing tubular structures remains poorly understood. An in vitro liposome co-sedimentation assay revealed that EHD3 interacted with phosphatidic acid through its helical domain and this interaction induced liposomal tubulations. Additionally, inhibiting phosphatidic acid synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor significantly reduced the number of EHD3-containing tubules and impaired their trafficking from early endosomes. These results suggest that EHD3 and phosphatidic acid cooperatively regulate membrane deformation and trafficking from early endosomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Promyelocytic leukemia bodies tether to early endosomes during mitosis.

PubMed

Palibrk, Vuk; Lång, Emma; Lång, Anna; Schink, Kay Oliver; Rowe, Alexander D; Bøe, Stig Ove

2014-01-01

During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

True and masked three-coordinate T-shaped platinum(II) intermediates.

PubMed

Ortuño, Manuel A; Conejero, Salvador; Lledós, Agustí

2013-01-01

Although four-coordinate square-planar geometries, with a formally 16-electron counting, are absolutely dominant in isolated Pt(II) complexes, three-coordinate, 14-electron Pt(II) complexes are believed to be key intermediates in a number of platinum-mediated organometallic transformations. Although very few authenticated three-coordinate Pt(II) complexes have been characterized, a much larger number of complexes can be described as operationally three-coordinate in a kinetic sense. In these compounds, which we have called masked T-shaped complexes, the fourth position is occupied by a very weak ligand (agostic bond, solvent molecule or counteranion), which can be easily displaced. This review summarizes the structural features of the true and masked T-shaped Pt(II) complexes reported so far and describes synthetic strategies employed for their formation. Moreover, recent experimental and theoretical reports are analyzed, which suggest the involvement of such intermediates in reaction mechanisms, particularly C-H bond-activation processes.

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies

PubMed Central

Fuchs, Hendrik; Niesler, Nicole; Trautner, Alexandra; Sama, Simko; Jerz, Gerold; Panjideh, Hossein; Weng, Alexander

2017-01-01

Protein-based targeted toxins play an increasingly important role in targeted tumor therapies. In spite of their high intrinsic toxicity, their efficacy in animal models is low. A major reason for this is the limited entry of the toxin into the cytosol of the target cell, which is required to mediate the fatal effect. Target receptor bound and internalized toxins are mostly either recycled back to the cell surface or lysosomally degraded. This might explain why no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date although more than 500 targeted toxins have been developed within the last decades. To overcome the problem of insufficient endosomal escape, a number of strategies that make use of diverse chemicals, cell-penetrating or fusogenic peptides, and light-induced techniques were designed to weaken the membrane integrity of endosomes. This review focuses on glycosylated triterpenoids as endosomal escape enhancers and throws light on their structure, the mechanism of action, and on their efficacy in cell culture and animal models. Obstacles, challenges, opportunities, and future prospects are discussed. PMID:28536357

The functional interplay of Rab11, FIP3 and Rho proteins on the endosomal recycling pathway controls cell shape and symmetry.

PubMed

Bouchet, Jérôme; McCaffrey, Mary W; Graziani, Andrea; Alcover, Andrés

2018-07-04

Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.

Cellular trafficking of low molecular weight heparin incorporated in layered double hydroxide nanoparticles in rat vascular smooth muscle cells.

PubMed

Gu, Zi; Rolfe, Barbara E; Thomas, Anita C; Campbell, Julie H; Lu, G Q Max; Xu, Zhi P

2011-10-01

This paper reports a clear elucidation of the pathway for the cellular delivery of layered double hydroxide (LDH) nanoparticles intercalated with anti-restenotic low molecular weight heparin (LMWH). Cellular uptake of LMWH-LDH conjugates into cultured rat vascular smooth muscle cells (SMCs) measured via flow cytometry was more than ten times greater than that of LMWH alone. Confocal and transmission electron microscopy showed LMWH-LDH conjugates taken up by endosomes, then released into the cytoplasm. We propose that LMWH-LDH is taken up via a unique 'modified endocytic' pathway, whereby the conjugate is internalized by SMCs in early endosomes, sorted in late endosomes, and quickly released from late endosomes/lysosomes, avoiding degradation. Treatment of cells with LMWH-LDH conjugates suppressed the activation of ERK1/2 in response to foetal calf serum (FCS) for up to 24h, unlike unconjugated LMWH which had no significant effect at 24h. Improved understanding of the intracellular pathway of LMWH-LDH nanohybrids in SMC will allow for refinement of design for LDH nanomedicine applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking

PubMed Central

Iles, Lakesla R.; Bartholomeusz, Geoffrey

2017-01-01

Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor–guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors. PMID:28883040

Uncommon endocytic and trafficking pathway of the natural killer cell CD94/NKG2A inhibitory receptor.

