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Sample records for actin regulatory protein

  1. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins.

    PubMed

    Xiao, Xiang; Mruk, Dolores D; Tang, Elizabeth I; Wong, Chris K C; Lee, Will M; John, Constance M; Turek, Paul J; Silvestrini, Bruno; Cheng, C Yan

    2014-06-01

    Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood-testis barrier (BTB)? Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. We examined the effects of two environmental toxicants: cadmium chloride (0.5-20 µM) and bisphenol A (0.4-200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by mislocalization of actin filament barbed end capping and bundling protein Eps8, and branched actin polymerization protein Arp3. Besides impeding actin dynamics

  2. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed

    Fujita, H; Ishiwata, S

    1998-09-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.

  3. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed Central

    Fujita, H; Ishiwata, S

    1998-01-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself. PMID:9726945

  4. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

  5. Actin binding proteins and spermiogenesis

    PubMed Central

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  6. Study of actin and its interactions with heavy meromyosin and the regulatory proteins by the pulse fluorimetry in polarized light of a fluorescent probe attached to an actin cysteine.

    PubMed

    Tawada, K; Wahl, P; Auchet, J C

    1978-08-01

    The decay of anisotropy of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine fluorescence attached to cysteine-373 of actin can be characterized by two correlation times theta1 and theta2. theta1 has a value of several nanoseconds and is thought to represent some local protein motion. theta2 is of the order of several hundreds of nanoseconds. Its value increases with actin concentration. It represents an average of the G and F actin correlation times. When actin interacts with heavy meromyosin, theta2 increases and becomes infinite at a molar ratio of one heavy meromyosin molecule per four actin protomers. It is concluded that a definite complex is then formed between F actin and heavy meromyosin. In the same time, G actin concentration becomes equal to zero. Finally, when F actin forms a complex with the regulatory proteins tropomyosin and troponin, the value of theta2 is greater in the absence than in the presence of Ca2+. This result indicates that micromolar concentrations of Ca2+ induces a conformation change of the complex of F actin with the regulatory proteins.

  7. Actin and Actin-Binding Proteins.

    PubMed

    Pollard, Thomas D

    2016-08-01

    Organisms from all domains of life depend on filaments of the protein actin to provide structure and to support internal movements. Many eukaryotic cells use forces produced by actin polymerization for their motility, and myosin motor proteins use ATP hydrolysis to produce force on actin filaments. Actin polymerizes spontaneously, followed by hydrolysis of a bound adenosine triphosphate (ATP). Dissociation of the γ-phosphate prepares the polymer for disassembly. This review provides an overview of the properties of actin and shows how dozens of proteins control both the assembly and disassembly of actin filaments. These players catalyze nucleotide exchange on actin monomers, initiate polymerization, promote phosphate dissociation, cap the ends of polymers, cross-link filaments to each other and other cellular components, and sever filaments. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

  8. Regulatory role of the second gelsolin-like domain of Caenorhabditis elegans gelsolin-like protein 1 (GSNL-1) in its calcium-dependent conformation and actin-regulatory activities

    PubMed Central

    Liu, Zhongmei; Ono, Shoichiro

    2013-01-01

    Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is an unconventional member of the gelsolin family of actin-regulatory proteins. Unlike typical gelsolin-related proteins with three or six G domains, GSNL-1 has four gelsolin-like (G) domains (G1–G4) and exhibits calcium-dependent actin filament severing and capping activities. The first G domain (G1) of GSNL-1 is necessary for its actin-regulatory activities. However, how other domains in GSNL-1 participate in regulation of its functions is not understood. Here, we report biochemical evidence that the second G domain (G2) of GSNL-1 has a regulatory role in its calcium-dependent conformation and actin-regulatory activities. Comparison of the sequences of gelsolin-related proteins from various species indicates that sequences of G2 are highly conserved. Among the conserved residues in G2, we focused on D162 of GSNL-1, since equivalent residues in gelsolin and severin are part of the calcium-binding sites and is a pathogenic mutation site in human gelsolin causing familial amyloidosis, Finnish-type. The D162N mutation does not alter the inactive and fully calcium-activated states of GSNL-1 for actin filament severing (at 20 nM GSNL-1) and capping activities (at 50 nM GSNL-1). However, under these conditions, the mutant shows reduced calcium sensitivity for activation. By contrast, the D162N mutation strongly enhances susceptibility of GSNL-1 to chymotrypsin digestion only at high calcium concentrations but not at low calcium concentrations. The mutation also reduces affinity of GSNL-1 with actin monomers. These results suggest that G2 of GSNL-1 functions as a regulatory domain for its calcium-dependent actin-regulatory activities by mediating conformational changes of the GSNL-1 molecule. PMID:23475707

  9. Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges.

