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.

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

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

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

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).

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.

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.

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

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

PubMed

Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

2009-05-01

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

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

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

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

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

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

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.

Dictyostelium LvsB has a regulatory role in endosomal vesicle fusion

PubMed Central

Falkenstein, Kristin; De Lozanne, Arturo

2014-01-01

ABSTRACT Defects in human lysosomal-trafficking regulator (Lyst) are associated with the lysosomal disorder Chediak–Higashi syndrome. The absence of Lyst results in the formation of enlarged lysosome-related compartments, but the mechanism for how these compartments arise is not well established. Two opposing models have been proposed to explain Lyst function. The fission model describes Lyst as a positive regulator of fission from lysosomal compartments, whereas the fusion model identifies Lyst as a negative regulator of fusion between lysosomal vesicles. Here, we used assays that can distinguish between defects in vesicle fusion versus fission. We compared the phenotype of Dictyostelium discoideum cells defective in LvsB, the ortholog of Lyst, with that of two known fission defect mutants (μ3- and WASH-null mutants). We found that the temporal localization characteristics of the post-lysosomal marker vacuolin, as well as vesicular acidity and the fusion dynamics of LvsB-null cells are distinct from those of both μ3- and WASH-null fission defect mutants. These distinctions are predicted by the fusion defect model and implicate LvsB as a negative regulator of vesicle fusion. PMID:25086066

Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

PubMed Central

Wold, Mitchell S.; Gong, Shiaoching; Phillips, Greg R.; Dou, Zhixun; Zhao, Yanxiang; Heintz, Nathaniel; Zong, Wei-Xing; Yue, Zhenyu

2014-01-01

Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis. PMID:25275521

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

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.

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

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

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

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.

Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

PubMed Central

Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

2015-01-01

Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

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.

Insulin-regulated Aminopeptidase Is a Key Regulator of GLUT4 Trafficking by Controlling the Sorting of GLUT4 from Endosomes to Specialized Insulin-regulated Vesicles

PubMed Central

Jordens, Ingrid; Molle, Dorothee; Xiong, Wenyong; Keller, Susanna R.

2010-01-01

Insulin stimulates glucose uptake by regulating translocation of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane. In the absence of insulin GLUT4 is actively sequestered away from the general endosomes into GLUT4-specialized compartments, thereby controlling the amount of GLUT4 at the plasma membrane. Here, we investigated the role of the aminopeptidase IRAP in GLUT4 trafficking. In unstimulated IRAP knockdown adipocytes, plasma membrane GLUT4 levels are elevated because of increased exocytosis, demonstrating an essential role of IRAP in GLUT4 retention. Current evidence supports the model that AS160 RabGAP, which is required for basal GLUT4 retention, is recruited to GLUT4 compartments via an interaction with IRAP. However, here we show that AS160 recruitment to GLUT4 compartments and AS160 regulation of GLUT4 trafficking were unaffected by IRAP knockdown. These results demonstrate that AS160 is recruited to membranes by an IRAP-independent mechanism. Consistent with a role independent of AS160, we showed that IRAP functions in GLUT4 sorting from endosomes to GLUT4-specialized compartments. This is revealed by the relocalization of GLUT4 to endosomes in IRAP knockdown cells. Although IRAP knockdown has profound effects on GLUT4 traffic, GLUT4 knockdown does not affect IRAP trafficking, demonstrating that IRAP traffics independent of GLUT4. In sum, we show that IRAP is both cargo and a key regulator of the insulin-regulated pathway. PMID:20410133

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

PubMed

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-07-22

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

PubMed Central

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-01-01

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

Heterotypic endosomal fusion as an initial trigger for insulin-induced glucose transporter 4 (GLUT4) translocation in skeletal muscle.

PubMed

Hatakeyama, Hiroyasu; Kanzaki, Makoto

2017-08-15

Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot-based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving 'static retention' and 'stimulus-dependent liberation'. Vital imaging analyses and super-resolution microscopy-based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4-containing vesicles in skeletal myofibres. Prior exposure to exercise-mimetic stimuli potentiated this insulin-responsive endomembrane fusion event involving GLUT4-containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise. Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin-induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4-containing vesicles with a subset of transferrin receptor-containing endosomes. Importantly, pretreatment with exercise-mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe-like localization close to sarcomeric α-actinin and that insulin induced a reduction of the stripe-like localization accompanying changes in its detergent solubility. The results of the present study thus provide a

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

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

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

Par3 and aPKC regulate BACE1 endosome-to-TGN trafficking through PACS1.

PubMed

Sun, Miao; Zhang, Huaye

2017-12-01

The cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) is the rate-limiting step in beta amyloid generation during Alzheimer's disease (AD) pathogenesis. In AD brains, BACE1 is abnormally accumulated in endocytic compartments, where the acidic pH is optimal for its activity. However, mechanisms regulating the endosome-to-trans-Golgi network (TGN) retrieval of BACE1 remain unclear. Here, we show that partitioning defective 3 (Par3) facilitates BACE1 retrograde trafficking from endosomes to the TGN. Par3 functions through aPKC-mediated phosphorylation of BACE1 on Ser498, which in turn promotes the interaction between BACE1 and phosphofurin acidic cluster sorting protein 1 and facilitates the retrograde trafficking of BACE1 to the TGN. In human AD brains, there is a significant decrease in Ser498 phosphorylation of BACE1 suggesting that defective phosphorylation-dependent retrograde transport of BACE1 is important in AD pathogenesis. Together, our studies provide mechanistic insight into a novel role for Par3 and aPKC in regulating the retrograde endosome-to-TGN trafficking of BACE1 and shed light on the mechanisms of AD pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Mahogunin regulates fusion between amphisomes/MVBs and lysosomes via ubiquitination of TSG101

PubMed Central

Majumder, P; Chakrabarti, O

2015-01-01

Aberrant metabolic forms of the prion protein (PrP), membrane-associated CtmPrP and cytosolic (cyPrP) interact with the cytosolic ubiquitin E3 ligase, Mahogunin Ring Finger-1 (MGRN1) and affect lysosomes. MGRN1 also interacts with and ubiquitinates TSG101, an ESCRT-I protein, involved in endocytosis. We report that MGRN1 modulates macroautophagy. In cultured cells, functional depletion of MGRN1 or overexpression of CtmPrP and cyPrP blocks autophagosome–lysosome fusion, alleviates the autophagic flux and its degradative competence. Concurrently, the degradation of cargo from the endo-lysosomal pathway is also affected. This is significant because catalytic inactivation of MGRN1 alleviates fusion of lysosomes with either autophagosomes (via amphisomes) or late endosomes (either direct or mediated through amphisomes), without drastically perturbing maturation of late endosomes, generation of amphisomes or lysosomal proteolytic activity. The compromised lysosomal fusion events are rescued by overexpression of TSG101 and/or its monoubiquitination in the presence of MGRN1. Thus, for the first time we elucidate that MGRN1 simultaneously modulates both autophagy and heterophagy via ubiquitin-mediated post-translational modification of TSG101. PMID:26539917

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.

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.

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

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

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

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

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.

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

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

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.

PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site

PubMed Central

Bothe, Ingo; Deng, Su; Baylies, Mary

2014-01-01

Cell-cell fusion is a regulated process that requires merging of the opposing membranes and underlying cytoskeletons. However, the integration between membrane and cytoskeleton signaling during fusion is not known. Using Drosophila, we demonstrate that the membrane phosphoinositide PI(4,5)P2 is a crucial regulator of F-actin dynamics during myoblast fusion. PI(4,5)P2 is locally enriched and colocalizes spatially and temporally with the F-actin focus that defines the fusion site. PI(4,5)P2 enrichment depends on receptor engagement but is upstream or parallel to actin remodeling. Regulators of actin branching via Arp2/3 colocalize with PI(4,5)P2 in vivo and bind PI(4,5)P2 in vitro. Manipulation of PI(4,5)P2 availability leads to impaired fusion, with a reduction in the F-actin focus size and altered focus morphology. Mechanistically, the changes in the actin focus are due to a failure in the enrichment of actin regulators at the fusion site. Moreover, improper localization of these regulators hinders expansion of the fusion interface. Thus, PI(4,5)P2 enrichment at the fusion site encodes spatial and temporal information that regulates fusion progression through the localization of activators of actin polymerization. PMID:24821989

Insulin-regulated Glut4 Translocation

PubMed Central

Brewer, Paul Duffield; Habtemichael, Estifanos N.; Romenskaia, Irina; Mastick, Cynthia Corley; Coster, Adelle C. F.

2014-01-01

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min−1 versus 0.6 min−1). Differentiation decreases Glut4 ken 40% (ken = 0.12 min−1). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025–0.038 min−1), not through the fast Tf receptor pathway (kex = 0.2 min−1). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min−1). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs. PMID:24778187

Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1.

PubMed

Wang, Han; Shi, Yi; Song, Jian; Qi, Jianxun; Lu, Guangwen; Yan, Jinghua; Gao, George F

2016-01-14

Filoviruses, including Ebola and Marburg, cause fatal hemorrhagic fever in humans and primates. Understanding how these viruses enter host cells could help to develop effective therapeutics. An endosomal protein, Niemann-Pick C1 (NPC1), has been identified as a necessary entry receptor for this process, and priming of the viral glycoprotein (GP) to a fusion-competent state is a prerequisite for NPC1 binding. Here, we have determined the crystal structure of the primed GP (GPcl) of Ebola virus bound to domain C of NPC1 (NPC1-C) at a resolution of 2.3 Å. NPC1-C utilizes two protruding loops to engage a hydrophobic cavity on head of GPcl. Upon enzymatic cleavage and NPC1-C binding, conformational change in the GPcl further affects the state of the internal fusion loop, triggering membrane fusion. Our data therefore provide structural insights into filovirus entry in the late endosome and the molecular basis for design of therapeutic inhibitors of viral entry. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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

Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

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

Blanchette, C D; Woo, Y; Thomas, C

2008-12-22

Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) andmore » Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification process to

The tetraspanin CD63 regulates ESCRT-independent and dependent endosomal sorting during melanogenesis

PubMed Central

van Niel, Guillaume; Charrin, Stéphanie; Simoes, Sabrina; Romao, Maryse; Rochin, Leila; Saftig, Paul; Marks, Michael S.; Rubinstein, Eric; Raposo, Graça

2011-01-01

Summary Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for numerous physiological processes including lysosome-related organelle (LRO) biogenesis. PMEL – a component of melanocyte LROs (melanosomes) – is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis. PMID:21962903

Regulators of Lysosome Function and Dynamics in Caenorhabditis elegans

PubMed Central

Gee, Kevin; Zamora, Danniel; Horm, Teresa; George, Laeth; Upchurch, Cameron; Randall, Justin; Weaver, Colby; Sanford, Caitlin; Miller, Austin; Hernandez, Sebastian; Dang, Hope; Fares, Hanna

2017-01-01

Lysosomes, the major membrane-bound degradative organelles, have a multitude of functions in eukaryotic cells. Lysosomes are the terminal compartments in the endocytic pathway, though they display highly dynamic behaviors, fusing with each other and with late endosomes in the endocytic pathway, and with the plasma membrane during regulated exocytosis and for wound repair. After fusing with late endosomes, lysosomes are reformed from the resulting hybrid organelles through a process that involves budding of a nascent lysosome, extension of the nascent lysosome from the hybrid organelle, while remaining connected by a membrane bridge, and scission of the membrane bridge to release the newly formed lysosome. The newly formed lysosomes undergo cycles of homotypic fusion and fission reactions to form mature lysosomes. In this study, we used a forward genetic screen in Caenorhabditis elegans to identify six regulators of lysosome biology. We show that these proteins function in different steps of lysosome biology, regulating lysosome formation, lysosome fusion, and lysosome degradation. PMID:28122949

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.

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

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

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.

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.

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

Dysregulated Arl1, a regulator of post-Golgi vesicle tethering, can inhibit endosomal transport and cell proliferation in yeast

PubMed Central

Benjamin, Jeremy J. R.; Poon, Pak P.; Drysdale, John D.; Wang, Xiangmin; Singer, Richard A.; Johnston, Gerald C.

2011-01-01

Small monomeric G proteins regulated in part by GTPase-activating proteins (GAPs) are molecular switches for several aspects of vesicular transport. The yeast Gcs1 protein is a dual-specificity GAP for ADP-ribosylation factor (Arf) and Arf-like (Arl)1 G proteins, and also has GAP-independent activities. The absence of Gcs1 imposes cold sensitivity for growth and endosomal transport; here we present evidence that dysregulated Arl1 may cause these impairments. We show that gene deletions affecting the Arl1 or Ypt6 vesicle-tethering pathways prevent Arl1 activation and membrane localization, and restore growth and trafficking in the absence of Gcs1. A mutant version of Gcs1 deficient for both ArfGAP and Arl1GAP activity in vitro still allows growth and endosomal transport, suggesting that the function of Gcs1 that is required for these processes is independent of GAP activity. We propose that, in the absence of this GAP-independent regulation by Gcs1, the resulting dysregulated Arl1 prevents growth and impairs endosomal transport at low temperatures. In cells with dysregulated Arl1, an increased abundance of the Arl1 effector Imh1 restores growth and trafficking, and does so through Arl1 binding. Protein sequestration at the trans-Golgi membrane by dysregulated, active Arl1 may therefore be the mechanism of inhibition. PMID:21562219

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

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

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

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

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.

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

PubMed

Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min; Melikyan, Gregory B; Liu, Shan-Lu; Cohen, Fredric S

2016-01-01

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

PubMed Central

Zheng, Yi-Min; Melikyan, Gregory B.; Liu, Shan-Lu; Cohen, Fredric S.

2016-01-01

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

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.

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.

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

On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

PubMed Central

Carvalho, Carlos A.M.; Silva, Jerson L.; Oliveira, Andréa C.

2017-01-01

Mayaro virus (MAYV) is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell. PMID:28462045

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

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.

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.

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 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.

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.

GABARAP-mediated targeting of PI4K2A/PI4KIIα to autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion.

PubMed

Albanesi, Joseph; Wang, Hanzhi; Sun, Hui-Qiao; Levine, Beth; Yin, Helen

2015-11-02

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2 ). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.

Drosophila VAMP7 regulates Wingless intracellular trafficking.

PubMed

Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

2017-01-01

Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

Influenza Virus-Mediated Membrane Fusion: Determinants of Hemagglutinin Fusogenic Activity and Experimental Approaches for Assessing Virus Fusion

PubMed Central

Hamilton, Brian S.; Whittaker, Gary R.; Daniel, Susan

2012-01-01

Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future. PMID:22852045

The ubiquitin–proteasome system regulates membrane fusion of yeast vacuoles

PubMed Central

Kleijnen, Maurits F; Kirkpatrick, Donald S; Gygi, Steven P

2007-01-01

Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin–proteasome regulation may be involved in membrane fusion elsewhere. PMID:17183369

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

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

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

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.

Viral membrane fusion

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

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formedmore » draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.« less

The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation

PubMed Central

Deng, Su; Bothe, Ingo; Baylies, Mary K.

2015-01-01

The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease. PMID:26295716

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

Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

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

Hu, Dong, E-mail: austhudong@126.com; Wu, Jing, E-mail: wujing8008@126.com; Wang, Wan

The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domainmore » and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy.« less

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

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

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.

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.