PubMed

Masilamani, Madhan; Narayanan, Sriram; Prieto, Martha; Borrego, Francisco; Coligan, John E

2008-06-01

The CD94/NKG2A inhibitory receptor, expressed by natural killer and T cells, is constantly exposed to its HLA-E ligand expressed by surrounding cells. Ligand exposure often induces receptor downregulation. For CD94/NKG2A, this could potentiate activation receptor(s) induced responses to normal bystander cells. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent macropinocytic-like process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1(+), Rab5(+) early endosomes. It does appear in Rab4(+) early/sorting endosome, but, in the time period examined, fails to reach Rab11(+) recycling or Rab7(+) late endosomes or lysosome-associated membrane protein-1(+) lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression.

Improving the in vivo therapeutic index of siRNA polymer conjugates through increasing pH responsiveness.

PubMed

Guidry, Erin N; Farand, Julie; Soheili, Arash; Parish, Craig A; Kevin, Nancy J; Pipik, Brenda; Calati, Kathleen B; Ikemoto, Nori; Waldman, Jacob H; Latham, Andrew H; Howell, Bonnie J; Leone, Anthony; Garbaccio, Robert M; Barrett, Stephanie E; Parmar, Rubina Giare; Truong, Quang T; Mao, Bing; Davies, Ian W; Colletti, Steven L; Sepp-Lorenzino, Laura

2014-02-19

Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.

Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking.

PubMed

Selyunin, Andrey S; Iles, Lakesla R; Bartholomeusz, Geoffrey; Mukhopadhyay, Somshuvra

2017-10-02

Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors. © 2017 Selyunin et al.

The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome

PubMed Central

Xie, Shuwei; Bahl, Kriti; Reinecke, James B.; Hammond, Gerald R. V.; Naslavsky, Naava; Caplan, Steve

2016-01-01

The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some but not all ERC vesicles are tethered by contiguous “membrane bridges.” Furthermore, tubular recycling endosomes preferentially traffic CIE cargo and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC. PMID:26510502

Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases

PubMed Central

2017-01-01

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface. PMID:28768685

Synaptic activity regulates AMPA receptor trafficking through different recycling pathways

PubMed Central

Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

2015-01-01

Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. DOI: http://dx.doi.org/10.7554/eLife.06878.001 PMID:25970033

Development of a Kinetic Assay for Late Endosome Movement.

PubMed

Esner, Milan; Meyenhofer, Felix; Kuhn, Michael; Thomas, Melissa; Kalaidzidis, Yannis; Bickle, Marc

2014-08-01

Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering. © 2014 Society for Laboratory Automation and Screening.

A bis(amido) ligand set that supports two-coordinate chromium in the +1, +2, and +3 oxidation states†

PubMed Central

Cai, Irene C.; Lipschutz, Michael I.

2014-01-01

The amido ligand –N(SiiPr3)DIPP (DIPP = 2,6-diisopropylphenyl) has been used to prepare two-coordinate complexes of CrI, CrII, and CrIII. The two-coordinate CrII complex has also been used to prepare a three-coordinate CrIII iodide complex, which can be used to access a stable CrIII methyl species. PMID:25222516

ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments

PubMed Central

Conzemius, Rick; Ganjian, Haleh; Blaas, Dieter

2016-01-01

ABSTRACT Human rhinovirus A89 (HRV-A89) and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here, we show by immunofluorescence microscopy that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major-group HRVs productively uncoat in distinct endosomal compartments. Overexpression of constitutively active (Rab11-GTP) and dominant negative (Rab11-GDP) mutants revealed that uncoating of HRV-A89 depends on functional Rab11. Thus, two ICAM-1 binding HRVs are routed into distinct endosomal compartments for productive uncoating. IMPORTANCE Based on similarity of their RNA genomic sequences, the more than 150 currently known common cold virus serotypes were classified as species A, B, and C. The majority of HRV-A viruses and all HRV-B viruses use ICAM-1 for cell attachment and entry. Our results highlight important differences of two ICAM-1 binding HRVs with respect to their intracellular trafficking and productive uncoating; they demonstrate that serotypes belonging to species A and B, but entering the cell via the same receptors, direct the endocytosis machinery to ferry them along distinct pathways toward different endocytic compartments for uncoating. PMID:27334586

Calcium-dependent antigen binding as a novel modality for antibody recycling by endosomal antigen dissociation

PubMed Central

Hironiwa, N; Ishii, S; Kadono, S; Iwayanagi, Y; Mimoto, F; Habu, K; Igawa, T; Hattori, K

2016-01-01

The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody. PMID:26496237

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

PubMed

Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

2016-12-01

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

Venezuelan equine encephalitis virus entry mechanism requires late endosome formation and resists cell membrane cholesterol depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kolokoltsov, Andrey A.; Fleming, Elisa H.; Davey, Robert A.