    PubMed

    Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz; Hammer, John A

    2014-05-13

    Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there.

  10. Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

    PubMed Central

    Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz; Hammer, John A.

    2014-01-01

    Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there. PMID:24778263

  11. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  12. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  13. The G2019S LRRK2 mutation increases myeloid cell chemotactic responses and enhances LRRK2 binding to actin-regulatory proteins.

    PubMed

    Moehle, Mark S; Daher, João Paulo Lima; Hull, Travis D; Boddu, Ravindra; Abdelmotilib, Hisham A; Mobley, James; Kannarkat, George T; Tansey, Malú G; West, Andrew B

    2015-08-01

    The Leucine rich repeat kinase 2 (LRRK2) gene is genetically and biochemically linked to several diseases that involve innate immunity. LRRK2 protein is highly expressed in phagocytic cells of the innate immune system, most notably in myeloid cells capable of mounting potent pro-inflammatory responses. Knockdown of LRRK2 protein in these cells reduces pro-inflammatory responses. However, the effect of LRRK2 pathogenic mutations that cause Parkinson's disease on myeloid cell function is not clear but could provide insight into LRRK2-linked disease. Here, we find that rats expressing G2019S LRRK2 have exaggerated pro-inflammatory responses and subsequent neurodegeneration after lipopolysaccharide injections in the substantia nigra, with a marked increase in the recruitment of CD68 myeloid cells to the site of injection. While G2019S LRRK2 expression did not affect immunological homeostasis, myeloid cells expressing G2019S LRRK2 show enhanced chemotaxis both in vitro in two-chamber assays and in vivo in response to thioglycollate injections in the peritoneum. The G2019S mutation enhanced the association between LRRK2 and actin-regulatory proteins that control chemotaxis. The interaction between G2019S LRRK2 and actin-regulatory proteins can be blocked by LRRK2 kinase inhibitors, although we did not find evidence that LRRK2 phosphorylated these interacting proteins. These results suggest that the primary mechanism of G2019S LRRK2 with respect to myeloid cell function in disease may be related to exaggerated chemotactic responses. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    PubMed

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  15. Unconventional actins and actin-binding proteins in human protozoan parasites.

    PubMed

    Gupta, C M; Thiyagarajan, S; Sahasrabuddhe, A A

    2015-06-01

    Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites. Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  16. Shear stress-mediated changes in the expression of complement regulatory protein CD59 on human endothelial progenitor cells by ECM-integrinαVβ3-F-actin pathway in vitro.

    PubMed

    Cui, Xiaodong; Zhang, Xiaoyun; Bu, Hongnan; Liu, Na; Li, Hong; Guan, Xiumei; Yan, Hong; Wang, Yuzhen; Zhang, Hua; Ding, Yuzhen; Cheng, Min

    2017-09-21

    Membrane regulatory proteins, such as CD46, CD55, and CD59, prevent excess complement activation and to protect cells from damage. Previous investigations confirmed that shear stress in the physiological range was more favorable for endothelial progenitor cells (EPCs) to repair injured vascular endothelial cells and operates mainly in atheroprotective actions. However, detailed events that contribute to shear stress-induced protection in EPCs, particularly the mechanisms of signal transduction, remain poorly understood. In this study, we observed shear stress-mediated changes in the expression of complement regulatory proteins CD46, CD55, and CD59 on human EPCs and focused on the mechanical transmission mechanism in transformed cells in response to the ECM-F-actin pathway in vitro. Shear stress was observed to promote the expression of complement regulatory protein CD59, but not CD46 or CD55, on EPCs. In addition, the shear stress-induced CD59 expression was confirmed to be associated with the ECM components and was alleviated in EPCs pretreated with GRGDSP, which inhibits ECM components-integrin interaction. Furthermore, shear stress also promotes the rearrangement and polymerization of F-actin. However, shear stress-induced CD59 expression was reduced when the F-actin stress fiber formation process was delayed by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) or destroyed by cytochalasin D (Cyto D), while Jasplakinolide (JAS) reversed the expression of CD59 through promotion of F-actin polymerization and its stabilizing capacities. Our results indicates that shear stress is an important mediator in EPC expression of CD59 regulated by the ECM-F-actin pathway, which is a key factor in preventing membrane attack complex (MAC) -mediated cell autolysis. Copyright © 2017. Published by Elsevier Inc.