The early endosome: a busy sorting station for proteins at the crossroads

PubMed Central

Jovic, Marko; Sharma, Mahak; Rahajeng, Juliati; Caplan, Steve

2010-01-01

Summary Endocytosis marks the entry of internalized receptors into the complex network of endocytic trafficking pathways. Endocytic vesicles are rapidly targeted to a distinct membrane-bound endocytic organelle referred to as the early endosome. Despite the existence of numerous internalization routes, early endosomes (EE) serve as a focal point of the endocytic pathway. Sorting events initiated at this compartment determine the subsequent fate of internalized proteins and lipids, destining them either for recycling to the plasma membrane, degradation in lysosomes or delivery to the trans-Golgi network. Sorting of endocytic cargo to the latter compartments is accomplished through the formation of distinct microdomains within early endosomes, through the coordinate recruitment and assembly of the sorting machinery. An elaborate network of interactions between endocytic regulatory proteins ensures synchronized sorting of cargo to microdomains followed by morphological changes at the early endosomal membranes. Consequently, the cargo targeted either for recycling back to the plasma membrane, or for retrograde transport to the trans-Golgi network, localizes to newly-formed tubular membranes. With a high ratio of membrane surface to lumenal volume, these tubules effectively concentrate the recycling cargo, ensuring efficient transport out of the EE. Conversely, receptors sorted for degradation cluster at the flat clathrin lattices involved in invaginations of the limiting membrane, associating with newly formed intralumenal vesicles. In this review we will discuss the characteristics of early endosomes, their role in the regulation of endocytic transport, and their aberrant function in a variety of diseases. PMID:19924646

GRASP1 regulates synaptic plasticity and learning through endosomal recycling of AMPA receptors

PubMed Central

Chiu, Shu-Ling; Diering, Graham Hugh; Ye, Bing; Takamiya, Kogo; Chen, Chih-Ming; Jiang, Yuwu; Niranjan, Tejasvi; Schwartz, Charles E.; Wang, Tao; Huganir, Richard L.

2017-01-01

Summary Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects on AMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescues spine loss in hippocampal CA1 neurons of Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity in vivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders. PMID:28285821

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

PubMed Central

Puchner, Elias M.; Walter, Jessica M.; Kasper, Robert; Huang, Bo; Lim, Wendell A.

2013-01-01

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes. PMID:24043832

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

MiR-153 Regulates Amelogenesis by Targeting Endocytotic and Endosomal/lysosomal Pathways–Novel Insight into the Origins of Enamel Pathologies

PubMed Central

Yin, Kaifeng; Lin, Wenting; Guo, Jing; Sugiyama, Toshihiro; Snead, Malcolm L.; Hacia, Joseph G.; Paine, Michael L.

2017-01-01

Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3′-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins. Finally, microinjection of miR-153 in the region of mouse first mandibular molar at postnatal day 8 (PN8) induced AI-like pathologies when the enamel development reached maturity (PN12). In conclusion, miR-153 regulates maturation-stage amelogenesis by targeting key genes involved in the endocytotic and endosomal/lysosomal pathways, and disruption of miR-153 expression is a potential candidate etiologic factor contributing to the occurrence of AI. PMID:28287144

MiR-153 Regulates Amelogenesis by Targeting Endocytotic and Endosomal/lysosomal Pathways-Novel Insight into the Origins of Enamel Pathologies.

PubMed

Yin, Kaifeng; Lin, Wenting; Guo, Jing; Sugiyama, Toshihiro; Snead, Malcolm L; Hacia, Joseph G; Paine, Michael L

2017-03-13

Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3'-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins. Finally, microinjection of miR-153 in the region of mouse first mandibular molar at postnatal day 8 (PN8) induced AI-like pathologies when the enamel development reached maturity (PN12). In conclusion, miR-153 regulates maturation-stage amelogenesis by targeting key genes involved in the endocytotic and endosomal/lysosomal pathways, and disruption of miR-153 expression is a potential candidate etiologic factor contributing to the occurrence of AI.

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

Endocytosis regulates membrane localization and function of the fusogen EFF-1.

PubMed

Smurova, Ksenia; Podbilewicz, Benjamin

2017-07-03

Cell fusion is essential for sexual reproduction and formation of muscles, bones, and placenta. Two families of cell fusion proteins (Syncytins and FFs) have been identified in eukaryotes. Syncytins have been shown to form the giant syncytial trophoblasts in the placenta. The FFs are essential to fuse cells in the skin, reproductive, excretory, digestive and nervous systems in nematodes. EFF-1 (Epithelial Fusion Failure 1), a member of the FF family, is a type I membrane glycoprotein that is essential for most cell fusions in C. elegans. The crystal structure of EFF-1 ectodomain reveals striking structural similarity to class II fusion glycoproteins from enveloped viruses (e.g. dengue and rubella) that mediate virus to cell fusion. We found EFF-1 to be present on the plasma membrane and in RAB-5-positive early endosomes, with EFF-1 recycling between these 2 cell compartments. Only when EFF-1 proteins transiently arrive to the surfaces of 2 adjacent cells do they dynamically interact in trans and mediate membrane fusion. EFF-1 is continuously internalized by receptor-mediated endocytosis via the activity of 2 small GTPases: RAB-5 and Dynamin. Here we propose a model that explains how EFF-1 endocytosis together with interactions in trans can control cell-cell fusion. Kontani et al. showed that vacuolar ATPase (vATPase) mutations result in EFF-1-dependent hyperfusion. 1 We propose that vATPase is required for normal degradation of EFF-1. Failure to degrade EFF-1 results in delayed hyperfusion and mislocalization to organelles that appear to be recycling endosomes. EFF-1 is also required to fuse neurons as part of the repair mechanism following injury and to prune dendrites. We speculate that EFF-1 may regulate neuronal tree like structures via endocytosis. Thus, endocytosis of cell-cell fusion proteins functions to prevent merging of cells and to sculpt organs and neurons.

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

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.

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

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

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

PubMed Central

Wu, Zhenyong; Thiyagarajan, Sathish; O’Shaughnessy, Ben; Karatekin, Erdem

2017-01-01

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically

The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

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

Huang, Claire Y.-H., E-mail: CHuang1@cdc.go; Butrapet, Siritorn; Moss, Kelly J.

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could notmore » re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.« less

Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.

PubMed

Bale, Shridhar; Liu, Tong; Li, Sheng; Wang, Yuhao; Abelson, Dafna; Fusco, Marnie; Woods, Virgil L; Saphire, Erica Ollmann

2011-11-01

Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2)) on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2) is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP(1,2) trimeric, viral surface spike. After entry into host endosomes, GP(1,2) is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown. We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS) of glycoproteins from two different ebolaviruses that although enzymatic priming of GP(1,2) is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP(1,2). Further, ELISA binding data of primed GP(1,2) to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion. The results reveal that the ebolavirus GP(1,2) ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP(1,2) and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.

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

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

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.

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

A histidine residue of the influenza virus hemagglutinin controls the pH dependence of the conformational change mediating membrane fusion.

PubMed

Mair, Caroline M; Meyer, Tim; Schneider, Katjana; Huang, Qiang; Veit, Michael; Herrmann, Andreas

2014-11-01

The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKas match the pHs of late endosomes (pK(a) of ∼ 6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pK(a). The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pK(a)-modulating effect of neighboring residues. The main pathogenic determinant of influenza viruses, the hemagglutinin (HA) protein, triggers a key step of the infection process: the fusion of the virus envelope with the endosomal membrane releasing the viral genome. Whereas essential aspects of the fusion-inducing mechanism of HA at low pH are well understood, the molecular trigger of the pH-dependent conformational change inducing fusion has been unclear. We provide evidence that His184 regulates the pH dependence of the HA

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes

PubMed Central

Tzafriri, A. Rami; Edelman, Elazer R.

2006-01-01

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor–receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor–receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor–receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered. PMID:17117924

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.

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

PubMed

Lee, Jinwoo; Nyenhuis, David A; Nelson, Elizabeth A; Cafiso, David S; White, Judith M; Tamm, Lukas K

2017-09-19

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

PubMed Central

Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

2008-01-01

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

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

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

Regulation of exocytotic fusion by cell inflation.

PubMed Central

Solsona, C; Innocenti, B; Fernández, J M

1998-01-01

We have inflated patch-clamped mast cells by 3.8 +/- 1.6 times their volume by applying a hydrostatic pressure of 5-15 cm H2O to the interior of the patch pipette. Inflation did not cause changes in the cell membrane conductance and caused only a small reversible change in the cell membrane capacitance (36 +/- 5 fF/cm H2O). The specific cell membrane capacitance of inflated cells was found to be 0.5 microF/cm2. High-resolution capacitance recordings showed that inflation reduced the frequency of exocytotic fusion events by approximately 70-fold, with the remaining fusion events showing an unusual time course. Shortly after the pressure was returned to 0 cm H2O, mast cells regained their normal size and appearance and degranulated completely, even after remaining inflated for up to 60 min. We interpret these observations as an indication that inflated mast cells reversibly disassemble the structures that regulate exocytotic fusion. Upon returning to its normal size, the cell cytosol reassembles the fusion pore scaffolds and allows exocytosis to proceed, suggesting that exocytotic fusion does not require soluble proteins. Reassembly of the fusion pore can be prevented by inflating the cells with solutions containing the protease pronase, which completely blocked exocytosis. We also interpret these results as evidence that the activity of the fusion pore is sensitive to the tension of the plasma membrane. PMID:9533718

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

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

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

DOE PAGES

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

2015-01-02

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

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

Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

PubMed

Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

2016-03-01

Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion.

PubMed

Karim, Mahmoud Abdul; Brett, Christopher Leonard

2018-02-01

Loss-of-function mutations in human endosomal Na + (K + )/H + exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function. © 2018 Karim and Brett. 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).

Residues in the Hendra Virus Fusion Protein Transmembrane Domain Are Critical for Endocytic Recycling

PubMed Central

Popa, Andreea; Carter, James R.; Smith, Stacy E.; Hellman, Lance; Fried, Michael G.

2012-01-01

Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking. PMID:22238299

Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion

PubMed Central

den Brave, Fabian

2018-01-01

Cdc48/p97, a ubiquitin-selective chaperone, orchestrates the function of E3 ligases and deubiquitylases (DUBs). Here, we identify a new function of Cdc48 in ubiquitin-dependent regulation of mitochondrial dynamics. The DUBs Ubp12 and Ubp2 exert opposing effects on mitochondrial fusion and cleave different ubiquitin chains on the mitofusin Fzo1. We demonstrate that Cdc48 integrates the activities of these two DUBs, which are themselves ubiquitylated. First, Cdc48 promotes proteolysis of Ubp12, stabilizing pro-fusion ubiquitylation on Fzo1. Second, loss of Ubp12 stabilizes Ubp2 and thereby facilitates removal of ubiquitin chains on Fzo1 inhibiting fusion. Thus, Cdc48 synergistically regulates the ubiquitylation status of Fzo1, allowing to control the balance between activation or repression of mitochondrial fusion. In conclusion, we unravel a new cascade of ubiquitylation events, comprising Cdc48 and two DUBs, fine-tuning the fusogenic activity of Fzo1. PMID:29309037

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

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 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.

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).

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.

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

PubMed

Tani, Motohiro; Kuge, Osamu

2012-12-01

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. © 2012 Blackwell Publishing Ltd.

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

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics.

PubMed

O'Loughlin, Thomas; Masters, Thomas A; Buss, Folma

2018-04-01

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine-tune motor activity in time and space. These motor-adaptor-cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling-based proteomics strategy to map the interactome of the unique minus end-directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6-adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG-Induced F-actin for Tethering) complex that controls endosome positioning and motility through RHO-driven actin polymerisation; and the DISP (DOCK7-Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Association of p60c-src with endosomal membranes in mammalian fibroblasts

PubMed Central

1992-01-01

We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three- dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI- MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking. PMID:1378446

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.

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

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.

Dual wavelength imaging allows analysis of membrane fusion of influenza virus inside cells.

PubMed

Sakai, Tatsuya; Ohuchi, Masanobu; Imai, Masaki; Mizuno, Takafumi; Kawasaki, Kazunori; Kuroda, Kazumichi; Yamashina, Shohei

2006-02-01

Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion.

Determination of Rab5 activity in the cell by effector pull-down assay.

PubMed

Qi, Yaoyao; Liang, Zhimin; Wang, Zonghua; Lu, Guodong; Li, Guangpu

2015-01-01

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State

PubMed Central

Russell, Charles J.; Jardetzky, Theodore S.; Lamb, Robert A.

2004-01-01

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation. PMID:15564482

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.

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.

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

Two Endosomal NHX-type Na+/ H+ Antiporters are Involved in Auxin Mediated Development in Arabidopsis thaliana.

PubMed

Dragwidge, Jonathan Michael; Ford, Brett Andrew; Ashnest, Joanne Rachel; Das, Partha; Gendall, Anthony Richard

2018-05-16

In Arabidopsis thaliana, the endosomal localised Na+/H+ antiporters NHX5 and NHX6 regulate ion and pH homeostasis and are important for plant growth and development. However, the mechanism of how these endosomal NHXs function in plant development is not well understood. Auxin modulates plant growth and development through the formation of concentration gradients in plant tissue to control cell division and expansion. Here, we identified a role for NHX5 and NHX6 in the establishment and maintenance of auxin gradients in embryo and root tissues. We observed developmental impairment and abnormal cell division in embryo and root tissues in the double knockout nhx5 nhx6, consistent with these tissues showing high expression of NHX5 and NHX6. Through confocal microscopy imaging with the DR5::GFP auxin reporter, we identify defects to the perception, accumulation, and redistribution of auxin in nhx5 nhx6 cells. Furthermore, we find that the steady state levels of the PIN-FORMED (PIN) auxin efflux carriers PIN1 and PIN2 are reduced in nhx5 nhx6 root cells. Our results demonstrate that NHX5 and NHX6 function in auxin mediated plant development by maintaining PIN abundance at the plasma membrane, and provides new insight into the regulation of plant development by endosomal NHX antiporters.

Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion*

PubMed Central

Kondo, Naoyuki; Marin, Mariana; Kim, Jeong Hwa; Desai, Tanay M.; Melikyan, Gregory B.

2015-01-01

Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as “fusion-from-without” (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4+ T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes. PMID:25589785

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

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

PubMed

James, Declan J; Khodthong, Chuenchanok; Kowalchyk, Judith A; Martin, Thomas F J

2008-07-28

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

PubMed

Zhang, Xiaoyan; Wang, Leibin; Lak, Behnam; Li, Jie; Jokitalo, Eija; Wang, Yanzhuang

2018-04-23

The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

Analysis of Snail1 function and regulation by Twist1 in palatal fusion.

PubMed

Yu, Wenli; Zhang, Yanping; Ruest, L Bruno; Svoboda, Kathy K H

2013-01-01

Palatal fusion is a tightly controlled process which comprises multiple cellular events, including cell movement and differentiation. Midline epithelial seam (MES) degradation is essential to palatal fusion. In this study, we analyzed the function of Snail1 during the degradation of the MES. We also analyzed the mechanism regulating the expression of the Snail1 gene in palatal shelves. Palatal explants treated with Snail1 siRNA did not degrade the MES and E-cadherin was not repressed leading to failure of palatal fusion. Transforming growth factor beta 3 (Tgfβ3) regulated Snail1 mRNA, as Snail1 expression decreased in response to Tgfβ3 neutralizing antibody and a PI-3 kinase (PI3K) inhibitor. Twist1, in collaboration with E2A factors, regulated the expression of Snail1. Twist1/E47 dimers bond to the Snail1 promoter to activate expression. Without E47, Twist1 repressed Snail1 expression. These results support the hypothesis that Tgfβ3 may signal through Twist1 and then Snail1 to downregulate E-cadherin expression during palatal fusion.

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

The role of fusion activity of influenza A viruses in their biological properties.