2006-04-10

Virus envelope proteins determine receptor utilization and host range. The choice of receptor not only permits specific targeting of cells that express it, but also directs the virus into specific endosomal trafficking pathways. Disrupting trafficking can result in loss of virus infectivity due to redirection of virions to non-productive pathways. Identification of the pathway or pathways used by a virus is, thus, important in understanding virus pathogenesis mechanisms and for developing new treatment strategies. Most of our understanding of alphavirus entry has focused on the Old World alphaviruses, such as Sindbis and Semliki Forest virus. In comparison, very little ismore » known about the entry route taken by more pathogenic New World alphaviruses. Here, we use a novel contents mixing assay to identify the cellular requirements for entry of a New World alphavirus, Venezuelan equine encephalitis virus (VEEV). Expression of dominant negative forms of key endosomal trafficking genes shows that VEEV must access clathrin-dependent endocytic vesicles for membrane fusion to occur. Unexpectedly, the exit point is different from Old World alphaviruses that leave from early endosomes. Instead, VEEV also requires functional late endosomes. Furthermore, unlike the Old World viruses, VEEV entry is insensitive to cholesterol sequestration from cell membranes and may reflect a need to access an endocytic compartment that lacks cholesterol. This indicates fundamental differences in the entry route taken by VEEV compared to Old World alphaviruses.« less

Increased expression of endosomal members of toll-like receptor family abrogates wound healing in patients with type 2 diabetes mellitus.

PubMed

Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Mohan, Gyanendra; Chaturvedi, Sunanda; Singh, Kiran

2016-10-01

The inflammatory phase of wound healing cascade is an important determinant of the fate of the wound. Acute inflammation is necessary to initiate proper wound healing, while chronic inflammation abrogates wound healing. Different endosomal members of toll-like receptor (TLR) family initiate inflammatory signalling via a range of different inflammatory mediators such as interferons, internal tissue damaged-associated molecular patterns (DAMPs) and hyperactive effector T cells. Sustained signalling of TLR9 and TLR7 contributes to chronic inflammation by activating the plasmacytoid dendritic cells. Diabetic wounds are also characterised by sustained inflammatory phase. The objective of this study was to analyse the differential expression of endosomal TLRs in human diabetic wounds compared with control wounds. We analysed the differential expression of TLR7 and TLR9 both at transcriptional and translational levels in wounds of 84 patients with type 2 diabetes mellitus (T2DM) and 6 control subjects without diabetes using quantitative real-time polymerase chain reaction (RT-PCR), western blot and immunohistochemistry. TLR7 and TLR9 were significantly up-regulated in wounds of the patients with T2DM compared with the controls and were dependent on the infection status of the diabetic wounds, and wounds with microbial infection exhibited lower expression levels of endosomal TLRs. Altered endosomal TLR expression in T2DM subjects might be associated with wound healing impairment. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Nipah virus fusion protein: Importance of the cytoplasmic tail for endosomal trafficking and bioactivity.

PubMed

Weis, Michael; Maisner, Andrea

2015-01-01

Nipah virus (NiV) is a highly pathogenic paramyxovirus which encodes two surface glycoproteins: the receptor-binding protein G and the fusion protein F. As for all paramyxoviruses, proteolytic activation of the NiV-F protein is an indispensable prerequisite for viral infectivity. Interestingly, proteolytic activation of NiV-F differs principally from other paramyxoviruses with respect to protease usage (cathepsins instead of trypsin- or furin-like proteases), and the subcellular localization where cleavage takes place (endosomes instead of Golgi or plasma membrane). To allow efficient F protein activation needed for productive virus replication and cell-to-cell fusion, the NiV-F cytoplasmic tail contains a classical tyrosine-based endocytosis signal (Y525RSL) that we have shown earlier to be needed for F uptake and proteolytic activation. In this report, we furthermore revealed that an intact endocytosis signal alone is not sufficient for full bioactivity. The very C-terminus of the cytoplasmic tail is needed in addition. Deletions of more than four residues did not affect F uptake or endosomal cleavage but downregulated the surface expression, likely by delaying the intracellular trafficking through endosomal-recycling compartments. Given that the NiV-F cytoplasmic tail is needed for timely and correct intracellular trafficking, endosomal cleavage and fusion activity, the influence of tail truncations on NiV-mediated cell-to-cell fusion and on pseudotyping lentiviral vectors is discussed. Copyright © 2015 Elsevier GmbH. All rights reserved.