  17. The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

    PubMed Central

    Goldschmidt-Clermont, P J; Furman, M I; Wachsstock, D; Safer, D; Nachmias, V T; Pollard, T D

    1992-01-01

    We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover. Images PMID:1330091

  18. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner*

    PubMed Central

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-01-01

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. PMID:26747606

  19. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

    PubMed

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-03-04

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  1. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  2. Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila.

    PubMed

    Fernández, Beatriz García; Gaspar, Pedro; Brás-Pereira, Catarina; Jezowska, Barbara; Rebelo, Sofia Raquel; Janody, Florence

    2011-06-01

    The conserved Hippo tumor suppressor pathway is a key kinase cascade that controls tissue growth by regulating the nuclear import and activity of the transcription co-activator Yorkie. Here, we report that the actin-Capping Protein αβ heterodimer, which regulates actin polymerization, also functions to suppress inappropriate tissue growth by inhibiting Yorkie activity. Loss of Capping Protein activity results in abnormal accumulation of apical F-actin, reduced Hippo pathway activity and the ectopic expression of several Yorkie target genes that promote cell survival and proliferation. Reduction of two other actin-regulatory proteins, Cofilin and the cyclase-associated protein Capulet, cause abnormal F-actin accumulation, but only the loss of Capulet, like that of Capping Protein, induces ectopic Yorkie activity. Interestingly, F-actin also accumulates abnormally when Hippo pathway activity is reduced or abolished, independently of Yorkie activity, whereas overexpression of the Hippo pathway component expanded can partially reverse the abnormal accumulation of F-actin in cells depleted for Capping Protein. Taken together, these findings indicate a novel interplay between Hippo pathway activity and actin filament dynamics that is essential for normal growth control.

  3. Effects of actin-binding proteins on the thermal stability of monomeric actin.

    PubMed

    Pivovarova, Anastasia V; Chebotareva, Natalia A; Kremneva, Elena V; Lappalainen, Pekka; Levitsky, Dmitrii I

    2013-01-08

    Differential scanning calorimetry (DSC) was applied to investigate the thermal unfolding of rabbit skeletal muscle G-actin in its complexes with actin-binding proteins, cofilin, twinfilin, and profilin. The results show that the effects of these proteins on the thermal stability of G-actin depend on the nucleotide, ATP or ADP, bound in the nucleotide-binding cleft between actin subdomains 2 and 4. Interestingly, cofilin binding stabilizes both ATP-G-actin and ADP-G-actin, whereas twinfilin increases the thermal stability of the ADP-G-actin but not that of the ATP-G-actin. By contrast, profilin strongly decreases the thermal stability of the ATP-G-actin but has no appreciable effect on the ADP-G-actin. Comparison of these DSC results with literature data reveals a relationship between the effects of actin-binding proteins on the thermal unfolding of G-actin, stabilization or destabilization, and their effects on the rate of nucleotide exchange in the nucleotide-binding cleft, decrease or increase. These results suggest that the thermal stability of G-actin depends, at least partially, on the conformation of the nucleotide-binding cleft: the actin molecule is more stable when the cleft is closed, while an opening of the cleft leads to significant destabilization of G-actin. Thus, DSC studies of the thermal unfolding of G-actin can provide new valuable information about the conformational changes induced by actin-binding proteins in the actin molecule.

  4. Toxofilin, a Novel Actin-binding Protein from Toxoplasma gondii, Sequesters Actin Monomers and Caps Actin Filaments

    PubMed Central

    Poupel, Olivier; Boleti, Haralabia; Axisa, Sophie; Couture-Tosi, Evelyne; Tardieux, Isabelle

    2000-01-01

    Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end. PMID:10637313

  5. Biallelic Truncating Mutations in FMN2, Encoding the Actin-Regulatory Protein Formin 2, Cause Nonsyndromic Autosomal-Recessive Intellectual Disability

    PubMed Central

    Law, Rosalind; Dixon-Salazar, Tracy; Jerber, Julie; Cai, Na; Abbasi, Ansar A.; Zaki, Maha S.; Mittal, Kirti; Gabriel, Stacey B.; Rafiq, Muhammad Arshad; Khan, Valeed; Nguyen, Maria; Ali, Ghazanfar; Copeland, Brett; Scott, Eric; Vasli, Nasim; Mikhailov, Anna; Khan, Muhammad Nasim; Andrade, Danielle M.; Ayaz, Muhammad; Ansar, Muhammad; Ayub, Muhammad; Vincent, John B.; Gleeson, Joseph G.