PubMed

Jakubcová, L; Hollý, J; Varečková, E

2016-06-01

Influenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with significant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, after their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors influencing the virulence and pathogenicity of IAV viruses in a given host. The virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. The fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. The HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specific. The process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity.

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

PubMed Central

Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

2014-01-01

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

Parkin Modulates Endosomal Organization and Function of the Endo-Lysosomal Pathway.

PubMed

Song, Pingping; Trajkovic, Katarina; Tsunemi, Taiji; Krainc, Dimitri

2016-02-24

Mutations in PARK2 (parkin), which encodes Parkin protein, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While several studies implicated Parkin in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration upon Parkin loss of function remains incompletely understood. In this study, we found that Parkin modulates the endocytic pathway through the regulation of endosomal structure and function. We showed that loss of Parkin function led to decreased endosomal tubulation and membrane association of vesicle protein sorting 35 (VPS35) and sorting nexin 1 (SNX1), as well as decreased mannose 6 phosphate receptor (M6PR), suggesting the impairment of retromer pathway in Parkin-deficient cells. We also found increased formation of intraluminal vesicles coupled with enhanced release of exosomes in the presence of mutant Parkin. To elucidate the molecular mechanism of these alterations in the endocytic pathway in Parkin-deficient cells, we found that Parkin regulates the levels and activity of Rab7 by promoting its ubiquitination on lysine 38 residue. Both endogenous Rab7 in Parkin-deficient cells and overexpressed K38 R-Rab7 mutant displayed decreased effector binding and membrane association. Furthermore, overexpression of K38R-Rab7 in HEK293 cells phenocopied the increased secretion of exosomes observed in Parkin-deficient cells, suggesting that Rab7 deregulation may be at least partially responsible for the endocytic phenotype observed in Parkin-deficient cells. These findings establish a role for Parkin in regulating the endo-lysosomal pathway and retromer function and raise the possibility that alterations in these pathways contribute to the development of pathology in Parkin-linked Parkinson's disease. Copyright © 2016 the authors 0270-6474/16/362425-13$15.00/0.

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

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

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.

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.

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

Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

PubMed

Yoon, June-Sun; Gurusamy, Dhandapani; Palli, Subba Reddy

2017-11-01

RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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.

Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop.

PubMed

Liu, Nina; Tao, Yisong; Brenowitz, Michael D; Girvin, Mark E; Lai, Jonathan R

2015-10-01

Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion. Here, we describe the first structural and functional studies of a protein corresponding to the MARV GP2 FL. We found that this protein undergoes a pH-dependent conformational change, as monitored by circular dichroism and nuclear magnetic resonance. Furthermore, we report that, under low pH conditions, the MARV GP2 FL can induce content leakage from liposomes. The general aspects of this pH-dependent structure and lipid-perturbing behavior are consistent with previous reports on Ebola virus GP2 FL. However, nuclear magnetic resonance studies in lipid bicelles and mutational analysis indicate differences in structure exist between MARV and Ebola virus GP2 FL. These results provide new insight into the mechanism of MARV GP2-mediated cell entry. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

Early Endosomal Escape of a Cyclic Cell-Penetrating Peptide Allows Effective Cytosolic Cargo Delivery

PubMed Central

2015-01-01

Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape. PMID:24896852

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.

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

PubMed Central

Hoogenraad, Casper C.; Popa, Ioana; Futai, Kensuke; Sanchez-Martinez, Emma; Wulf, Phebe S.; van Vlijmen, Thijs; Dortland, Bjorn R.; Oorschot, Viola; Govers, Roland; Monti, Maria; Heck, Albert J. R.; Sheng, Morgan; Klumperman, Judith; Rehmann, Holger; Jaarsma, Dick; Kapitein, Lukas C.; van der Sluijs, Peter

2010-01-01

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking. PMID:20098723

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

The tumorigenic FGFR3-TACC3 gene fusion escapes miR-99a regulation in glioblastoma.

PubMed

Parker, Brittany C; Annala, Matti J; Cogdell, David E; Granberg, Kirsi J; Sun, Yan; Ji, Ping; Li, Xia; Gumin, Joy; Zheng, Hong; Hu, Limei; Yli-Harja, Olli; Haapasalo, Hannu; Visakorpi, Tapio; Liu, Xiuping; Liu, Chang-Gong; Sawaya, Raymond; Fuller, Gregory N; Chen, Kexin; Lang, Frederick F; Nykter, Matti; Zhang, Wei

2013-02-01

Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Fusions can lead to production of oncogenic fusion proteins or to enhanced expression of oncogenes. Several recent studies have reported that some fusion genes can escape microRNA regulation via 3'-untranslated region (3'-UTR) deletion. We performed whole transcriptome sequencing to identify fusion genes in glioma and discovered FGFR3-TACC3 fusions in 4 of 48 glioblastoma samples from patients both of mixed European and of Asian descent, but not in any of 43 low-grade glioma samples tested. The fusion, caused by tandem duplication on 4p16.3, led to the loss of the 3'-UTR of FGFR3, blocking gene regulation of miR-99a and enhancing expression of the fusion gene. The fusion gene was mutually exclusive with EGFR, PDGFR, or MET amplification. Using cultured glioblastoma cells and a mouse xenograft model, we found that fusion protein expression promoted cell proliferation and tumor progression, while WT FGFR3 protein was not tumorigenic, even under forced overexpression. These results demonstrated that the FGFR3-TACC3 gene fusion is expressed in human cancer and generates an oncogenic protein that promotes tumorigenesis in glioblastoma.

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

In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5.

PubMed

Pagano, Adriana; Crottet, Pascal; Prescianotto-Baschong, Cristina; Spiess, Martin

2004-11-01

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

A Membrane-Destabilizing Peptide in Capsid Protein L2 Is Required for Egress of Papillomavirus Genomes from Endosomes

PubMed Central

Kämper, Nadine; Day, Patricia M.; Nowak, Thorsten; Selinka, Hans-Christoph; Florin, Luise; Bolscher, Jan; Hilbig, Lydia; Schiller, John T.; Sapp, Martin

2006-01-01

Papillomaviruses are internalized via clathrin-dependent endocytosis. However, the mechanism by which viral genomes pass endosomal membranes has not been elucidated. In this report we show that the minor capsid protein L2 is required for egress of viral genomes from endosomes but not for initial uptake and uncoating and that a 23-amino-acid peptide at the C terminus of L2 is necessary for this function. Pseudogenomes encapsidated by L1 and L2 lacking this peptide accumulated in vesicular compartments similar to that observed with L1-only viral particles, and these mutant pseudoviruses were noninfectious. This L2 peptide displayed strong membrane-disrupting activity, induced cytolysis of bacteria and eukaryotic cells in a pH-dependent manner, and permeabilized cells after exogenous addition. Fusions between green fluorescent protein and the L2 peptide integrated into cellular membranes like the wild type but not like C-terminal mutants of L2. Our data indicate that the L2 C terminus facilitates escape of viral genomes from the endocytic compartment and that this feature is conserved among papillomaviruses. Furthermore, the characteristic of this peptide differs from the classical virus-encoded membrane-penetrating peptides. PMID:16378978

Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

PubMed Central

Saw, Wan Ting; Eisenberg, Roselyn J.; Cohen, Gary H.

2016-01-01

ABSTRACT Receptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH2/gL2), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD1) and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing the activity of the mutants. Under low-pH conditions or when

Herpesvirus Entry into Host Cells Mediated by Endosomal Low pH.

PubMed

Nicola, Anthony V

2016-09-01

Herpesviral pathogenesis stems from infection of multiple cell types including the site of latency and cells that support lytic replication. Herpesviruses utilize distinct cellular pathways, including low pH endocytic pathways, to enter different pathophysiologically relevant target cells. This review details the impact of the mildly acidic milieu of endosomes on the entry of herpesviruses, with particular emphasis on herpes simplex virus 1 (HSV-1). Epithelial cells, the portal of primary HSV-1 infection, support entry via low pH endocytosis mechanisms. Mildly acidic pH triggers reversible conformational changes in the HSV-1 class III fusion protein glycoprotein B (gB). In vitro treatment of herpes simplex virions with a similar pH range inactivates infectivity, likely by prematurely activating the viral entry machinery in the absence of a target membrane. How a given herpesvirus mediates both low pH and pH-independent entry events is a key unresolved question. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The time-dependent distribution of 125I-asialo-orosomucoid-horseradish peroxidase and 131I-immunoglobulin A among three endosomal subfractions isolated from rat liver.

PubMed Central

Kennedy, G; Cooper, C

1988-01-01

Three discrete endosomal fractions showing a time-dependent uptake of radioactive ligand were partially purified from rat liver. The 3,3'-diaminobenzidine (DAB)-induced density-shift protocol of Courtoy, Quintart & Baudhuin [(1984) J. Cell Biol. 98, 870-876] was used to study the distribution among these three endosomal fractions of two ligands with different intracellular destinations. Rats received both 125I-asialo-orosomucoid-horseradish peroxidase (125I-ASOR-HRP) and 131I-dIgA simultaneously by intraportal injection. The liver was fractionated at various times after injection, the three ligand-containing endosomal fractions (A, B and C) were separated and each was subjected separately to the DAB-induced density-shift procedure in which only vesicles containing 125I-ASOR-HRP are increased in density. Information on whether 131I-dIgA was co-localized or segregated from 125I-ASOR-HRP was obtained. The two ligands in the A fraction were partly segregated and partly co-localized, and this distribution appeared to be relatively unchanged with time. The two ligands in the B fraction were co-localized at all times studied. We have tentatively identified the B fraction as a compartment in which vesicle fusion has occurred. The two ligands in the C fraction were also partly co-localized and partly segregated, but the 131I-dIgA became increasingly segregated with time. This represents the first report of the purification of an endosomal subfraction specifically involved in the accumulation of multiple ligands. Images Fig. 7. PMID:3421920

Phosphatidylcholine Membrane Fusion Is pH-Dependent.

PubMed

Akimov, Sergey A; Polynkin, Michael A; Jiménez-Munguía, Irene; Pavlov, Konstantin V; Batishchev, Oleg V

2018-05-03

Membrane fusion mediates multiple vital processes in cell life. Specialized proteins mediate the fusion process, and a substantial part of their energy is used for topological rearrangement of the membrane lipid matrix. Therefore, the elastic parameters of lipid bilayers are of crucial importance for fusion processes and for determination of the energy barriers that have to be crossed for the process to take place. In the case of fusion of enveloped viruses (e.g., influenza) with endosomal membrane, the interacting membranes are in an acidic environment, which can affect the membrane's mechanical properties. This factor is often neglected in the analysis of virus-induced membrane fusion. In the present work, we demonstrate that even for membranes composed of zwitterionic lipids, changes of the environmental pH in the physiologically relevant range of 4.0 to 7.5 can affect the rate of the membrane fusion notably. Using a continual model, we demonstrated that the key factor defining the height of the energy barrier is the spontaneous curvature of the lipid monolayer. Changes of this parameter are likely to be caused by rearrangements of the polar part of lipid molecules in response to changes of the pH of the aqueous solution bathing the membrane.

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.

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.

The tumorigenic FGFR3-TACC3 gene fusion escapes miR-99a regulation in glioblastoma

PubMed Central

Parker, Brittany C.; Annala, Matti J.; Cogdell, David E.; Granberg, Kirsi J.; Sun, Yan; Ji, Ping; Li, Xia; Gumin, Joy; Zheng, Hong; Hu, Limei; Yli-Harja, Olli; Haapasalo, Hannu; Visakorpi, Tapio; Liu, Xiuping; Liu, Chang-gong; Sawaya, Raymond; Fuller, Gregory N.; Chen, Kexin; Lang, Frederick F.; Nykter, Matti; Zhang, Wei

2013-01-01

Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Fusions can lead to production of oncogenic fusion proteins or to enhanced expression of oncogenes. Several recent studies have reported that some fusion genes can escape microRNA regulation via 3′–untranslated region (3′-UTR) deletion. We performed whole transcriptome sequencing to identify fusion genes in glioma and discovered FGFR3-TACC3 fusions in 4 of 48 glioblastoma samples from patients both of mixed European and of Asian descent, but not in any of 43 low-grade glioma samples tested. The fusion, caused by tandem duplication on 4p16.3, led to the loss of the 3′-UTR of FGFR3, blocking gene regulation of miR-99a and enhancing expression of the fusion gene. The fusion gene was mutually exclusive with EGFR, PDGFR, or MET amplification. Using cultured glioblastoma cells and a mouse xenograft model, we found that fusion protein expression promoted cell proliferation and tumor progression, while WT FGFR3 protein was not tumorigenic, even under forced overexpression. These results demonstrated that the FGFR3-TACC3 gene fusion is expressed in human cancer and generates an oncogenic protein that promotes tumorigenesis in glioblastoma. PMID:23298836

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.

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes*

PubMed Central

Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

2016-01-01

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. PMID:26987901

Insulin Recruits GLUT4 from Specialized VAMP2-carrying Vesicles as well as from the Dynamic Endosomal/Trans-Golgi Network in Rat Adipocytes.

PubMed Central

Ramm, Georg; Slot, Jan Willem; James, David E.; Stoorvogel, Willem

2000-01-01

Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-d resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system. PMID:11102509

A novel chimeric cell-penetrating peptide with membrane-disruptive properties for efficient endosomal escape.

PubMed

Salomone, Fabrizio; Cardarelli, Francesco; Di Luca, Mariagrazia; Boccardi, Claudia; Nifosì, Riccardo; Bardi, Giuseppe; Di Bari, Lorenzo; Serresi, Michela; Beltram, Fabio

2012-11-10

Efficient endocytosis into a wide range of target cells and low toxicity make the arginine-rich Tat peptide (Tat(11): YGRKKRRQRRR, residues 47-57 of HIV-1 Tat protein) an excellent transporter for delivery purposes. Unfortunately, molecules taken up by endocytosis undergo endosomal entrapment and possible metabolic degradation. Escape from the endosome is therefore actively researched. In this context, antimicrobial peptides (AMPs) provide viable templates for the design of new membrane-disruptive motifs. In particular the Cecropin-A and Melittin hybrids (CMs) are among the smallest and most effective peptides with membrane-perturbing abilities. Here we present a novel chimeric peptide in which the Tat(11) motif is fused to the CM(18) hybrid (KWKLFKKIGAVLKVLTTG, residues 1-7 of Cecropin-A and 2-12 of Melittin). When administered to cells, CM(18)-Tat(11) combines the two desired functionalities: efficient uptake and destabilization of endocytotic-vesicle membranes. We show that this chimeric peptide effectively increases cargo-molecule cytoplasm availability and allows the subsequent intracellular localization of diverse membrane-impermeable molecules (i.e. Tat(11)-EGFP fusion protein, calcein, dextrans, and plasmidic DNA) with no detectable cytotoxicity. The present results open the way to the rational engineering of "modular" cell-penetrating peptides (CPPs) that combine (i) efficient translocation from the extracellular milieu into vesicles and (ii) efficient release of molecules from vesicles into the cytoplasm. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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.

The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

PubMed

Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

2015-03-01

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. © 2015. Published by The Company of Biologists Ltd.

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

PubMed Central

Chen, Xu-Qiao; Wang, Bin; Wu, Chengbiao; Pan, Jin; Yuan, Bo; Su, Yuan-Yuan; Jiang, Xing-Yu; Zhang, Xu; Bao, Lan

2012-01-01

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X3 receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X3 receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X3 receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X3 receptors. The α, β-MeATP-induced Ca2+ influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X3 receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X3 receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X3 receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels. PMID:22157653

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons.

PubMed

Chen, Xu-Qiao; Wang, Bin; Wu, Chengbiao; Pan, Jin; Yuan, Bo; Su, Yuan-Yuan; Jiang, Xing-Yu; Zhang, Xu; Bao, Lan

2012-04-01

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.