Effect of the side chain spacer structure on the pH-responsive properties of polycarboxylates.

PubMed

Harada, Atsushi; Teranishi, Ryoma; Yuba, Eiji; Kono, Kenji

The properties of stimuli-responsive polymers change significantly with changes to their environment, such as temperature and pH. This behavior can be utilized for the preparation of stimuli-responsive carriers for efficient cytosolic delivery of active drugs. Among the possible environmental conditions, pH is one of the most useful stimuli because the pH in an endosome is lower than under physiological conditions, depending on endosomal development. This pH difference is an important factor in the design of pH-responsive polymers, which can be used to enhance the transport of endocytosed drugs from the endosomal compartment to the cytoplasm. Such polymers can destabilize the endosomal bilayer under mildly acidic conditions and be nondisruptive at pH 7.4 not only for efficient endosomal escape but also for the suppression of nonspecific interaction with lipids existing under physiological conditions. In this study, we developed polycarboxylates with well-controlled pH-responsive properties bearing various spacer structures with different hydrophobicity. 3-methyl glutarylated polyallylamine and 2-carboxy-cyclohexanoylated polyallylamine were synthesized through the reaction between primary amine of PAA and acid anhydrides. Side chain spacers with higher hydrophobicity induced significant interactions with liposomal membranes at higher pH. pH-destabilizing liposomes could be modulated through the changing the composition of spacer structures with different hydrophobicity. Such formulations may represent an attractive strategy for the improvement of cytosolic delivery of active molecules.

Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

PubMed

Sun, Ying-Jie; Nishikawa, Kaori; Yuda, Hideki; Wang, Yu-Lai; Osaka, Hitoshi; Fukazawa, Nobuna; Naito, Akira; Kudo, Yoshihisa; Wada, Keiji; Aoki, Shunsuke

2006-09-01

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

Mechanism of Diphtheria Toxin Catalytic Domain Delivery to the Eukaryotic Cell Cytosol and the Cellular Factors that Directly Participate in the Process

PubMed Central

Murphy, John R.

2011-01-01

Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism. PMID:22069710

Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors

PubMed Central

Sumner, Jonathan C.; Pickering, Suzanne; Neil, Stuart J. D.

2015-01-01

HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu’s mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism. PMID:26317613

The V-ATPase a2-subunit as a putative endosomal pH-sensor.

PubMed

Marshansky, V

2007-11-01

V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14

PubMed Central

Roosterman, Dirk; Kempkes, Cordula; Cottrell, Graeme S.; Padilla, Benjamin E.; Bunnett, Nigel W.; Turck, Christoph W.; Steinhoff, Martin

2008-01-01

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with β-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized 125I-Tyr1-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A1. ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis. PMID:18276747

The coordination structure of the extracted copper(II) complex with a synergistic mixture containing dinonylnaphthalene sulfonic acid and n-hexyl 3-pyridinecarboxylate ester

NASA Astrophysics Data System (ADS)

Zhu, Shan; Hu, Huiping; Hu, Jiugang; Li, Jiyuan; Hu, Fang; Wang, Yongxi

2017-09-01

In continuation of our interest in the coordination structure of the nickel(II) complex with dinonylnaphthalene sulfonic acid (HDNNS) and 2-ethylhexyl 4-pyridinecarboxylate ester (4PC), it was observed that the coordination sphere was completed by the coordination of two N atoms of pyridine rings in ligands 4PC and four water molecules while no direct interaction between Ni(II) and deprotonated HDNNS was observed. To investigate whether the coordination structure of nickel(II) with the synergistic mixture containing HDNNS and 4PC predominates or not in the copper(II) complex with the synergistic mixtures containing HDNNS and pyridinecarboxylate esters, a copper(II) synergist complex with n-hexyl 3-pyridinecarboxylate ester (L) and naphthalene-2-sulfonic acid (HNS, the short chain analogue of HDNNS), was prepared and studied by X-ray single crystal diffraction, elemental analyses and thermo gravimetric analysis (TGA), respectively. It was shown that the composition of the copper(II) synergist complex was [Cu(H2O)2(L)2(NS)2] and formed a trans-form distorted octahedral coordination structure. Two oxygen atoms of the two coordinated water molecules and two N atoms of the pyridine rings in the ligands L defined the basal plane while two O atoms from two sulfonate anions of the deprotonated HNS ligands occupied the apical positions by direct coordination with Cu(II), which was distinguished from the coordination structure of the nickel(II) synergist complex as reported in our previous work. In the crystal lattice, neighboring molecules [Cu(H2O)2L2(NS)2] were linked through the intermolecular hydrogen bonds between the hydrogen atoms of the coordinated water molecules and the oxygen atoms of the sulfonate anions in the copper(II) synergist complex to form a 2D plane. In order to bridge the gap between the solid state structure of the copper(II) synergist complex and the solution structure of the extracted copper(II) complex with the actual synergistic mixture containing L and HDNNS in the non-polar organic phase, the structures of the two copper(II) complexes were further investigated by Fourier transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectrometry (ESI-MS), and the results indicated that the extracted copper(II) complex in the non-polar organic phase might possess a similar coordination structure as the copper(II) synergist complex.