    2014-01-01

    Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density. PMID:25480035

  6. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.

    PubMed

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2015-03-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

  7. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  8. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes.

  9. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein

    PubMed Central

    1983-01-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three- dimensional network resembling the peripheral cytoskeleton of motile cells. PMID:6682423

  10. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  11. Actin-related proteins in Actinobacillus pleuropneumoniae and their interactions with actin-binding proteins.

    PubMed

    Guerrero-Barrera, A L; de la Garza, M; Mondragón, R; García-Cuéllar, C; Segura-Nieto, M

    1999-11-01

    A group of prokaryotic actin-related proteins (PARP) with an Mr of 43000 was detected in Actinobacillus pleuropneumoniae. These proteins were enriched by a depolymerization/polymerization cycle, under similar conditions to those used to polymerize muscle actin, and purified by affinity chromatography on a DNase I-Sepharose column. Three isoforms of A. pleuropneumoniae PARP (Ap-PARP) with pI values of 5.8, 6.15 and 6.2 were detected. Ap-PARP were recognized by four different anti-actin antibodies (one anti-muscle and three anti-cytoplasmic isoforms). Ap-PARP were also recognized by antibodies against Anabaena variabilis PARP (Av-PARP) and against actin-binding proteins such as alpha-actinin and spectrin, and also by a monoclonal antibody against heat-shock cognate protein 70 (Hsc70). Specific binding of phalloidin to Ap-PARP was detected both in permeabilized cells and in vitro. Purified Ap-PARP can polymerize under similar conditions to those required for skeletal muscle actin polymerization and the filaments formed appear to be decorated with myosin subfragment-1(S1) as observed by transmission electron microscopy. The amino acid composition of Ap-PARP revealed more similarities to muscle gamma-actin and the cytoplasmic beta-actin isoform than to eukaryotic actin-related proteins.

  12. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  13. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

    PubMed

    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

  14. Regulatory mimicry in Listeria monocytogenes actin-based motility

    PubMed Central

    Chong, Ryan; Swiss, Rachel; Briones, Gabriel; Stone, Kathryn L.; Gulcicek, Erol E.; Agaisse, Hervé

    2009-01-01

    Summary The actin-based motility of the intracellular pathogen Listeria monocytogenes relies on ActA, a bacterial factor mimicking the activity of host cell nucleation-promoting factors of the WASP/WAVE family. The activity of WASP and WAVE is tightly regulated in cells. However, it is not known whether the activity of ActA is regulated upon L. monocytogenes infection. Here, we used an RNAi-based genetic approach in combination with computer-assisted image analysis to investigate the role of host factors in L. monocytogenes spread from cell to cell. We showed that the host cell serine/threonine kinase CK2 is required for efficient actin tail formation. We demonstrated that, similar to WASP and WAVE, the affinity of ActA for the ARP2/3 complex is regulated by CK2-mediated phosphorylation. We also demonstrated the importance of this regulatory mechanism in a mouse model of infection. Our work suggests that ActA is a bacterial virulence factor that not only displays a structural mimic of the VCA domain of WASP/WAVE family members, but also co-opted CK2 as the host cell factor regulating its activity, a form of mimicry that we refer to as regulatory mimicry. We present comparative evidence supporting the notion that unrelated pathogens displaying actin-based motility may have evolved a similar strategy. PMID:19748468

  15. In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins.

    PubMed

    Zimmermann, Dennis; Morganthaler, Alisha N; Kovar, David R; Suarez, Cristian

    2016-01-01

    Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) "bulk" pyrene actin assembly/disassembly assays, and (3) "bulk" sedimentation actin filament binding and bundling assays.

  16. Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types

    PubMed Central

    Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.