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

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

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.

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

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.

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.

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.

Deleting the DAG Kinase Dgk1 Augments Yeast Vacuole Fusion Through In-creased Ypt7 Activity and Altered Membrane Fluidity

PubMed Central

Miner, Gregory E.; Starr, Matthew L.; Hurst, Logan R.; Fratti, Rutilio A.

2017-01-01

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2, and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1, however deleting both genes did not rescue the pah1Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity. PMID:28276191

Regulation of cell protrusions by small GTPases during fusion of the neural folds

PubMed Central

Rolo, Ana; Savery, Dawn; Escuin, Sarah; de Castro, Sandra C; Armer, Hannah EJ; Munro, Peter MG; Molè, Matteo A; Greene, Nicholas DE; Copp, Andrew J

2016-01-01

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development. DOI: http://dx.doi.org/10.7554/eLife.13273.001 PMID:27114066

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.

A residue located at the junction of the head and stalk regions of measles virus fusion protein regulates membrane fusion by controlling conformational stability.

PubMed

Satoh, Yuto; Yonemori, Saeka; Hirose, Mitsuhiro; Shogaki, Hiroko; Wakimoto, Hiroshi; Kitagawa, Yoshinori; Gotoh, Bin; Shirai, Tsuyoshi; Takahashi, Ken-Ichi; Itoh, Masae

2017-02-01

The fusion (F) protein of measles virus performs refolding from the thermodynamically metastable prefusion form to the highly stable postfusion form via an activated unstable intermediate stage, to induce membrane fusion. Some amino acids involved in the fusion regulation cluster in the heptad repeat B (HR-B) domain of the stalk region, among which substitution of residue 465 by various amino acids revealed that fusion activity correlates well with its side chain length from the Cα (P<0.01) and van der Waals volume (P<0.001), except for Phe, Tyr, Trp, Pro and His carrying ring structures. Directed towards the head region, longer side chains of the non-ring-type 465 residues penetrate more deeply into the head region and may disturb the hydrophobic interaction between the stalk and head regions and cause destabilization of the molecule by lowering the energy barrier for refolding, which conferred the F protein enhanced fusion activity. Contrarily, the side chain of ring-type 465 residues turned away from the head region, resulting in not only no contact with the head region but also extensive coverage of the HR-B surface, which may prevent the dissociation of the HR-B bundle for initiation of membrane fusion and suppress fusion activity. Located in the HR-B domain just at the junction between the head and stalk regions, amino acid 465 is endowed with a possible ability to either destabilize or stabilize the F protein depending on its molecular volume and the direction of the side chain, regulating fusion activity of measles virus F protein.

Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

PubMed

Ahmad, Waqas; Li, Yingying; Guo, Yidi; Wang, Xinyu; Duan, Ming; Guan, Zhenhong; Liu, Zengshan; Zhang, Maolin

2017-06-01

Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

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).

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.

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.

LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity.

PubMed

Gómez-Suaga, Patricia; Rivero-Ríos, Pilar; Fdez, Elena; Blanca Ramírez, Marian; Ferrer, Isidro; Aiastui, Ana; López De Munain, Adolfo; Hilfiker, Sabine

2014-12-20

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD), and sequence variations at the LRRK2 locus are associated with increased risk for sporadic PD. LRRK2 contains both GTPase and kinase domains flanked by protein interaction motifs, and mutations associated with familial PD have been described for both catalytic domains. LRRK2 has been implicated in diverse cellular processes, and recent evidence pinpoints to an important role for LRRK2 in modulating a variety of intracellular membrane trafficking pathways. However, the underlying mechanisms are poorly understood. Here, by studying the classical, well-understood, degradative trafficking pathway of the epidermal growth factor receptor (EGFR), we show that LRRK2 regulates endocytic membrane trafficking in an Rab7-dependent manner. Mutant LRRK2 expression causes a slight delay in early-to-late endosomal trafficking, and a pronounced delay in trafficking out of late endosomes, which become aberrantly elongated into tubules. This is accompanied by a delay in EGFR degradation. The LRRK2-mediated deficits in EGFR trafficking and degradation can be reverted upon coexpression of active Rab7 and of a series of proteins involved in bridging the EGFR to Rab7 on late endosomes. Effector pulldown assays indicate that pathogenic LRRK2 decreases Rab7 activity both in cells overexpressing LRRK2, as well as in fibroblasts from pathogenic mutant LRRK2 PD patients when compared with healthy controls. Together, these findings provide novel insights into a previously unknown regulation of Rab7 activity by mutant LRRK2 which impairs membrane trafficking at very late stages of the endocytic pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain.

PubMed

Haug, Gerd; Wilde, Christian; Leemhuis, Jost; Meyer, Dieter K; Aktories, Klaus; Barth, Holger

2003-12-30

The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

PubMed

Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes.

PubMed

Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

2016-05-13

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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

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.

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.

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

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

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

Jeong, Jaemin, E-mail: jmj1103@kirams.re.kr; Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul; Conboy, Irina M., E-mail: iconboy@berkeley.edu

2011-10-14

Highlights: {yields} PS broadly and persistently trans-locates to the outer leaflet of plasma membrane during myoblast fusion into myotubes. {yields} Robust myotubes are formed when PS liposomes are added exogenously. {yields} PS increases the width of de novo myotubes and the numbers of myonuclei, but not the myotube length. {yields} Annexin V or PS antibody inhibits myotube formation by masking exposed PS. -- Abstract: Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generallymore » but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell-cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.« less

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

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

Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

PubMed

Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

2010-02-15

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

Visualization of endosome dynamics in living nerve terminals with four-dimensional fluorescence imaging.

PubMed

Stewart, Richard S; Kiss, Ilona M; Wilkinson, Robert S

2014-04-16

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Long-distance endosome trafficking drives fungal effector production during plant infection

PubMed Central

Bielska, Ewa; Higuchi, Yujiro; Schuster, Martin; Steinberg, Natascha; Kilaru, Sreedhar; Talbot, Nicholas J.; Steinberg, Gero

2014-01-01

To cause plant disease, pathogenic fungi can secrete effector proteins into plant cells to suppress plant immunity and facilitate fungal infection. Most fungal pathogens infect plants using very long strand-like cells, called hyphae, that secrete effectors from their tips into host tissue. How fungi undergo long-distance cell signalling to regulate effector production during infection is not known. Here we show that long-distance retrograde motility of early endosomes (EEs) is necessary to trigger transcription of effector-encoding genes during plant infection by the pathogenic fungus Ustilago maydis. We demonstrate that motor-dependent retrograde EE motility is necessary for regulation of effector production and secretion during host cell invasion. We further show that retrograde signalling involves the mitogen-activated kinase Crk1 that travels on EEs and participates in control of effector production. Fungal pathogens therefore undergo long-range signalling to orchestrate host invasion. PMID:25283249

Long-distance endosome trafficking drives fungal effector production during plant infection.

PubMed

Bielska, Ewa; Higuchi, Yujiro; Schuster, Martin; Steinberg, Natascha; Kilaru, Sreedhar; Talbot, Nicholas J; Steinberg, Gero

2014-10-06

To cause plant disease, pathogenic fungi can secrete effector proteins into plant cells to suppress plant immunity and facilitate fungal infection. Most fungal pathogens infect plants using very long strand-like cells, called hyphae, that secrete effectors from their tips into host tissue. How fungi undergo long-distance cell signalling to regulate effector production during infection is not known. Here we show that long-distance retrograde motility of early endosomes (EEs) is necessary to trigger transcription of effector-encoding genes during plant infection by the pathogenic fungus Ustilago maydis. We demonstrate that motor-dependent retrograde EE motility is necessary for regulation of effector production and secretion during host cell invasion. We further show that retrograde signalling involves the mitogen-activated kinase Crk1 that travels on EEs and participates in control of effector production. Fungal pathogens therefore undergo long-range signalling to orchestrate host invasion.

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.

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

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.

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

Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.

PubMed

Rappa, Germana; Santos, Mark F; Green, Toni M; Karbanová, Jana; Hassler, Justin; Bai, Yongsheng; Barsky, Sanford H; Corbeil, Denis; Lorico, Aurelio

2017-02-28

Extracellular membrane vesicles (EVs) function as vehicles of intercellular communication, but how the biomaterials they carry reach the target site in recipient cells is an open question. We report that subdomains of Rab7+ late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, where biomaterials associated with CD9+ EVs are delivered. EV-derived biomaterials were also found in the nuclei of host cells. The inhibition of nuclear import and export pathways abrogated the nuclear localization of EV-derived biomaterials or led to their accumulation therein, respectively, suggesting that their translocation is dependent on nuclear pores. Nuclear envelope invagination-associated late endosomes were observed in ex vivo biopsies in both breast carcinoma and associated stromal cells. The transcriptome of stromal cells exposed to cancer cell-derived CD9+ EVs revealed that the regulation of eleven genes, notably those involved in inflammation, relies on the nuclear translocation of EV-derived biomaterials. Our findings uncover a new cellular pathway used by EVs to reach nuclear compartment.

Capturing a flavivirus pre-fusion intermediate.

PubMed

Kaufmann, Bärbel; Chipman, Paul R; Holdaway, Heather A; Johnson, Syd; Fremont, Daved H; Kuhn, Richard J; Diamond, Michael S; Rossmann, Michael G

2009-11-01

During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting approximately 60 A-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements.

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.

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

Full-Length Trimeric Influenza Virus Hemagglutinin II Membrane Fusion Protein and Shorter Constructs Lacking the Fusion Peptide or Transmembrane Domain: Hyperthermostability of the Full-Length Protein and the Soluble Ectodomain and Fusion Peptide Make Significant Contributions to Fusion of Membrane Vesicles†

PubMed Central

Ratnayake, Punsisi U.; Ekanayaka, E. A. Prabodha; Komanduru, Sweta S.; Weliky, David P.

2015-01-01

Influenza virus is a Class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5–6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ~25, ~160, ~25, and ~10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP + SE, and SHA2-TM ≡ SE + TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm > 90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM. PMID:26297995

Full-length trimeric influenza virus hemagglutinin II membrane fusion protein and shorter constructs lacking the fusion peptide or transmembrane domain: Hyperthermostability of the full-length protein and the soluble ectodomain and fusion peptide make significant contributions to fusion of membrane vesicles.

PubMed

Ratnayake, Punsisi U; Prabodha Ekanayaka, E A; Komanduru, Sweta S; Weliky, David P

2016-01-01

Influenza virus is a class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5-6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ∼ 25, ∼ 160, ∼ 25, and ∼ 10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP+SE, and SHA2-TM ≡ SE+TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm>90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM. Copyright © 2015 Elsevier Inc. All rights reserved.

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

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.

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

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.

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 mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

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

Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared

2006-03-15

The Nipah virus fusion (F) protein is proteolytically processed to F{sub 1} + F{sub 2} subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsinsmore » can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.« less

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.

Enhancing Endosomal Escape of Transduced Proteins by Photochemical Internalisation

PubMed Central

Mellert, Kevin; Lamla, Markus; Scheffzek, Klaus; Wittig, Rainer; Kaufmann, Dieter

2012-01-01

Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro. PMID:23285056

Enhancing endosomal escape of transduced proteins by photochemical internalisation.

PubMed

Mellert, Kevin; Lamla, Markus; Scheffzek, Klaus; Wittig, Rainer; Kaufmann, Dieter

2012-01-01

Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.

Rotation of endosomes demonstrates coordination of molecular motors during axonal transport.

PubMed

Kaplan, Luke; Ierokomos, Athena; Chowdary, Praveen; Bryant, Zev; Cui, Bianxiao

2018-03-01

Long-distance axonal transport is critical to the maintenance and function of neurons. Robust transport is ensured by the coordinated activities of multiple molecular motors acting in a team. Conventional live-cell imaging techniques used in axonal transport studies detect this activity by visualizing the translational dynamics of a cargo. However, translational measurements are insensitive to torques induced by motor activities. By using gold nanorods and multichannel polarization microscopy, we simultaneously measure the rotational and translational dynamics for thousands of axonally transported endosomes. We find that the rotational dynamics of an endosome provide complementary information regarding molecular motor activities to the conventionally tracked translational dynamics. Rotational dynamics correlate with translational dynamics, particularly in cases of increased rotation after switches between kinesin- and dynein-mediated transport. Furthermore, unambiguous measurement of nanorod angle shows that endosome-contained nanorods align with the orientation of microtubules, suggesting a direct mechanical linkage between the ligand-receptor complex and the microtubule motors.

Rotation of endosomes demonstrates coordination of molecular motors during axonal transport

PubMed Central

Kaplan, Luke; Ierokomos, Athena; Chowdary, Praveen; Bryant, Zev; Cui, Bianxiao

2018-01-01

Long-distance axonal transport is critical to the maintenance and function of neurons. Robust transport is ensured by the coordinated activities of multiple molecular motors acting in a team. Conventional live-cell imaging techniques used in axonal transport studies detect this activity by visualizing the translational dynamics of a cargo. However, translational measurements are insensitive to torques induced by motor activities. By using gold nanorods and multichannel polarization microscopy, we simultaneously measure the rotational and translational dynamics for thousands of axonally transported endosomes. We find that the rotational dynamics of an endosome provide complementary information regarding molecular motor activities to the conventionally tracked translational dynamics. Rotational dynamics correlate with translational dynamics, particularly in cases of increased rotation after switches between kinesin- and dynein-mediated transport. Furthermore, unambiguous measurement of nanorod angle shows that endosome-contained nanorods align with the orientation of microtubules, suggesting a direct mechanical linkage between the ligand-receptor complex and the microtubule motors. PMID:29536037

Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage.

PubMed

Voiniciuc, Catalin; Dean, Gillian H; Griffiths, Jonathan S; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L; Estelle, Mark; Haughn, George W

2013-03-01

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

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.

Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion.

PubMed

Zeng, Xiancheng; Mukhopadhyay, Suchetana; Brooks, Charles L

2015-02-17

Alphavirus envelope proteins, organized as trimers of E2-E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.

Histone deacetylase-mediated regulation of endolysosomal pH.

PubMed

Prasad, Hari; Rao, Rajini

2018-05-04

The pH of the endolysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover, and signaling functions in the cell. Dysregulation of endolysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders, including Alzheimer's disease. Therefore, understanding the mechanisms that regulate luminal pH may be key to identifying new targets for managing these disorders. Meta-analysis of yeast microarray databases revealed that nutrient-limiting conditions inhibited the histone deacetylase (HDAC) Rpd3 and thereby up-regulated transcription of the endosomal Na + /H + exchanger Nhx1, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of the Nhx1 orthologs DmNHE3 and NHE6, respectively, is also mediated by HDACs. We show that NHE6 is a target of the transcription factor cAMP-response element-binding protein (CREB), a known regulator of cellular responses to low-nutrient conditions, providing a molecular mechanism for nutrient- and HDAC-dependent regulation of endosomal pH. Of note, pharmacological targeting of the CREB pathway to increase NHE6 expression helped regulate endosomal pH and correct defective clearance of amyloid Aβ in an apoE4 astrocyte model of Alzheimer's disease. These observations from yeast, fly, mouse, and cell culture models point to an evolutionarily conserved mechanism for HDAC-mediated regulation of endosomal NHE expression. Our insights offer new therapeutic strategies for modulation of endolysosomal pH in fungal infection and human disease. © 2018 Prasad and Rao.

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.

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2[S

PubMed Central

Oninla, Vincent O.; Breiden, Bernadette; Babalola, Jonathan O.; Sandhoff, Konrad

2014-01-01

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion. PMID:25339683

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.