The GARP complex is required for cellular sphingolipid homeostasis.

PubMed

Fröhlich, Florian; Petit, Constance; Kory, Nora; Christiano, Romain; Hannibal-Bach, Hans-Kristian; Graham, Morven; Liu, Xinran; Ejsing, Christer S; Farese, Robert V; Walther, Tobias C

2015-09-10

Sphingolipids are abundant membrane components and important signaling molecules in eukaryotic cells. Their levels and localization are tightly regulated. However, the mechanisms underlying this regulation remain largely unknown. In this study, we identify the Golgi-associated retrograde protein (GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation analogous to a VPS53 allele causing progressive cerebello-cerebral atrophy type 2 (PCCA2) in humans exhibits similar, albeit weaker, phenotypes in yeast, providing mechanistic insights into disease pathogenesis. Inhibition of the first step of de novo sphingolipid synthesis is sufficient to mitigate many of the phenotypes of GARP-deficient yeast or mammalian cells. Together, these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2.

Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes

PubMed Central

Chuang, Jen-Zen; Vega, Carrie; Jun, Wenjin; Sung, Ching-Hwa

2004-01-01

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations. PMID:15232620

Tropomyosin Tm5NM1 Spatially Restricts Src Kinase Activity through Perturbation of Rab11 Vesicle Trafficking

PubMed Central

Bach, Cuc T.; Murray, Rachael Z.; Owen, Dylan; Gaus, Kat

2014-01-01

In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules. PMID:25288639

Reactivity of the Donor-Stabilized Silylenes [iPrNC(Ph)NiPr]2 Si and [iPrNC(NiPr2 )NiPr]2 Si: Activation of CO2 and CS2.

PubMed

Mück, Felix M; Baus, Johannes A; Nutz, Marco; Burschka, Christian; Poater, Jordi; Bickelhaupt, F Matthias; Tacke, Reinhold

2015-11-09

Activation of CO2 by the bis(amidinato)silylene 1 and the analogous bis(guanidinato)silylene 2 leads to the structurally analogous six-coordinate silicon(IV) complexes 4 (previous work) and 8, respectively, the first silicon compounds with a chelating carbonato ligand. Likewise, CS2 activation by silylene 1 affords the analogous six-coordinate silicon(IV) complex 10, the first silicon compound with a chelating trithiocarbonato ligand. CS2 activation by silylene 2, however, yields the five-coordinate silicon(IV) complex 13 with a carbon-bound CS2 (2-) ligand, which also represents an unprecedented coordination mode in silicon coordination chemistry. Treatment of the dinuclear silicon(IV) complexes 5 and 6 with CO2 also affords the six-coordinate carbonatosilicon(IV) complexes 4 and 8, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Four coordination polymers based on 5-tert-butyl isophthalic acid and rigid bis(imidazol-1yl)benzene linkers: Synthesis, luminescence detection of acetone and optical properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arıcı, Mürsel, E-mail: marici@ogu.edu.tr; Zafer Yeşilel, Okan; Büyükgüngör, Orhan