    2012-01-01

    Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942

  17. Actin-binding proteins take the reins in growth cones.

    PubMed

    Pak, Chi W; Flynn, Kevin C; Bamburg, James R

    2008-02-01

    Higher-order actin-based networks (actin superstructures) are important for growth-cone motility and guidance. Principles for generating, organizing and remodelling actin superstructures have emerged from recent findings in cell-free systems, non-neuronal cells and growth cones. This Review examines how actin superstructures are initiated de novo at the leading-edge membrane and how the spontaneous organization of actin superstructures is driven by ensembles of actin-binding proteins. How the regulation of actin-binding proteins can affect growth-cone turning and axonal regeneration is also discussed.

  18. Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes

    PubMed Central

    Delage, Elise; Cervantes, Diégo Cordero; Pénard, Esthel; Schmitt, Christine; Syan, Sylvie; Disanza, Andrea; Scita, Giorgio; Zurzolo, Chiara

    2016-01-01

    Tunneling Nanotubes (TNTs) are actin enriched filopodia-like protrusions that play a pivotal role in long-range intercellular communication. Different pathogens use TNT-like structures as “freeways” to propagate across cells. TNTs are also implicated in cancer and neurodegenerative diseases, making them promising therapeutic targets. Understanding the mechanism of their formation, and their relation with filopodia is of fundamental importance to uncover their physiological function, particularly since filopodia, differently from TNTs, are not able to mediate transfer of cargo between distant cells. Here we studied different regulatory complexes of actin, which play a role in the formation of both these structures. We demonstrate that the filopodia-promoting CDC42/IRSp53/VASP network negatively regulates TNT formation and impairs TNT-mediated intercellular vesicle transfer. Conversely, elevation of Eps8, an actin regulatory protein that inhibits the extension of filopodia in neurons, increases TNT formation. Notably, Eps8-mediated TNT induction requires Eps8 bundling but not its capping activity. Thus, despite their structural similarities, filopodia and TNTs form through distinct molecular mechanisms. Our results further suggest that a switch in the molecular composition in common actin regulatory complexes is critical in driving the formation of either type of membrane protrusion. PMID:28008977

  19. The actin cytoskeleton may control the polar distribution of an auxin transport protein.

    PubMed

    Muday, G K; Hu, S; Brady, S R

    2000-06-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  20. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    NASA Technical Reports Server (NTRS)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  1. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    NASA Technical Reports Server (NTRS)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  2. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the “status” of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  3. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    PubMed Central

    Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  4. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-12-01

    Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

  5. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin

    PubMed Central

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-01-01

    ABSTRACT Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation. PMID:25315833

  6. Microscopy basics and the study of actin-actin-binding protein interactions.

    PubMed

    Thomasson, Maggie S; Macnaughtan, Megan A

    2013-12-15

    Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Novel anti-HIV therapeutics targeting chemokine receptors and actin regulatory pathways.

    PubMed

    Spear, Mark; Guo, Jia; Wu, Yuntao

    2013-11-01

    The human immunodeficiency virus-1 (HIV-1) infects helper CD4(+) T cells, and causes CD4(+) T-cell depletion and immunodeficiency. In the past 30 years, significant progress has been made in antiretroviral therapy, and the disease has become manageable. Nevertheless, an effective vaccine is still nowhere in sight, and a cure or a functional cure awaits discovery. Among possible curative therapies, traditional antiretroviral therapy, mostly targeting viral proteins, has been proven ineffective. It is possible that targeting HIV-dependent host cofactors may offer alternatives, both for preventing HIV transmission and for forestalling disease progression. Recently, the actin cytoskeleton and its regulators in blood CD4(+) T cells have emerged as major host cofactors that could be targeted. The novel concept that the cortical actin is a barrier to viral entry and early post-entry migration has led to the nascent model of virus-host interaction at the cortical actin layer. Deciphering the cellular regulatory pathways has manifested exciting prospects for future therapeutics. In this review, we describe the study of HIV interactions with actin cytoskeleton. We also examine potential pharmacological targets that emerge from this interaction. In addition, we briefly discuss several actin pathway-based anti-HIV drugs that are currently in development or testing.