The dynamics and regulation of mesenchymal cell fusion in the sea urchin embryo.

PubMed

Hodor, P G; Ettensohn, C A

1998-07-01

Cell-cell fusion occurs in a wide variety of developmental contexts, yet the mechanisms involved are just beginning to be elucidated. In the sea urchin embryo, primary mesenchyme cells (PMCs) fuse to form syncytial filopodial cables within which skeletal spicules are deposited. Taking advantage of the optical transparency and ease of micromanipulation of sea urchin embryos, we have developed methods for directly observing the dynamics of PMC fusion in vivo. A fraction of the PMCs was labeled with fluorescent dextran and transfer of the dye to unlabeled PMCs was followed by time-lapse, fluorescence microscopy. Fusion was first detected about 2 h after PMCs began to migrate within the blastocoel. Fusion proceeded in parallel with the assembly of the PMC ring pattern and was complete by the early gastrula stage. The formation of a single, extensive PMC syncytium was confirmed by DiI labeling of fixed embryos. When single micromeres were isolated and cultured in unsupplemented seawater, they divided and their progeny underwent fusion. This shows that the capacity to fuse is autonomously programmed in the micromere-PMC lineage by the 16-cell stage. PMC transplantations at late embryonic stages revealed that these cells remain fusion-competent long after their fusion is complete. At late stages, other mesenchyme cells (blastocoelar cells) are also present within the blastocoel and are migrating and fusing with one another. Fusion-competent blastocoelar cells and PMCs come into contact but do not fuse with one another, indicating that these two cell types fuse by distinct mechanisms. When secondary mesenchyme cells convert to a skeletogenic fate they alter their fusogenic properties and join the PMC syncytium, as shown by transfer of fluorescent dextran. Our analysis has provided a detailed picture of the cellular basis and regulation of mesodermal cell fusion and has important implications regarding molecular mechanisms that underlie fusion.

Vacuolar ATPase in Phagosome-Lysosome Fusion

PubMed Central

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-01-01

The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

Vacuolar ATPase in phagosome-lysosome fusion.

PubMed

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-05-29

The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamin Regulates Specific Membrane Fusion Events Necessary for Acrosomal Exocytosis in Mouse Spermatozoa

PubMed Central

Reid, Andrew T.; Lord, Tessa; Stanger, Simone J.; Roman, Shaun D.; McCluskey, Adam; Robinson, Phillip J.; Aitken, R. John; Nixon, Brett

2012-01-01

Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signaling and scaffolding proteins. Here we show that two different members of the dynamin GTPase family localize to the developing acrosome of maturing mouse germ cells. Both dynamin 1 and 2 also remain within the periacrosomal region of mature mouse spermatozoa and are thus well positioned to regulate the acrosome reaction. Two pharmacological inhibitors of dynamin, dynasore and Dyngo-4a, blocked the in vitro induction of acrosomal exocytosis by progesterone, but not by the calcium ionophore A23187, and elicited a concomitant reduction of in vitro fertilization. In vivo treatment with these inhibitors also resulted in spermatozoa displaying reduced acrosome reaction potential. Dynamin 1 and 2 phosphorylation increased on progesterone treatment, and this was also selectively blocked by dynasore. On the basis of our collective data, we propose that dynamin could regulate specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa. PMID:22977254

Na+/H+ Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation.

PubMed

Beydoun, Rami; Hamood, Mohamed A; Gomez Zubieta, Daniela M; Kondapalli, Kalyan C

2017-03-10

Iron is essential for brain function, with loss of iron homeostasis in the brain linked to neurological diseases ranging from rare syndromes to more common disorders, such as Parkinson's and Alzheimer's diseases. Iron entry into the brain is regulated by the blood-brain barrier (BBB). Molecular mechanisms regulating this transport are poorly understood. Using an in vitro model of the BBB, we identify NHE9, an endosomal cation/proton exchanger, as a novel regulator of this system. Human brain microvascular endothelial cells (hBMVECs) that constitute the BBB receive brain iron status information via paracrine signals from ensheathing astrocytes. In hBMVECs, we show that NHE9 expression is up-regulated very early in a physiological response invoked by paracrine signals from iron-starved astrocytes. Ectopic expression of NHE9 in hBMVECs without external cues induced up-regulation of the transferrin receptor (TfR) and down-regulation of ferritin, leading to an increase in iron uptake. Mechanistically, we demonstrate that NHE9 localizes to recycling endosomes in hBMVECs where it raises the endosomal pH. The ensuing alkalization of the endosomal lumen increased translocation of TfRs to the hBMVEC membrane. TfRs on the membrane were previously shown to facilitate both recycling-dependent and -independent iron uptake. We propose that NHE9 regulates TfR-dependent, recycling-independent iron uptake in hBMVECs by fine-tuning the endosomal pH in response to paracrine signals and is therefore an important regulator in iron mobilization pathway at the BBB. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol

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

Kadam, Rameshwar U.; Wilson, Ian A.

The broad-spectrum antiviral drug Arbidol shows efficacy against influenza viruses by targeting the hemagglutinin (HA) fusion machinery. However, the structural basis of the mechanism underlying fusion inhibition by Arbidol has remained obscure, thereby hindering its further development as a specific and optimized influenza therapeutic. We determined crystal structures of Arbidol in complex with influenza virus HA from pandemic 1968 H3N2 and recent 2013 H7N9 viruses. Arbidol binds in a hydrophobic cavity in the HA trimer stem at the interface between two protomers. This cavity is distal to the conserved epitope targeted by broadly neutralizing stem antibodies and is ~16 Åmore » from the fusion peptide. Arbidol primarily makes hydrophobic interactions with the binding site but also induces some conformational rearrangements to form a network of inter- and intraprotomer salt bridges. By functioning as molecular glue, Arbidol stabilizes the prefusion conformation of HA that inhibits the large conformational rearrangements associated with membrane fusion in the low pH of the endosome. This unique binding mode compared with the small-molecule inhibitors of other class I fusion proteins enhances our understanding of how small molecules can function as fusion inhibitors and guides the development of broad-spectrum therapeutics against influenza virus.« less

Caspase-Activated Cell-Penetrating Peptides Reveal Temporal Coupling Between Endosomal Release and Apoptosis in an RGC-5 Cell Model

PubMed Central

Johnson, James R.; Kocher, Brandon; Barnett, Edward M.; Marasa, Jayne; Piwnica-Worms, David

2012-01-01

Caspase-activatable cell-penetrating peptide (CPP) probes, designed for efficient cell uptake and specificity via cleavable intramolecular quenched-fluorophore strategies, show promise for identifying and imaging retinal ganglion cell apoptosis in vivo. However, initial cell uptake and trafficking events cannot be visualized because the probes are designed to be optically quenched in the intact state. To visualize subcellular activation events in real-time during apoptosis, a new series of matched quenched and non-quenched CPP probes were synthesized. In both native and staurosporine-differentiated RGC-5 cells, probe uptake was time- and concentration-dependent through clathrine-, caveolin- and pinocytosis-mediated endocytic mechanisms. During apoptosis, KcapTR488, a novel dual fluorophore CPP probe, revealed by multi-spectral imaging a temporal coupling of endosomal release and effector caspase activation in RGC-5 cells. The novel CPPs described herein provide new tools to study spatial and temporal regulation of endosomal permeability during apoptosis. PMID:22900707

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

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

Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our datamore » strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.« less

Specific regulation of thermosensitive lipid droplet fusion by a nuclear hormone receptor pathway

PubMed Central

Li, Shiwei; Li, Qi; Kong, Yuanyuan; Wu, Shuang; Cui, Qingpo; Zhang, Mingming; Zhang, Shaobing O.

2017-01-01

Nuclear receptors play important roles in regulating fat metabolism and energy production in humans. The regulatory functions and endogenous ligands of many nuclear receptors are still unidentified, however. Here, we report that CYP-37A1 (ortholog of human cytochrome P450 CYP4V2), EMB-8 (ortholog of human P450 oxidoreductase POR), and DAF-12 (homolog of human nuclear receptors VDR/LXR) constitute a hormone synthesis and nuclear receptor pathway in Caenorhabditis elegans. This pathway specifically regulates the thermosensitive fusion of fat-storing lipid droplets. CYP-37A1, together with EMB-8, synthesizes a lipophilic hormone not identical to Δ7-dafachronic acid, which represses the fusion-promoting function of DAF-12. CYP-37A1 also negatively regulates thermotolerance and lifespan at high temperature in a DAF-12–dependent manner. Human CYP4V2 can substitute for CYP-37A1 in C. elegans. This finding suggests the existence of a conserved CYP4V2-POR–nuclear receptor pathway that functions in converting multilocular lipid droplets to unilocular ones in human cells; misregulation of this pathway may lead to pathogenic fat storage. PMID:28760992

Specific regulation of thermosensitive lipid droplet fusion by a nuclear hormone receptor pathway.

PubMed

Li, Shiwei; Li, Qi; Kong, Yuanyuan; Wu, Shuang; Cui, Qingpo; Zhang, Mingming; Zhang, Shaobing O

2017-08-15

Nuclear receptors play important roles in regulating fat metabolism and energy production in humans. The regulatory functions and endogenous ligands of many nuclear receptors are still unidentified, however. Here, we report that CYP-37A1 (ortholog of human cytochrome P450 CYP4V2), EMB-8 (ortholog of human P450 oxidoreductase POR), and DAF-12 (homolog of human nuclear receptors VDR/LXR) constitute a hormone synthesis and nuclear receptor pathway in Caenorhabditis elegans This pathway specifically regulates the thermosensitive fusion of fat-storing lipid droplets. CYP-37A1, together with EMB-8, synthesizes a lipophilic hormone not identical to Δ7-dafachronic acid, which represses the fusion-promoting function of DAF-12. CYP-37A1 also negatively regulates thermotolerance and lifespan at high temperature in a DAF-12-dependent manner. Human CYP4V2 can substitute for CYP-37A1 in C. elegans This finding suggests the existence of a conserved CYP4V2-POR-nuclear receptor pathway that functions in converting multilocular lipid droplets to unilocular ones in human cells; misregulation of this pathway may lead to pathogenic fat storage.

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2.

PubMed

Oninla, Vincent O; Breiden, Bernadette; Babalola, Jonathan O; Sandhoff, Konrad

2014-12-01

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747-1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

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

Cystatin F as a regulator of immune cell cytotoxicity.

PubMed

Kos, Janko; Nanut, Milica Perišić; Prunk, Mateja; Sabotič, Jerica; Dautović, Esmeralda; Jewett, Anahid

2018-05-10

Cysteine cathepsins are lysosomal peptidases involved in the regulation of innate and adaptive immune responses. Among the diverse processes, regulation of granule-dependent cytotoxicity of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells during cancer progression has recently gained significant attention. The function of cysteine cathepsins is regulated by endogenous cysteine protease inhibitors-cystatins. Whereas other cystatins are generally cytosolic or extracellular proteins, cystatin F is present in endosomes and lysosomes and is thus able to regulate the activity of its target directly. It is delivered to endosomal/lysosomal vesicles as an inactive, disulphide-linked dimer. Proteolytic cleavage of its N-terminal part leads to the monomer, the only form that is a potent inhibitor of cathepsins C, H and L, involved in the activation of granzymes and perforin. In NK cells and CTLs the levels of active cathepsin C and of granzyme B are dependent on the concentration of monomeric, active cystatin F. In tumour microenvironment, inactive dimeric cystatin F can be secreted from tumour cells or immune cells and further taken up by the cytotoxic cells. Subsequent monomerization and inhibition of cysteine cathepsins within the endosomal/lysosomal vesicles impairs granzyme and perforin activation, and provokes cell anergy. Further, the glycosylation pattern has been shown to be important in controlling secretion of cystatin F from target cells, as well as internalization by cytotoxic cells and trafficking to endosomal/lysosomal vesicles. Cystatin F is therefore an important mediator used by bystander cells to reduce NK and T-cell cytotoxicity.

Pax2 regulates a fadd-dependent molecular switch that drives tissue fusion during eye development.

PubMed

Viringipurampeer, Ishaq A; Ferreira, Todd; DeMaria, Shannon; Yoon, Jookyung J; Shan, Xianghong; Moosajee, Mariya; Gregory-Evans, Kevin; Ngai, John; Gregory-Evans, Cheryl Y

2012-05-15

Tissue fusion is an essential morphogenetic mechanism in development, playing a fundamental role in developing neural tube, palate and the optic fissure. Disruption of genes associated with the tissue fusion can lead to congenital malformations, such as spina bifida, cleft lip/palate and ocular coloboma. For instance, the Pax2 transcription factor is required for optic fissure closure, although the mechanism of Pax2 action leading to tissue fusion remains elusive. This lack of information defining how transcription factors drive tissue morphogenesis at the cellular level is hampering new treatments options. Through loss- and gain-of-function analysis, we now establish that pax2 in combination with vax2 directly regulate the fas-associated death domain (fadd) gene. In the presence of fadd, cell proliferation is restricted in the developing eye through a caspase-dependent pathway. However, the loss of fadd results in a proliferation defect and concomitant activation of the necroptosis pathway through RIP1/RIP3 activity, leading to an abnormal open fissure. Inhibition of RIP1 with the small molecule drug necrostatin-1 rescues the pax2 eye fusion defect, thereby overcoming the underlying genetic defect. Thus, fadd has an essential physiological function in protecting the developing optic fissure neuroepithelium from RIP3-dependent necroptosis. This study demonstrates the molecular hierarchies that regulate a cellular switch between proliferation and the apoptotic and necroptotic cell death pathways, which in combination drive tissue morphogenesis. Furthermore, our data suggest that future therapeutic strategies may be based on small molecule drugs that can bypass the gene defects causing common congenital tissue fusion defects.

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

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

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

CED-10/Rac1 Regulates Endocytic Recycling through the RAB-5 GAP TBC-2

PubMed Central

Sun, Lin; Liu, Ou; Desai, Jigar; Karbassi, Farhad; Sylvain, Marc-André; Shi, Anbing; Zhou, Zheng; Rocheleau, Christian E.; Grant, Barth D.

2012-01-01

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane. PMID:22807685

FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification in Arabidopsis Seed Mucilage[W

PubMed Central

Voiniciuc, Cătălin; Dean, Gillian H.; Griffiths, Jonathan S.; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L.; Estelle, Mark; Haughn, George W.

2013-01-01

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca2+ ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca2+ or completely rescued using alkaline Ca2+ chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1–yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells. PMID:23482858

Small Molecules for Early Endosome-Specific Patch Clamping.

PubMed

Chen, Cheng-Chang; Butz, Elisabeth S; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian

2017-07-20

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

PubMed

Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

2016-02-01

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH.

PubMed

Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

2015-04-24

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH*

PubMed Central

Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S.; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

2015-01-01

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. PMID:25673693

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.

Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells.

PubMed

Millet, Jean Kaoru; Whittaker, Gary R

2018-04-01

During viral entry, enveloped viruses require the fusion of their lipid envelope with host cell membranes. For coronaviruses, this critical step is governed by the virally-encoded spike (S) protein, a class I viral fusion protein that has several unique features. Coronavirus entry is unusual in that it is often biphasic in nature, and can occur at or near the cell surface or in late endosomes. Recent advances in structural, biochemical and molecular biology of the coronavirus S protein has shed light on the intricacies of coronavirus entry, in particular the molecular triggers of coronavirus S-mediated membrane fusion. Furthermore, characterization of the coronavirus fusion peptide (FP), the segment of the fusion protein that inserts to a target lipid bilayer during membrane fusion, has revealed its particular attributes which imparts some of the unusual properties of the S protein, such as Ca 2+ -dependency. These unusual characteristics can explain at least in part the biphasic nature of coronavirus entry. In this review, using severe acute respiratory syndrome coronavirus (SARS-CoV) as model virus, we give an overview of advances in research on the coronavirus fusion peptide with an emphasis on its role and properties within the biological context of host cell entry. Copyright © 2017 Elsevier Inc. All rights reserved.

Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability.

PubMed

Hardwick, Jean C; Clason, Todd A; Tompkins, John D; Girard, Beatrice M; Baran, Caitlin N; Merriam, Laura A; May, Victor; Parsons, Rodney L

2017-08-01

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels ( I h ) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability. Copyright © 2017 the American Physiological Society.

The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

PubMed Central

Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J.; Baldari, Cosima T.

2015-01-01

ABSTRACT IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11+ endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR+ endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis. PMID:26034069

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.

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

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

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

Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru; Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru; St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time ofmore » slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.« less

Unconventional secretion of FABP4 by endosomes and secretory lysosomes.

PubMed

Villeneuve, Julien; Bassaganyas, Laia; Lepreux, Sebastien; Chiritoiu, Marioara; Costet, Pierre; Ripoche, Jean; Malhotra, Vivek; Schekman, Randy

2018-02-05

An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion. © 2018 Villeneuve et al.

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

A sorting nexin 17-binding domain within the LRP1 cytoplasmic tail mediates receptor recycling through the basolateral sorting endosome

PubMed Central

Farfán, Pamela; Lee, Jiyeon; Larios, Jorge; Sotelo, Pablo; Bu, Guojun; Marzolo, María-Paz

2013-01-01

Sorting nexin 17 (SNX17) is an adaptor protein present in 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 MDCK 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. PMID:23593972

AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

PubMed Central

Glyvuk, Nataliya; Tsytsyura, Yaroslav; Geumann, Constanze; D'Hooge, Rudi; Hüve, Jana; Kratzke, Manuel; Baltes, Jennifer; Böning, Daniel; Klingauf, Jürgen; Schu, Peter

2010-01-01

Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation. PMID:20203623

Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein

PubMed Central

Sato, Miyuki; Sato, Ken; Liou, Willisa; Pant, Saumya; Harada, Akihiro; Grant, Barth D

2008-01-01

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP–RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP–RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling. PMID:18354496

Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses.

PubMed

Chen, Hui-Wen; Cheng, Jenna Xiao; Liu, Ming-Tsan; King, Kevin; Peng, Ju-Yi; Zhang, Xin-Quan; Wang, Ching-Ho; Shresta, Sujan; Schooley, Robert T; Liu, Yu-Tsueng

2013-09-01

An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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

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

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

Ca2+ homeostasis in microvascular endothelial cells from an insulin-dependent diabetic model: role of endosomes/lysosomes

NASA Astrophysics Data System (ADS)

Sanka, Shankar C.; Bennett, David C.; Rojas, Jose D.; Tasby, Geraldine B.; Meininger, Cynthia J.; Wu, Guoyao; Wesson, Donald E.; Pfarr, Curtis M.; Martinez-Zaguilan, Raul

2000-04-01

Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

TPC Proteins Are Phosphoinositide-activated Sodium-selective Ion Channels in Endosomes and Lysosomes

PubMed Central

Wang, Xiang; Zhang, Xiaoli; Dong, Xian-ping; Samie, Mohammad; Li, Xinran; Cheng, Xiping; Goschka, Andrew; Shen, Dongbiao; Zhou, Yandong; Harlow, Janice; Zhu, Michael X.; Clapham, David E.; Ren, Dejian; Xu, Haoxing

2012-01-01

Summary Mammalian Two-Pore Channels (TPC1, 2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P2, and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na+, not K+, as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes, and may explain the specificity of PI(3,5)P2 in regulating the fusogenic potential of intracellular organelles. PMID:23063126

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.

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

PubMed

Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

2003-08-01

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

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

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.

Role of cytoskeleton in regulating fusion of nucleoli: a study using the activated mouse oocyte model.

PubMed

Lian, Hua-Yu; Jiao, Guang-Zhong; Wang, Hui-Li; Tan, Xiu-Wen; Wang, Tian-Yang; Zheng, Liang-Liang; Kong, Qiao-Qiao; Tan, Jing-He

2014-09-01

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²⁺-free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF. © 2014 by the Society for the Study of Reproduction, Inc.

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.

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

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.

The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection

PubMed Central

Arvin, Ann M.; Oliver, Stefan L.

2016-01-01

ABSTRACT The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (−1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a −0.5-log10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt. IMPORTANCE Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

PubMed

Heyward, Catherine A; Pettitt, Trevor R; Leney, Sophie E; Welsh, Gavin I; Tavaré, Jeremy M; Wakelam, Michael J O

2008-05-20

Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

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

Regulation of endocytosis via the oxygen-sensing pathway.

PubMed

Wang, Yi; Roche, Olga; Yan, Mathew S; Finak, Greg; Evans, Andrew J; Metcalf, Julie L; Hast, Bridgid E; Hanna, Sara C; Wondergem, Bill; Furge, Kyle A; Irwin, Meredith S; Kim, William Y; Teh, Bin T; Grinstein, Sergio; Park, Morag; Marsden, Philip A; Ohh, Michael

2009-03-01

Tumor hypoxia is associated with disease progression, resistance to conventional cancer therapies and poor prognosis. Hypoxia, by largely unknown mechanisms, leads to deregulated accumulation of and signaling via receptor tyrosine kinases (RTKs) that are critical for driving oncogenesis. Here, we show that hypoxia or loss of von Hippel-Lindau protein--the principal negative regulator of hypoxia-inducible factor (HIF)--prolongs the activation of epidermal growth factor receptor that is attributable to lengthened receptor half-life and retention in the endocytic pathway. The deceleration in endocytosis is due to the attenuation of Rab5-mediated early endosome fusion via HIF-dependent downregulation of a critical Rab5 effector, rabaptin-5, at the level of transcription. Primary kidney and breast tumors with strong hypoxic signatures show significantly lower expression of rabaptin-5 RNA and protein. These findings reveal a general role of the oxygen-sensing pathway in endocytosis and support a model in which tumor hypoxia or oncogenic activation of HIF prolongs RTK-mediated signaling by delaying endocytosis-mediated deactivation of receptors.

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

PubMed

Cook, Jonathan D; Soto-Montoya, Hazel; Korpela, Markus K; Lee, Jeffrey E

2015-07-24

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation*

PubMed Central

Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob; Gether, Ulrik; Jørgensen, Trine Nygaard

2014-01-01

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation. PMID:24973209

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

LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

PubMed Central

Bennett, Terry L.; Kraft, Shannon M.; Reaves, Barbara J.; Mima, Joji; O’Brien, Kevin M.; Starai, Vincent J.

2013-01-01

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis. PMID:23437241

Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by down-regulating PKA and CREB activation

PubMed Central

Saryu Malhotra, Sudha; Suman, Pankaj; Kumar Gupta, Satish

2015-01-01

The aim of the present study is to delineate the role of human chorionic gonadotropin (hCG) in trophoblast fusion. In this direction, using shRNA lentiviral particles, α- and β-hCG silenced ‘BeWo’ cell lines were generated. Treatment of both α- and β-hCG silenced BeWo cells with either forskolin or exogenous hCG showed a significant reduction in cell fusion as compared with control shRNA treated cells. Studies by qRT-PCR, Western blotting and immunofluorescence revealed down-regulation of fusion-associated proteins such as syncytin-1 and syndecan-1 in the α- and β-hCG silenced cells. Delineation of downstream signaling pathways revealed that phosphorylation of PKA and CREB were compromised in the silenced cells whereas, no significant changes in p38MAPK and ERK1/2 phosphorylation were observed. Moreover, β-catenin activation was unaffected by either α- or β-hCG silencing. Further, inhibition of PKA by H89 inhibitor led to a significant decrease in BeWo cell fusion but had no effect on β-catenin activation suggesting the absence of non-canonical β-catenin stabilization via PKA. Interestingly, canonical activation of β-catenin was associated with the up-regulation of Wnt 10b expression. In summary, this study establishes the significance of hCG in the fusion of trophoblastic BeWo cells, but there may be additional factors involved in this process. PMID:26053549

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

PubMed

Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

2016-07-01

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

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

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

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

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

Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers.

PubMed

Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

2016-07-01

The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

PubMed Central

Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

2016-01-01

The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments. PMID:27376327

Spatiotemporal regulation of cAMP signaling controls the human trophoblast fusion

PubMed Central

Gerbaud, Pascale; Taskén, Kjetil; Pidoux, Guillaume

2015-01-01

During human placentation, mononuclear cytotrophoblasts fuse to form multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. Syncytial formation is essential for the maintenance of pregnancy and for fetal growth. The cAMP signaling pathway is the major route to trigger trophoblast fusion and its activation results in phosphorylation of specific intracellular target proteins, in transcription of fusogenic genes and assembly of macromolecular protein complexes constituting the fusogenic machinery at the plasma membrane. Specificity in cAMP signaling is ensured by generation of localized pools of cAMP controlled by cAMP phosphodiesterases (PDEs) and by discrete spatial and temporal activation of protein kinase A (PKA) in supramolecular signaling clusters inside the cell organized by A-kinase-anchoring proteins (AKAPs) and by organization of signal termination by protein phosphatases (PPs). Here we present original observations on the available components of the cAMP signaling pathway in the human placenta including PKA, PDE, and PP isoforms as well as AKAPs. We continue to discuss the current knowledge of the spatiotemporal regulation of cAMP signaling triggering trophoblast fusion. PMID:26441659

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

Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

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

Nayak, Vinod; Dessau, Moshe; Kucera, Kaury

Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flavivirusesmore » and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.« less

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.

Multivesicular bodies: co-ordinated progression to maturity

PubMed Central

Woodman, Philip G; Futter, Clare E

2008-01-01

Multivesicular endosomes/bodies (MVBs) sort endocytosed proteins to different destinations. Many lysosomally directed membrane proteins are sorted onto intralumenal vesicles, whilst recycling proteins remain on the perimeter membrane from where they are removed via tubular extensions. MVBs move to the cell centre during this maturation process and, when all recycling proteins have been removed, fuse with lysosomes. Recent advances have identified endosomal-sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways in intralumenal vesicle formation and mechanisms for sorting recycling cargo into tubules. Cytoskeletal motors, through interactions with these machineries and by regulating MVB movement, help to co-ordinate events leading to a mature, fusion-competent MVB. PMID:18502633

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

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

PubMed Central

Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

2015-01-01

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

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.

Trans-Golgi network/early endosome: a central sorting station for cargo proteins in plant immunity.

PubMed

LaMontagne, Erica D; Heese, Antje

2017-12-01

In plants, the trans-Golgi network (TGN) functionally overlaps with the early endosome (EE), serving as a central sorting hub to direct newly synthesized and endocytosed cargo to the cell surface or vacuole. Here, we focus on the emerging role of the TGN/EE in sorting of immune cargo proteins for effective plant immunity against pathogenic bacteria and fungi. Specific vesicle coat and regulatory components at the TGN/EE ensure that immune cargoes are correctly sorted and transported to the location of their cellular functions. Our understanding of the identity of immune cargoes and the underlying cellular mechanisms regulating their sorting are still rudimentary, but this knowledge is essential to understanding the physiological contribution of the TGN/EE to effective immune responses. Copyright © 2017. Published by Elsevier Ltd.

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

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.

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

PubMed

Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

2016-02-12

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

PubMed Central

Stith, Bradley J.

2015-01-01

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

PubMed Central

Connor, Michael G.; Pulsifer, Amanda R.; Ceresa, Brian K.

2018-01-01

ABSTRACT Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV. PMID:29463656

Cytomegalovirus immune evasion by perturbation of endosomal trafficking

PubMed Central

Lučin, Pero; Mahmutefendić, Hana; Blagojević Zagorac, Gordana; Ilić Tomaš, Maja

2015-01-01

Cytomegaloviruses (CMVs), members of the herpesvirus family, have evolved a variety of mechanisms to evade the immune response to survive in infected hosts and to establish latent infection. They effectively hide infected cells from the effector mechanisms of adaptive immunity by eliminating cellular proteins (major histocompatibility Class I and Class II molecules) from the cell surface that display viral antigens to CD8 and CD4 T lymphocytes. CMVs also successfully escape recognition and elimination of infected cells by natural killer (NK) cells, effector cells of innate immunity, either by mimicking NK cell inhibitory ligands or by downregulating NK cell-activating ligands. To accomplish these immunoevasion functions, CMVs encode several proteins that function in the biosynthetic pathway by inhibiting the assembly and trafficking of cellular proteins that participate in immune recognition and thereby, block their appearance at the cell surface. However, elimination of these proteins from the cell surface can also be achieved by perturbation of their endosomal route and subsequent relocation from the cell surface into intracellular compartments. Namely, the physiological route of every cellular protein, including immune recognition molecules, is characterized by specific features that determine its residence time at the cell surface. In this review, we summarize the current understanding of endocytic trafficking of immune recognition molecules and perturbations of the endosomal system during infection with CMVs and other members of the herpesvirus family that contribute to their immune evasion mechanisms. PMID:25263490

The longitudinal effects of emotion regulation on physical and psychological health: A latent growth analysis exploring the role of cognitive fusion in inflammatory bowel disease.

PubMed

Trindade, Inês A; Ferreira, Cláudia; Pinto-Gouveia, José

2018-02-01

This study thus aims to test differences between patients with inflammatory bowel disease (IBD) regarding IBD symptomatology, cognitive fusion, and psychological and physical health, as well as to explore whether the maladaptive emotion regulation process of cognitive fusion longitudinally impacts on the baseline and evolution of these outcomes over a period of 18 months. Participants include 116 IBD patients with a mean age of 36.76 (SD = 11.39) of both genders (69.83% females) that completed the self-report measures of interest in three different times, equally spaced 9 months apart, over a period of 18 months. Latent growth curve models were conducted using structural equation modelling to estimate the growth trajectory of the variables in study. Inflammatory bowel disease symptomatology and cognitive fusion's levels were negatively associated with psychological health and physical health's baseline levels. Furthermore, IBD symptomatology did not influence the growth of psychological health, while cognitive fusion did (β = .30, p = .007). The same result was found for physical health (β = .26, p = .024). These findings indicate that individuals with higher levels of cognitive fusion present lower levels of psychological health and physical health that tend to further decrease over the time through the effects of this maladaptive emotion regulation process. This study implies that it is of crucial importance to include psychotherapeutic interventions in the health care of patients with IBD. If successful, these interventions could represent decreases in the cost of IBD treatment and in the use of drugs with adverse side effects, in addition to improving patients' mental health and quality of life. Further implications for clinical and research work are discussed. Statement of contribution What is already known on this subject? Research has demonstrated the impact of emotion regulation on both physical and mental health. Nevertheless, the

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

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

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

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.

The Role of TSC2 Phosphorylation in the Regulation of TSC2 Localization and mTOR Signaling

DTIC Science & Technology

2015-05-01

lysosomal system. During the first year of this project we have successfully identified 6 phosphorylation sites on TSC2 that are regulated by...GAP, Insulin, LAMP2, Late Endosome, Lysosome , Mass Spectrometry, Mechanical, mTOR, p70S6k, Phosphorylation, Rheb, Transfection, TSC2, Tuberous...Electroporation, GAP, Insulin, LAMP2, Late Endosome, Lysosome , Mass Spectrometry, Mechanical, mTOR, p70S6k, Phosphorylation, Rheb, Transfection, TSC2

Local electrostatic interactions determine the diameter of fusion pores

PubMed Central

Guček, Alenka; Jorgačevski, Jernej; Górska, Urszula; Rituper, Boštjan; Kreft, Marko; Zorec, Robert

2015-01-01

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition. PMID:25835258

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

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

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton

2010-08-15

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmicmore » tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.« less

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

PubMed Central

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Dutch, Rebecca Ellis; McCann, Richard O.