Four coordination polymers including, [Co(µ-Htbip){sub 2}(µ-dib)]{sub n} (1), [Co(µ-tbip)(µ-dmib){sub 0.5}]{sub n} (2), [Zn{sub 2}(µ-tbip)(µ{sub 3}-tbip)(µ-dmib){sub 1.5}]{sub n} (3) and [Cd(µ{sub 3}-tbip)(µ-dib){sub 0.5} (H{sub 2}O)]{sub n} (4) (tbip: 5-tert-butylisophthalate, dib: 1,4-bis(imidazol-1yl)benzene, dmib: 1,4-bis(imidazol-1yl)-2,5-dimethylbenzene), were hydrothermally synthesized and characterized by elemental analysis, IR spectra, single crystal and powder X-ray diffraction and thermal analysis (TG/DTA). The structural diversity is observed depending on ligands and coordination number of metal centers in the synthesized complexes. The tbip ligand displayed five different coordination modes in its complexes. In 1 and 2, complex 1 is 3D framework with the dia topology while complex 2 has 2D structuremore » with the sql topology depending on coordination geometries of Co ions. Complex 3 is 3D framework with the fsh 4,6-conn topology and complex 4 has 2D 4-connected sql topology. Photoluminescent properties of complex 3 dispersed in various organic solvents were investigated and the results showed that 3 dispersed in methanol could be used as a fluorescent sensor for the detection of acetone. Moreover, thermal and optical properties of the complexes were also studied. - Graphical abstract: Four coordination polymers were hydrothermally synthesized and characterized by various techniques. The complexes showed the structural diversity depending on ligands and coordination number of metal centers. The tbip ligand displayed four different coordination modes in its complexes. In 1 and 2, complexes 1 and 2 are 3D and 2D structures with the dia and sql topologies depending on coordination geometries of Co ions, respectively. Complexes 3 and 4 are 3D and 2D structures with the fsh 4,6-conn and sql topology, respectively. Photoluminescent properties of complex 3 dispersed in various organic solvents were investigated and the results showed that 3 dispersed in methanol could be used as a fluorescent sensor for the detection of acetone. Moreover, thermal and optical properties of the complexes were also studied. - Highlights: • Four new 2D and 3D coordination polymers with 5-tert-butyl isophthalic acid and rigid bis(imidazol-1yl)benzene linkers. • The structural diversity depending on ligands and coordination number of metal centers. • Fluorescent sensor for the detection of acetone.« less

Polymer complexes.. XXXX. Supramolecular assembly on coordination models of mixed-valence-ligand poly[1-acrylamido-2-(2-pyridyl)ethane] complexes

NASA Astrophysics Data System (ADS)

El-Sonbati, A. Z.; El-Bindary, A. A.; Diab, M. A.

2003-02-01

The build-up of polymer metallic supramolecules based on homopolymer (1-acrylamido-2-(2-pyridyl)ethane (AEPH)) and ruthenium, rhodium, palladium as well as platinum complexes has been pursued with great interest. The homopolymer shows three types of coordination behaviour. In the mixed valence paramagnetic trinuclear polymer complexes [( 11)+( 12)] in the paper and in mononuclear polymer complexes ( 1)-( 5) it acts as a neutral bidentate ligand coordinating through the N-pyridine and NH-imino atoms, while in the mixed ligand diamagnetic poly-chelates, which are obtained from the reaction of AEPH with PdX 2 and KPtCl 4 in the presence of N-heterocyclic base consisting of polymer complexes ( 9)+( 10), and in monouclear compounds ( 6)-( 8), it behaves as a monobasic bidentate ligand coordinating through the same donor atoms. In mononuclear compounds ( 13)+( 14) it acts as a monobasic and neutral bidentate ligand coordinating only through the same donor atoms. Monomeric distorted octahedral or trimeric chlorine-bridged, approximately octahedral structures are proposed for these polymer complexes. The poly-chelates are of 1:1, 1:2 and 3:2 (metal-homopolymer) stoichiometry and exhibit six coordination. The values of ligand field parameters were calculated. The homopolymer and their polymer complexes have been characterized physicochemically.

Polymer complexes. XXXX. Supramolecular assembly on coordination models of mixed-valence-ligand poly[1-acrylamido-2-(2-pyridyl)ethane] complexes.

PubMed

El-Sonbati, A Z; El-Bindary, A A; Diab, M A

2003-02-01

The build-up of polymer metallic supramolecules based on homopolymer (1-acrylamido-2-(2-pyridyl)ethane (AEPH)) and ruthenium, rhodium, palladium as well as platinum complexes has been pursued with great interest. The homopolymer shows three types of coordination behaviour. In the mixed valence paramagnetic trinuclear polymer complexes [(11)+(12)] in the paper and in mononuclear polymer complexes (1)-(5) it acts as a neutral bidentate ligand coordinating through the N-pyridine and NH-imino atoms, while in the mixed ligand diamagnetic poly-chelates, which are obtained from the reaction of AEPH with PdX2 and KPtCl4 in the presence of N-heterocyclic base consisting of polymer complexes (9)+(10), and in monouclear compounds (6)-(8), it behaves as a monobasic bidentate ligand coordinating through the same donor atoms. In mononuclear compounds (13)+(14) it acts as a monobasic and neutral bidentate ligand coordinating only through the same donor atoms. Monomeric distorted octahedral or trimeric chlorine-bridged, approximately octahedral structures are proposed for these polymer complexes. The poly-chelates are of 1:1, 1:2 and 3:2 (metal-homopolymer) stoichiometry and exhibit six coordination. The values of ligand field parameters were calculated. The homopolymer and their polymer complexes have been characterized physicochemically.