  8. Novel anti-HIV therapeutics targeting chemokine receptors and actin regulatory pathways

    PubMed Central

    Spear, Mark; Guo, Jia; Wu, Yuntao

    2013-01-01

    Summary The human immunodeficiency virus-1 (HIV-1) infects helper CD4+ T cells, and causes CD4+ T-cell depletion and immunodeficiency. In the past 30 years, significant progress has been made in antiretroviral therapy, and the disease has become manageable. Nevertheless, an effective vaccine is still nowhere in sight, and a cure or a functional cure awaits discovery. Among possible curative therapies, traditional antiretroviral therapy, mostly targeting viral proteins, has been proven ineffective. It is possible that targeting HIV-dependent host cofactors may offer alternatives, both for preventing HIV transmission and for forestalling disease progression. Recently, the actin cytoskeleton and its regulators in blood CD4+ T cells have emerged as major host cofactors that could be targeted. The novel concept that the cortical actin is a barrier to viral entry and early post-entry migration has led to the nascent model of virus-host interaction at the cortical actin layer. Deciphering the cellular regulatory pathways has manifested exciting prospects for future therapeutics. In this review, we describe the study of HIV interactions with actin cytoskeleton. We also examine potential pharmacological targets that emerge from this interaction. In addition, we briefly discuss several actin pathway-based anti-HIV drugs that are currently in development or testing. PMID:24117829

  9. Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

    PubMed Central

    Whitten, Andrew E.; Jeffries, Cy M.; Harris, Samantha P.; Trewhella, Jill

    2008-01-01

    Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin. PMID:19011110

  10. Identification of a novel regulatory sequence of actin nucleation promoting factor encoded by Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Wang, Yun; Zhang, Yongli; Han, Shili; Hu, Xue; Zhou, Yuan; Mu, Jingfang; Pei, Rongjuan; Wu, Chunchen; Chen, Xinwen

    2015-04-10

    Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.

  11. Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

    NASA Astrophysics Data System (ADS)

    Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

    1990-05-01

    THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

  12. Nuclear actin and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Zheng, Bin; Han, Mei; Bernier, Michel; Wen, Jin-kun

    2010-01-01

    Nuclear actin is involoved in transcription of all three RNA polymerases, chromatin remodeling, and formation of hnRNP complexes as well as recruitment of histone modifier to the active gene. In addition, actin-binding proteins (ABPs) control actin nucleation, bundling, filament capping, fragmentation, and monomer availability in the cytoplasm. In recent years, more and more attention is on the role of actin and ABPs in the modulation of the subcellular localization of transcriptional regulators. This review focuses on the recent developments about transcription and transcriptional regulation by nuclear actin, regulation of muscle-specific gene expression, nuclear receptor and transcription complexes by ABPs. Among them, STARS and ABLIM regulate actin dynamics and SRF-dependent muscle-specific gene expression. Functionally and structurally unrelated cytoplasmic ABPs interact cooperatively with nuclear receptor and regulate its transactivation. Furthermore, ABPs also participate in the formation of transcription complexes. PMID:19459931

  13. Cysteine-rich protein 2 accelerates actin filament cluster formation

    PubMed Central

    Shinohara, Satoko; Takaoka, Shunpei; Miyake, Jun

    2017-01-01

    Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation. PMID:28813482

  14. Actin binding proteins, spermatid transport and spermiation*

    PubMed Central

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  15. Time-resolved studies of actin organization by multivalent ions and actin-binding proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Purdy, Kirstin; Bartles, James R.; Chee Lai Wong, Gerard

    2007-03-01

    Actin is one of the principal components in the eukaryotic cytoskeleton, the architecture of which is highly regulated for a wide range of biological functions. In the presence of multivalent salts or actin-binding proteins, it is known that F-actin can organize into bundles or networks. In this work, we use time-resolved confocal microscopy to study the dynamics of actin bundle growth induced by multivalent ions and by espin, a prototypical actin binding protein that is known to induce bundles. For divalent ion induced bundles, we observe a rapid lateral saturation followed by longitudinal growth of bundles, in sharp contrast to the bundling mechanism of espin, which favors finite length bundles.

  16. PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins

    PubMed Central

    Leyton-Puig, Daniela; Kedziora, Katarzyna M.; Isogai, Tadamoto; van den Broek, Bram; Jalink, Kees

    2016-01-01

    ABSTRACT Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins. PMID:27378434

  17. New Insights into Mechanism and Regulation of Actin Capping Protein

    PubMed Central

    Cooper, John A.; Sept, David

    2008-01-01

    The heterodimeric actin capping protein, referred to here as “CP,” is an essential element of the actin cytoskeleton, binding to the barbed ends of actin filaments and regulating their polymerization. In vitro, CP has a critical role in the dendritic nucleation process of actin assembly mediated by Arp2/3 complex, and in vivo, CP is important for actin assembly and actin-based process of morphogenesis and differentiation. Recent studies have provided new insight into the mechanism of CP binding the barbed end, which raises new possibilities for the dynamics of CP and actin in cells. In addition, a number of molecules that bind and regulate CP have been discovered, suggesting new ideas for how CP may integrate into diverse processes of cell physiology. PMID:18544499