2010-01-01

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger. PMID:20537366

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

PubMed

Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

PubMed

Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

2018-03-01

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

Analysis of human soft palate morphogenesis supports regional regulation of palatal fusion

PubMed Central

Danescu, Adrian; Mattson, Melanie; Dool, Carly; Diewert, Virginia M; Richman, Joy M

2015-01-01

It is essential to complete palate closure at the correct time during fetal development, otherwise a serious malformation, cleft palate, will ensue. The steps in palate formation in humans take place between the 7th and 12th week and consist of outgrowth of palatal shelves from the paired maxillary prominences, reorientation of the shelves from vertical to horizontal, apposition of the medial surfaces, formation of a bilayered seam, degradation of the seam and bridging of mesenchyme. However, in the soft palate, the mechanism of closure is unclear. In previous studies it is possible to find support for both fusion and the alternative mechanism of merging. Here we densely sample the late embryonic-early fetal period between 54 and 74 days post-conception to determine the timing and mechanism of soft palate closure. We found the epithelial seam extends throughout the soft palates of 57-day specimens. Cytokeratin antibody staining detected the medial edge epithelium and distinguished clearly that cells in the midline retained their epithelial character. Compared with the hard palate, the epithelium is more rapidly degraded in the soft palate and only persists in the most posterior regions at 64 days. Our results are consistent with the soft palate following a developmentally more rapid program of fusion than the hard palate. Importantly, the two regions of the palate appear to be independently regulated and have their own internal clocks regulating the timing of seam removal. Considering data from human genetic and mouse studies, distinct anterior-posterior signaling mechanisms are likely to be at play in the human fetal palate. PMID:26299693

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

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

Neurolastin, a dynamin family GTPase, regulates excitatory synapses and spine density

PubMed Central

Madan Lomash, Richa; Gu, Xinglong; Youle, Richard J.; Lu, Wei; Roche, Katherine W.

2015-01-01

SUMMARY Membrane trafficking and spinogenesis contribute significantly to changes in synaptic strength during development and in various paradigms of synaptic plasticity. GTPases of the dynamin family are key players regulating membrane trafficking. Here, we identify a brain-specific dynamin family GTPase, neurolastin (RNF112/Znf179), with closest homology to atlastin. We demonstrate that neurolastin has functional GTPase and RING domains, making it a unique protein identified with this multi-enzymatic domain organization. We also show that neurolastin is a peripheral membrane protein, which localizes to endosomes and affects endosomal membrane dynamics via its RING domain. In addition, neurolastin knockout mice have fewer dendritic spines, and rescue of the wildtype phenotype requires both the GTPase and RING domains. Furthermore, we find fewer functional synapses and reduced paired pulse facilitation in neurolastin knockout mice. Thus, we identify neurolastin as a dynamin family GTPase that affects endosome size and spine density. PMID:26212327

Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

PubMed Central

Mayor, S; Sabharanjak, S; Maxfield, F R

1998-01-01

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins. PMID:9707422

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.

Crystal Structure of Dengue Type 1 Envelope Protein in the Postfusion Conformation and its Implication for Receptor Binding, Membrane Fusion and Antibody Recognition

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

Nayak, V.; Dessau, M; Kucera, K

Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flavivirusesmore » and are part of the pH sensor that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.« less

Lateral Membrane Heterogeneity Regulates Viral-Induced Membrane Fusion during HIV Entry

PubMed Central

Molotkovsky, Rodion J.; Alexandrova, Veronika V.; Galimzyanov, Timur R.; Jiménez-Munguía, Irene; Pavlov, Konstantin V.; Akimov, Sergey A.

2018-01-01

Sphingomyelin- and cholesterol- enriched membrane domains, commonly referred to as “rafts” play a crucial role in a large number of intra- and intercellular processes. Recent experiments suggest that not only the volumetric inhomogeneity of lipid distribution in rafts, but also the arrangement of the 1D boundary between the raft and the surrounding membrane is important for the membrane-associated processes. The reason is that the boundary preferentially recruits different peptides, such as HIV (human immunodeficiency virus) fusion peptide. In the present work, we report a theoretical investigation of mechanisms of influence of the raft boundary arrangement upon virus-induced membrane fusion. We theoretically predict that the raft boundary can act as an attractor for viral fusion peptides, which preferentially distribute into the vicinity of the boundary, playing the role of ‘line active components’ of the membrane (‘linactants’). We have calculated the height of the fusion energy barrier and demonstrated that, in the case of fusion between HIV membrane and the target cell, presence of the raft boundary in the vicinity of the fusion site facilitates fusion. The results we obtained can be further generalized to be applicable to other enveloped viruses. PMID:29772704

Lateral Membrane Heterogeneity Regulates Viral-Induced Membrane Fusion during HIV Entry.

PubMed

Molotkovsky, Rodion J; Alexandrova, Veronika V; Galimzyanov, Timur R; Jiménez-Munguía, Irene; Pavlov, Konstantin V; Batishchev, Oleg V; Akimov, Sergey A

2018-05-16

Sphingomyelin- and cholesterol- enriched membrane domains, commonly referred to as "rafts" play a crucial role in a large number of intra- and intercellular processes. Recent experiments suggest that not only the volumetric inhomogeneity of lipid distribution in rafts, but also the arrangement of the 1D boundary between the raft and the surrounding membrane is important for the membrane-associated processes. The reason is that the boundary preferentially recruits different peptides, such as HIV (human immunodeficiency virus) fusion peptide. In the present work, we report a theoretical investigation of mechanisms of influence of the raft boundary arrangement upon virus-induced membrane fusion. We theoretically predict that the raft boundary can act as an attractor for viral fusion peptides, which preferentially distribute into the vicinity of the boundary, playing the role of 'line active components' of the membrane ('linactants'). We have calculated the height of the fusion energy barrier and demonstrated that, in the case of fusion between HIV membrane and the target cell, presence of the raft boundary in the vicinity of the fusion site facilitates fusion. The results we obtained can be further generalized to be applicable to other enveloped viruses.

Nanoparticle delivery of miR-223 to attenuate macrophage fusion

PubMed Central

Moore, Laura Beth; Sawyer, Andrew J.; Saucier-Sawyer, Jennifer; Saltzman, W. Mark; Kyriakides, Themis R.

2016-01-01

The foreign body response (FBR) begins with injury acquired during implantation of a biomaterial (BM) and is detrimental due to the eventual encapsulation of the implant. Fusion of macrophages to form foreign body giant cells (FBGC), a hallmark of the FBR, is the consequence of a multistep mechanism induced by interleukin (IL)-4 that includes the acquisition of a fusion competent state and subsequent cytoskeletal rearrangements. However, the precise mechanism, regulation, and interplay among molecular mediators to generate FBGCs are insufficiently understood. Seeking novel mediators of fusion that might be regulated at the post-transcriptional level, we examined the role of microRNAs (miRs) in this process. A miR microarray was screened and identified miR-223 as a negative regulator of macrophage fusion. In addition, transfection of primary macrophages with a mir-223 mimic attenuated IL-4-induced fusion. Furthermore, miR-223 KO mice and mir-223 deficient cells displayed increased fusion in vivo and in vitro, respectively. Finally, we developed a method for in vivo delivery of miR-223 mimic utilizing PLGA nanoparticles, which inhibited FBGC formation in a biomaterial implant model. Our results identify miR-223 as a negative regulator of fusion and demonstrate miR-223 mimic-loaded nanoparticles as a therapeutic inhibitor of macrophage fusion. PMID:26967647

Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay

PubMed Central

Stockinger, Walter; Castoreno, Adam B.; Wang, Yan; Pagnon, Joanne C.; Nohturfft, Axel

2007-01-01

A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells. Mouse macrophages were cultured in the presence of tritiated cholesterol and scintillant microspheres. Microspheres were taken up by phagocytosis and stored in phagolysosomes. Absorption of tritium β particles by the scintillant produces light signals that can be measured in standard scintillation counters. Because of the short range of tritium β particles and for geometric reasons, scintillant microspheres detect only that fraction of tritiated cholesterol localized inside phagolysosomes or within a distance of ~600 nm. By incubating cultures in a temperature-controlled microplate reader, the kinetics of phagocytosis and cholesterol transport could be analyzed in near-real time. Scintillation signals were significantly increased in response to inhibitors of lysosomal cholesterol export. This method should prove a useful new tool for the study of endosomal trafficking of lipids and other molecules. PMID:15314094

Components of the SNARE-containing regulon are co-regulated in root cells undergoing defense

PubMed Central

Klink, Vincent P.; Sharma, Keshav; Pant, Shankar R.; McNeece, Brant; Niraula, Prakash; Lawrence, Gary W.

2017-01-01

ABSTRACT The term regulon has been coined in the genetic model plant Arabidopsis thaliana, denoting a structural and physiological defense apparatus defined genetically through the identification of the penetration (pen) mutants. The regulon is composed partially by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) syntaxin PEN1. PEN1 has homology to a Saccharomyces cerevisae gene that regulates a Secretion (Sec) protein, Suppressor of Sec 1 (Sso1p). The regulon is also composed of the β-glucosidase (PEN2) and an ATP binding cassette (ABC) transporter (PEN3). While important in inhibiting pathogen infection, limited observations have been made regarding the transcriptional regulation of regulon genes until now. Experiments made using the model agricultural Glycine max (soybean) have identified co-regulated gene expression of regulon components. The results explain the observation of hundreds of genes expressed specifically in the root cells undergoing the natural process of defense. Data regarding additional G. max genes functioning within the context of the regulon are presented here, including Sec 14, Sec 4 and Sec 23. Other examined G. max homologs of membrane fusion genes include an endosomal bromo domain-containing protein1 (Bro1), syntaxin6 (SYP6), SYP131, SYP71, SYP8, Bet1, coatomer epsilon (ε-COP), a coatomer zeta (ζ-COP) paralog and an ER to Golgi component (ERGIC) protein. Furthermore, the effectiveness of biochemical pathways that would function within the context of the regulon ave been examined, including xyloglucan xylosyltransferase (XXT), reticuline oxidase (RO) and galactinol synthase (GS). The experiments have unveiled the importance of the regulon during defense in the root and show how the deposition of callose relates to the process. PMID:28010187

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

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

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.

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

Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

PubMed Central

Verma, Jitender Kumar; Rastogi, Ruchir

2017-01-01

Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

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

Snx3 regulates recycling of the transferrin receptor and iron assimilation

PubMed Central

Chen, Caiyong; Garcia-Santos, Daniel; Ishikawa, Yuichi; Seguin, Alexandra; Li, Liangtao; Fegan, Katherine H.; Hildick-Smith, Gordon J.; Shah, Dhvanit I.; Cooney, Jeffrey D.; Chen, Wen; King, Matthew J.; Yien, Yvette Y.; Schultz, Iman J.; Anderson, Heidi; Dalton, Arthur J.; Freedman, Matthew L.; Kingsley, Paul D.; Palis, James; Hattangadi, Shilpa M.; Lodish, Harvey F.; Ward, Diane M.; Kaplan, Jerry; Maeda, Takahiro; Ponka, Prem; Paw, Barry H.

2013-01-01

SUMMARY Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc), and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism. PMID:23416069

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

Suppressor of fusion, a Fusarium oxysporum homolog of Ndt80, is required for nutrient-dependent regulation of anastomosis.

PubMed

Shahi, Shermineh; Fokkens, Like; Houterman, Petra M; Rep, Martijn

2016-10-01

Heterokaryon formation is an essential step in asexual recombination in Fusarium oxysporum. Filamentous fungi have an elaborate nonself recognition machinery to prevent formation and proliferation of heterokaryotic cells, called heterokaryon incompatibility (HI). In F. oxysporum the regulation of this machinery is not well understood. In Neurospora crassa, Vib-1, a putative transcription factor of the p53-like Ndt80 family of transcription factors, has been identified as global regulator of HI. In this study we investigated the role of the F. oxysporum homolog of Vib-1, called Suf, in vegetative hyphal and conidial anastomosis tube (CAT) fusion and HI. We identified a novel function for an Ndt80 homolog as a nutrient-dependent regulator of anastomosis. Strains carrying the SUF deletion mutation display a hyper-fusion phenotype during vegetative growth as well as germling development. In addition, conidial paring of incompatible SUF deletion strains led to more heterokaryon formation, which is independent of suppression of HI. Our data provides further proof for the divergence in the functions of different members Ndt80 family. We propose that Ndt80 homologs mediate responses to nutrient quality and quantity, with specific responses varying between species. Copyright © 2016 Elsevier Inc. All rights reserved.

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

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers

PubMed Central

Delevoye, Cédric; Romao, Maryse; Owen, David J.; Raposo, Graça

2016-01-01

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1–dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3–dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky–Pudlak syndrome variants. PMID:27482051

Phospholipase D function in Saccharomyces cerevisiae.

PubMed

Mendonsa, Rima; Engebrecht, JoAnne

2009-09-01

Phosphatidylinositol 4,5-bisphosphate-regulated phosphatidylcholine-specific phospholipase D is conserved from yeast to man. The essential role of this enzyme in yeast is to mediate the fusion of Golgi and endosome-derived vesicles to generate the prospore membrane during the developmental program of sporulation, through the production of the fusogenic lipid phosphatidic acid. In addition to recruiting proteins required for fusion, phosphatidic acid is believed to lower the energy barrier to stimulate membrane curvature. During mitotic growth, phospholipase D activity is dispensable unless the major phosphatidylinositol/phosphatidylcholine transfer protein is absent; it also appears to play a nonessential role in the mating signal transduction pathway. The regulation of phospholipase D activity during both sporulation and mitotic growth is still not fully understood and awaits further characterization.

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

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome.

PubMed

Liu, Rong; Zhi, Xiaoyong; Zhong, Qing

2015-01-01

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.

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

Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling

PubMed Central

Skorobogata, Olga; Meng, Jassy; Gauthier, Kimberley; Rocheleau, Christian E.

2016-01-01

Epidermal growth factor receptor (EGFR) signaling is essential for animal development, and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help to elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus end–directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell and genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants, consistent with mammalian cell studies in which dynein is shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking. PMID:27654944

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.

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.

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

PubMed Central

Li, Chengwen; He, Yi; Nicolson, Sarah; Hirsch, Matt; Weinberg, Marc S.; Zhang, Ping; Kafri, Tal; Samulski, R. Jude

2013-01-01

Adeno-associated virus (AAV) vectors are attractive for gene delivery-based therapeutics, but data from recent clinical trials have indicated that AAV capsids induce a cytotoxic T lymphocyte (CTL) response that eliminates transduced cells. In this study, we used traditional pharmacological agents and AAV mutants to elucidate the pathway of capsid cross-presentation in AAV-permissive cells. Endosomal acidification inhibitors blocked AAV2 antigen presentation by over 90%, while proteasome inhibitors completely abrogated antigen presentation. Using mutant viruses that are defective for nuclear entry, we observed a 90% decrease in capsid antigen presentation. Different antigen presentation efficiencies were achieved by selectively mutating virion nuclear localization signals. Low antigen presentation was demonstrated with basic region 1 (BR1) mutants, despite relatively high transduction efficiency, whereas there was no difference in antigen presentation between BR2 and BR3 mutants defective for transduction, as compared with wild-type AAV2. These results suggest that effective AAV2 capsid antigen presentation is dependent on AAV virion escape from the endosome/lysosome for antigen degradation by proteasomes, but is independent of nuclear uncoating. These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials. PMID:23454772

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.

Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

PubMed

Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

1996-08-01

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

Glycoprotein interactions in paramyxovirus fusion

PubMed Central

Iorio, Ronald M; Melanson, Vanessa R; Mahon, Paul J

2009-01-01

The Paramyxoviridae are enveloped, negative-stranded RNA viruses, some of which recognize sialic acid-containing receptors, while others recognize specific proteinaceous receptors. The major cytopathic effect of paramyxovirus infection is membrane fusion-induced syncytium formation. Paramyxoviruses are unusual in that the receptor-binding and fusion-promoting activities reside on two different spike structures, the attachment and fusion glycoproteins, respectively. For most paramyxoviruses, this distribution of functions requires a mechanism by which the two processes can be linked for the promotion of fusion. This is accomplished by a virus-specific interaction between the two proteins. An increasing body of evidence supports the notion that members of this family of viruses utilize this glycoprotein interaction in different ways in order to mediate the regulation of the fusion protein activation, depending on the type of receptor utilized by the virus. PMID:20161127

Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis.

PubMed

Cookson, Emma A; Conte, Ianina L; Dempster, John; Hannah, Matthew J; Carter, Tom

2013-12-01

Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

Investigation of fusion gene expression in HCT116 cells.

PubMed

Zhang, Yanmei; Ren, Juan; Fang, Mengdie; Wang, Xiaoju

2017-12-01

Colon cancer is the most common type of gastrointestinal cancer. A number of specific and sensitive biomarkers facilitate the diagnosis and monitoring of patients with colon cancer. Fusion genes are typically identified in cancer and a majority of the newly identified fusion genes are oncogenic in nature. Therefore, fusion genes are potential biomarkers and/or therapy targets in cancer. In the present study, the regulation of specific candidate fusion genes were investigated using Brother of the Regulator of Imprinted Sites (BORIS) in the HCT116 colon cancer cell line, which is a paralog of the fusion gene regulator CCCTC-binding factor (CTCF). The copy number of BORIS increased correspondingly to the progression of colorectal carcinoma from the M0 to the M1a stage. It was identified that EIF3E(e1)-RSPO2(e2) , EIF3E(e1)-RSPO2(e3) , PTPRK(e1)-RSPO3(e2) , PTPRK(e7)-RSPO3(e2), TADA2A-MEF2B and MED13L-CD4 are fusion transcripts present in the transcriptome of the HCT116 colon cancer cell line. CDC42SE2-KIAAO146 is a genomic fusion transcript, which originates from DNA arrangement in HCT116 cells. BORIS suppresses the expression of EIF3E , RSPO2 , PTPRK , RSPO3 , TADA2A and CD4 to inhibit the expression of fusion transcripts in HCT116 cells. It was hypothesized that the fusion transcripts investigated in the present study may not be oncogenic in HCT116 cells. As BORIS is not colorectal carcinoma-specific, the fusion genes investigated may be a biomarker assemblage for monitoring the progression of colorectal carcinoma.

Investigation of fusion gene expression in HCT116 cells

PubMed Central

Zhang, Yanmei; Ren, Juan; Fang, Mengdie; Wang, Xiaoju

2017-01-01

Colon cancer is the most common type of gastrointestinal cancer. A number of specific and sensitive biomarkers facilitate the diagnosis and monitoring of patients with colon cancer. Fusion genes are typically identified in cancer and a majority of the newly identified fusion genes are oncogenic in nature. Therefore, fusion genes are potential biomarkers and/or therapy targets in cancer. In the present study, the regulation of specific candidate fusion genes were investigated using Brother of the Regulator of Imprinted Sites (BORIS) in the HCT116 colon cancer cell line, which is a paralog of the fusion gene regulator CCCTC-binding factor (CTCF). The copy number of BORIS increased correspondingly to the progression of colorectal carcinoma from the M0 to the M1a stage. It was identified that EIF3E(e1)-RSPO2(e2), EIF3E(e1)-RSPO2(e3), PTPRK(e1)-RSPO3(e2), PTPRK(e7)-RSPO3(e2), TADA2A-MEF2B and MED13L-CD4 are fusion transcripts present in the transcriptome of the HCT116 colon cancer cell line. CDC42SE2-KIAAO146 is a genomic fusion transcript, which originates from DNA arrangement in HCT116 cells. BORIS suppresses the expression of EIF3E, RSPO2, PTPRK, RSPO3, TADA2A and CD4 to inhibit the expression of fusion transcripts in HCT116 cells. It was hypothesized that the fusion transcripts investigated in the present study may not be oncogenic in HCT116 cells. As BORIS is not colorectal carcinoma-specific, the fusion genes investigated may be a biomarker assemblage for monitoring the progression of colorectal carcinoma. PMID:29181107

A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face

PubMed Central

Hu, Diane; Young, Nathan M.; Li, Xin; Xu, Yanhua; Hallgrímsson, Benedikt; Marcucio, Ralph S.

2015-01-01

The mechanisms of morphogenesis are not well understood, yet shaping structures during development is essential for establishing correct organismal form and function. Here, we examine mechanisms that help to shape the developing face during the crucial period of facial primordia fusion. This period of development is a time when the faces of amniote embryos exhibit the greatest degree of similarity, and it probably results from the necessity for fusion to occur to establish the primary palate. Our results show that hierarchical induction mechanisms, consisting of iterative signaling by Sonic hedgehog (SHH) followed by Bone morphogenetic proteins (BMPs), regulate a dynamic expression pattern of Shh in the ectoderm covering the frontonasal (FNP) and maxillary (MxP) processes. Furthermore, this Shh expression domain contributes to the morphogenetic processes that drive the directional growth of the globular process of the FNP toward the lateral nasal process and MxP, in part by regulating cell proliferation in the facial mesenchyme. The nature of the induction mechanism that we discovered suggests that the process of fusion of the facial primordia is intrinsically buffered against producing maladaptive morphologies, such as clefts of the primary palate, because there appears to be little opportunity for variation to occur during expansion of the Shh expression domain in the ectoderm of the facial primordia. Ultimately, these results might explain why this period of development constitutes a phylotypic stage of facial development among amniotes. PMID:25605783

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.

Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells.

PubMed

Robben, J H; Knoers, N V A M; Deen, P M T

2004-12-01

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.

REGULATED VESICULAR TRAFFICKING OF SPECIFIC PCDH15 AND VLGR1 VARIANTS IN AUDITORY HAIR CELLS

PubMed Central

Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T.; Cosgrove, Dominic

2012-01-01

Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal co-localization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular sub-pools. The apically trafficked pool co-localized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associates with membrane microdomains and SNAP25. Moreover, co-immunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions. PMID:23035094

Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics.

PubMed

Rambold, Angelika S; Cohen, Sarah; Lippincott-Schwartz, Jennifer

2015-03-23

Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibition of Na+−H+ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum

PubMed Central

Gekle, Michael; Drumm, Karina; Mildenberger, Sigrid; Freudinger, Ruth; Gaßner, Birgit; Silbernagl, Stefan

1999-01-01

Receptor-mediated endocytosis is an important mechanism for transport of macromolecules and regulation of cell-surface receptor expression. In renal proximal tubules, receptor-mediated endocytosis mediates the reabsorption of filtered albumin. Acidification of the endocytic compartments is essential because it interferes with ligand-receptor dissociation, vesicle trafficking, fusion events and coat formation. Here we show that the activity of Na+−H+ exchanger isoform 3 (NHE3) is important for proper receptor-mediated endocytosis of albumin and endosomal pH homeostasis in a renal proximal tubular cell line (opossum kidney cells) which expresses NHE3 only. Depending on their inhibitory potency with respect to NHE3 and their lipophilicity, the NHE inhibitors EIPA, amiloride and HOE694 differentially reduced albumin endocytosis. The hydrophilic inhibitor HOE642 had no effect. Inhibition of NHE3 led to an alkalinization of early endosomes and to an acidification of the cytoplasm, indicating that Na+−H+ exchange contributes to the acidification of the early endosomal compartment due to the existence of a sufficient Na+ gradient across the endosomal membrane. Exclusive acidification of the cytoplasm with propionic acid or by removal of Na+ induced a significantly smaller reduction in endocytosis than that induced by inhibition of Na+−H+ exchange. Analysis of the inhibitory profiles indicates that in early endosomes and endocytic vesicles NHE3 is of major importance, whereas plasma membrane NHE3 plays a minor role. Thus, NHE3-mediated acidification along the first part of the endocytic pathway plays an important role in receptor-mediated endocytosis. Furthermore, the involvement of NHE3 offers new ways to explain the regulation of receptor-mediated endocytosis. PMID:10545138

Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes

NASA Astrophysics Data System (ADS)

Yamada, Yuma; Kawamura, Eriko; Harashima, Hideyoshi

2012-08-01

Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.

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

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Endosome-mediated autophagy

PubMed Central

Kondylis, Vangelis; van Nispen tot Pannerden, Hezder E.; van Dijk, Suzanne; ten Broeke, Toine; Wubbolts, Richard; Geerts, Willie J.; Seinen, Cor; Mutis, Tuna; Heijnen, Harry F.G.

2013-01-01

Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4BC74A mutant and atg4b−/− bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival. PMID:23481895

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.

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments

PubMed Central

1985-01-01

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin. PMID:2866191

HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

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

Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir

HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 ismore » a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses

PubMed Central

Gillespie, Eugene J.; Ho, Chi-Lee C.; Balaji, Kavitha; Clemens, Daniel L.; Deng, Gang; Wang, Yao E.; Elsaesser, Heidi J.; Tamilselvam, Batcha; Gargi, Amandeep; Dixon, Shandee D.; France, Bryan; Chamberlain, Brian T.; Blanke, Steven R.; Cheng, Genhong; de la Torre, Juan Carlos; Brooks, David G.; Jung, Michael E.; Colicelli, John; Damoiseaux, Robert; Bradley, Kenneth A.

2013-01-01

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease. PMID:24191014

Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors

PubMed Central

Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

2014-01-01

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75NTR) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75NTR available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. PMID:24920579

Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

PubMed

Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

2014-07-17

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans.

PubMed

Larsen, Morten K; Tuck, Simon; Faergeman, Nils J; Knudsen, Jens

2006-10-01

The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydrolysis of acyl-CoA lipid esters. The mechanisms by which these lipid esters are directed to the appropriate membranes in vivo, and their precise roles in vesicle biogenesis, are not yet understood. Here, we present the first report on membrane associated ACBP domain-containing protein-1 (MAA-1), a novel membrane-associated member of the acyl-CoA-binding protein family. We show that in Caenorhabditis elegans, MAA-1 localizes to intracellular membrane organelles in the secretory and endocytic pathway and that mutations in maa-1 reduce the rate of endosomal recycling. A lack of maa-1 activity causes a change in endosomal morphology. Although in wild type, many endosomal organelles have long tubular protrusions, loss of MAA-1 activity results in loss of the tubular domains, suggesting the maa-1 is required for the generation or maintenance of these domains. Furthermore, we demonstrate that MAA-1 binds fatty acyl-CoA in vitro and that this ligand-binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA-dependent process during vesicle formation.

Quantitative Analysis of Lipid Droplet Fusion: Inefficient Steady State Fusion but Rapid Stimulation by Chemical Fusogens

PubMed Central

Murphy, Samantha; Martin, Sally; Parton, Robert G.

2010-01-01

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types. PMID:21203462

Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

PubMed Central

Mundy, Dorothy I.; Li, Wei Ping; Luby-Phelps, Katherine; Anderson, Richard G. W.

2012-01-01

Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking. PMID:22238363

Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

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

Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert

2012-09-17

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pHmore » 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.« less

Case Study: Organotypic human in vitro models of embryonic morphogenetic fusion

EPA Science Inventory

Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell...

The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

PubMed

Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

2014-06-27

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33AD251E mutation

PubMed Central

Zhen, Yuanli; Li, Wei

2015-01-01

The HOPS (homotypic fusion and protein sorting) complex functions in endocytic and autophagic pathways in both lower eukaryotes and mammalian cells through its involvement in fusion events between endosomes and lysosomes or autophagosomes and lysosomes. However, the differential molecular mechanisms underlying these fusion processes are largely unknown. Buff (bf) is a mouse mutant that carries an Asp251-to-Glu point mutation (D251E) in the VPS33A protein, a tethering protein and a core subunit of the HOPS complex. Bf mice showed impaired spontaneous locomotor activity, motor learning, and autophagic activity. Although the gross anatomy of the brain was apparently normal, the number of Purkinje cells was significantly reduced. Furthermore, we found that fusion between autophagosomes and lysosomes was defective in bf cells without compromising the endocytic pathway. The direct association of mutant VPS33AD251E with the autophagic SNARE complex, STX17 (syntaxin 17)-VAMP8-SNAP29, was enhanced. In addition, the VPS33AD251E mutation enhanced interactions with other HOPS subunits, namely VPS41, VPS39, VPS18, and VPS11, except for VPS16. Reduction of the interactions between VPS33AY440D and several other HOPS subunits led to decreased association with STX17. These results suggest that the VPS33AD251E mutation plays dual roles by increasing the HOPS complex assembly and its association with the autophagic SNARE complex, which selectively affects the autophagosome-lysosome fusion that impairs basal autophagic activity and induces Purkinje cell loss. PMID:26259518

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 Pearling Transition Provides Evidence of Force-Driven Endosomal Tubulation during Salmonella Infection.

PubMed

Gao, Yunfeng; Spahn, Christoph; Heilemann, Mike; Kenney, Linda J

2018-06-19

Bacterial pathogens exploit eukaryotic pathways for their own end. Upon ingestion, Salmonella enterica serovar Typhimurium passes through the stomach and then catalyzes its uptake across the intestinal epithelium. It survives and replicates in an acidic vacuole through the action of virulence factors secreted by a type three secretion system located on Salmonella pathogenicity island 2 (SPI-2). Two secreted effectors, SifA and SseJ, are sufficient for endosomal tubule formation, which modifies the vacuole and enables Salmonella to replicate within it. Two-color, superresolution imaging of the secreted virulence factor SseJ and tubulin revealed that SseJ formed clusters of conserved size at regular, periodic intervals in the host cytoplasm. Analysis of SseJ clustering indicated the presence of a pearling effect, which is a force-driven, osmotically sensitive process. The pearling transition is an instability driven by membranes under tension; it is induced by hypotonic or hypertonic buffer exchange and leads to the formation of beadlike structures of similar size and regular spacing. Reducing the osmolality of the fixation conditions using glutaraldehyde enabled visualization of continuous and intact tubules. Correlation analysis revealed that SseJ was colocalized with the motor protein kinesin. Tubulation of the endoplasmic reticulum is driven by microtubule motors, and in the present work, we describe how Salmonella has coopted the microtubule motor kinesin to drive the force-dependent process of endosomal tubulation. Thus, endosomal tubule formation is a force-driven process catalyzed by Salmonella virulence factors secreted into the host cytoplasm during infection. IMPORTANCE This study represents the first example of using two-color, superresolution imaging to analyze the secretion of Salmonella virulence factors as they are secreted from the SPI-2 type three secretion system. Previous studies imaged effectors that were overexpressed in the host cytoplasm. The

Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

PubMed Central

Beznoussenko, Galina V.; Pilyugin, Sergei S.; Geerts, Willie J. C.; Kozlov, Michael M.; Burger, Koert N. J.; Luini, Alberto; Derganc, Jure; Mironov, Alexander A.

2015-01-01

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant. PMID:25761238

In Vitro Reconstitution of Autophagosome-Lysosome Fusion.