Electrostatically assembled dendrimer complex with a high-affinity protein binder for targeted gene delivery.

PubMed

Kim, Jong-Won; Lee, Joong-Jae; Choi, Joon Sig; Kim, Hak-Sung

2018-06-10

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas. Copyright © 2018 Elsevier B.V. All rights reserved.

Neurotrophin signaling endosomes; biogenesis, regulation, and functions

PubMed Central

Yamashita, Naoya; Kuruvilla, Rejji

2016-01-01

In the nervous system, communication between neurons and their post-synaptic target cells is critical for the formation, refinement and maintenance of functional neuronal connections. Diffusible signals secreted by target tissues, exemplified by the family of neurotrophins, impinge on nerve terminals to influence diverse developmental events including neuronal survival and axonal growth. Key mechanisms of action of target-derived neurotrophins include the cell biological processes of endocytosis and retrograde trafficking of their Trk receptors from growth cones to cell bodies. In this review, we summarize the molecular mechanisms underlying this endosome-mediated signaling, focusing on the instructive role of neurotrophin signaling itself in directing its own trafficking. Recent studies have linked impaired neurotrophin trafficking to neurodevelopmental disorders, highlighting the relevance of neurotrophin endosomes in human health. PMID:27327126

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein (GARP) Complex with the t-SNARE Syntaxin 6

PubMed Central

Abascal-Palacios, Guillermo; Schindler, Christina; Rojas, Adriana L; Bonifacino, Juan S.; Hierro, Aitor

2016-01-01

Summary The Golgi-Associated Retrograde Protein (GARP) is a tethering complex involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein we report the structural basis for the interaction of the human Ang2 subunit of GARP with Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a novel binding mode in which a di-tyrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction. The same binding determinants are found in other tethering proteins and syntaxins, suggesting a general interaction mechanism. PMID:23932592

The binding of Varp to VAMP7 traps VAMP7 in a closed, fusogenically inactive conformation

PubMed Central

Schäfer, Ingmar B.; Hesketh, Geoffrey G.; Bright, Nicholas A.; Gray, Sally R.; Pryor, Paul R.; Evans, Philip R; Luzio, J. Paul; Owen, David J.

2012-01-01

SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from Q-SNAREs embedded in one membrane and an R–SNARE embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp co-localises with and binds to VAMP7, an R-SNARE involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7 SNARE motif is trapped between Varp and the VAMP7 longin domain and hence Varp kinetically inhibits VAMP7’s ability to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as the VAMP7 such as Rab32:GTP. PMID:23104059

A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

PubMed Central

Eystathioy, Theophany; Chan, Edward K. L.; Tenenbaum, Scott A.; Keene, Jack D.; Griffith, Kevin; Fritzler, Marvin J.

2002-01-01

A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis. PMID:11950943

Assembly and Architecture of Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)*

PubMed Central

Lee, Hyung Ho; Nemecek, Daniel; Schindler, Christina; Smith, William J.; Ghirlando, Rodolfo; Steven, Alasdair C.; Bonifacino, Juan S.; Hurley, James H.

2012-01-01

BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting. PMID:22203680

Reversible five-coordinate ⇄ six-coordinate transformation in cobalt(II) complexes

NASA Astrophysics Data System (ADS)

Xiao, Linda; Bhadbhade, Mohan; Baker, Anthony T.

2018-04-01

The heterocyclic ligands 2,6-bis(pyrazol-1-yl)pyridine (L1) and 2,6-bis(benzimidazol-2-yl)pyridine (L2) and their cobalt(II) complexes were synthesized. The blue five-coordinate complex [Co(L1)Cl2] isolated initially from the reaction mixture rapidly absorbed water vapour from the atmosphere to yield the pink six-coordinate complex [Co(L1)(H2O)3]Cl2. This change is reversible upon desiccation or transferring [Co(L1)(H2O)3]Cl2 into acetonitrile. The five coordinate complex [Co(L2)Cl2], however, remains stable under similar conditions. The structures of the complexes [Co(L1)Cl2], [Co(L1)(H2O)3]Cl2 and [Co(L2)Cl2] have been determined by x-ray crystallography. The magnetic susceptibilities and the electronic spectra for [Co(L1)Cl2], [Co(L2)Cl2] and [Co(L1)(H2O)3]Cl2 are presented.

Transport According to GARP: Receiving Retrograde Cargo at the Trans-Golgi Network

PubMed Central

Bonifacino, Juan S.; Hierro, Aitor

2010-01-01

Tethering factors are large protein complexes that capture transport vesicles and enable their fusion with acceptor organelles at different stages of the endomembrane system. Recent studies have shed new light on the structure and function of a heterotetrameric tethering factor named Golgi-associated retrograde protein (GARP), which promotes fusion of endosome-derived, retrograde transport carriers to the trans-Golgi network (TGN). X-ray crystallography of the Vps53 and Vps54 subunits of GARP has revealed that this complex is structurally related to other tethering factors such as the exocyst, COG and Dsl1, indicating that they all might work by a similar mechanism. Loss of GARP function compromises the growth, fertility and/or viability of the defective organisms, underscoring the essential nature of GARP-mediated retrograde transport. PMID:21183348

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

PubMed

Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E

2017-05-01

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (k actual ) of 3.25 min -1 , threefold faster than α3 integrin (1.0 min -1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min -1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min -1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k actual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the k actual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells†

PubMed Central

Das, Lipsa; Anderson, Todd A.; Gard, Jaime M.C.; Sroka, Isis C.; Strautman, Stephanie R.; Nagle, Raymond B.; Morrissey, Colm; Knudsen, Beatrice S.; Cress, Anne E.

2017-01-01

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25min−1, 3-fold faster than α3 integrin (1.0 min−1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved PMID:27509031

Regulation of the collagenase-3 receptor and its role in intracellular ligand processing in rat osteoblastic cells

NASA Technical Reports Server (NTRS)

Walling, H. W.; Chan, P. T.; Omura, T. H.; Barmina, O. Y.; Fiacco, G. J.; Jeffrey, J. J.; Partridge, N. C.

1998-01-01

We have previously described a specific, saturable receptor for rat collagenase-3 in the rat osteosarcoma cell line, UMR 106-01. Binding of rat collagenase-3 to this receptor is coupled to the internalization and eventual degradation of the enzyme and correlates with observed extracellular levels of the enzyme. In this study we have shown that decreased binding, internalization, and degradation of 125I-rat collagenase-3 were observed in cells after 24 h of parathyroid hormone treatment; these activities returned to control values after 48 h and were increased substantially (twice control levels) after 96 h of treatment with the hormone. Subcellular fractionation studies to identify the route of uptake and degradation of collagenase-3 localized intracellular accumulation of 125I-rat collagenase-3 initially in Golgi-associated lysosomes and later in secondary lysosomes. Maximal lysosomal accumulation of the radiolabel and stimulation of general lysosomal activity occurred after 72 h of parathyroid hormone treatment. Preventing fusion of endosomes with lysosomes (by temperature shift, colchicine, or monensin) resulted in no internalized 125I-collagenase-3 in either lysosomal fraction. Treatment of UMR cells with the above agents or ammonium chloride decreased excretion of 125I-labeled degradation products of collagenase-3. These experiments demonstrated that degradation of collagenase-3 required receptor-mediated endocytosis and sequential processing by endosomes and lysosomes. Thus, parathyroid hormone regulates the expression and synthesis of collagenase-3 as well as the abundance and functioning of the collagenase-3 receptor and the intracellular degradation of its ligand. The coordinate changes in the secretion of collagenase-3 and expression of the receptor determine the net abundance of the enzyme in the extracellular space.

The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

PubMed Central

Martinez, Ruben; Schellenberger, Pascale; Vasishtan, Daven; Aknin, Cindy; Austin, Sisley; Dacheux, Denis; Rayne, Fabienne; Siebert, Alistair; Ruzsics, Zsolt; Gruenewald, Kay

2014-01-01

ABSTRACT Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle. PMID:25473051

Differential subcellular membrane recruitment of Src may specify its downstream signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah

2008-04-15

Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 andmore » flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ({approx} 70%) cholesterol extraction with methyl-{beta}-cyclodextrin (M{beta}CD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to M{beta}CD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.« less

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

PubMed Central

Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne; Patel, Sanjeevkumar R.; Shisheva, Assia

2016-01-01

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34–deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux. PMID:26825532

Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

PubMed

Nixon, Ralph A

2017-07-01

Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and β-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote β-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies β-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-β, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. © FASEB.

A sorting nexin 17-binding domain within the LRP1 cytoplasmic tail mediates receptor recycling through the basolateral sorting endosome.

PubMed

Farfán, Pamela; Lee, Jiyeon; Larios, Jorge; Sotelo, Pablo; Bu, Guojun; Marzolo, María-Paz

2013-07-01

Sorting nexin 17 (SNX17) is an adaptor protein present in early endosomal antigen 1 (EEA1)-positive sorting endosomes that promotes the efficient recycling of low-density lipoprotein receptor-related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1-positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17-binding domain, we generated chimeric proteins in which the SNX17-binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non-polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized Madin-Darby canine kidney cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17-binding receptors and the restricted function of SNX17 in the BSE. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.