  18. The chloroplast outer membrane protein CHUP1 interacts with actin and profilin.

    PubMed

    Schmidt von Braun, Serena; Schleiff, Enrico

    2008-04-01

    Chloroplasts accumulate in response to low light, whereas high light induces an actin-dependent avoidance movement. This is a long known process, but its molecular base is barely understood. Only recently first components of the blue light perceiving signal cascade initiating this process were described. Among these, a protein was identified by the analysis of a deletion mutant in the corresponding gene resulting in a chloroplast unusual positioning phenotype. The protein was termed CHUP1 and initial results suggested chloroplast localization. We demonstrate that the protein is indeed exclusively and directly targeted to the chloroplast surface. The analysis of the deletion mutant of CHUP1 using microarray analysis shows an influence on the expression of genes found to be up-regulated, but not on genes found to be down-regulated upon high light exposure in wild-type. Analyzing a putative role of CHUP1 as a linker between chloroplasts and the cytoskeleton, we demonstrate an interaction with actin, which is independent on the filamentation status of actin. Moreover, binding of CHUP1 to profilin -- an actin modifying protein -- could be shown and an enhancing effect of CHUP1 on the interaction of profilin to actin is demonstrated. Therefore, a role of CHUP1 in bridging chloroplasts to actin filaments and a regulatory function in actin polymerization can be discussed.

  19. Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Field, Jeffrey

    2014-01-01

    Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.

  20. The Actinome of Dictyostelium discoideum in Comparison to Actins and Actin-Related Proteins from Other Organisms

    PubMed Central

    Joseph, Jayabalan M.; Fey, Petra; Ramalingam, Nagendran; Liu, Xiao I.; Rohlfs, Meino; Noegel, Angelika A.; Müller-Taubenberger, Annette; Glöckner, Gernot; Schleicher, Michael

    2008-01-01

    Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes. PMID:18612387

  1. Differential functions of WAVE regulatory complex subunits in the regulation of actin-driven processes.

    PubMed

    Litschko, Christof; Linkner, Joern; Brühmann, Stefan; Stradal, Theresia E B; Reinl, Tobias; Jänsch, Lothar; Rottner, Klemens; Faix, Jan

    2017-09-04

    The WAVE regulatory complex (WRC) links upstream Rho-family GTPase signaling to the activation of the ARP2/3 complex in different organisms. WRC-induced and ARP2/3 complex-mediated actin nucleation beneath the plasma membrane is critical for actin assembly in the leading edge to drive efficient cell migration. The WRC is a stable heteropentamer composed of SCAR/WAVE, Abi, Nap, Pir and the small polypeptide Brk1/Hspc300. Functional interference with individual subunits of the complex frequently results in diminished amounts of the remaining polypeptides of the WRC complex, implying the complex to act as molecular entity. However, Abi was also found to associate with mammalian N-WASP, formins, Eps8/SOS1 or VASP, indicating additional functions of individual WRC subunits in eukaryotic cells. To address this issue systematically, we inactivated all WRC subunits, either alone or in combination with VASP in Dictyostelium cells and quantified the protein content of the remaining subunits in respective WRC knockouts. The individual mutants displayed highly differential phenotypes concerning various parameters, including cell morphology, motility, cytokinesis or multicellular development, corroborating the view of additional roles for individual subunits, beyond their established function in WRC-mediated Arp2/3 complex activation. Finally, our data uncover the interaction of the actin polymerase VASP with WRC-embedded Abi to mediate VASP accumulation in cell protrusions, driving efficient cell migration. Copyright © 2017 Elsevier GmbH. All rights reserved.

  2. Actin, actin-related proteins and profilin in diatoms: a comparative genomic analysis.

    PubMed

    Aumeier, Charlotte; Polinski, Ellen; Menzel, Diedrik

    2015-10-01

    Diatoms are heterokont unicellular algae with a widespread distribution throughout all aquatic habitats. Research on diatoms has advanced significantly over the last decade due to available genetic transformation methods and publicly available genome databases. Yet up to now, proteins involved in the regulation of the cytoskeleton in diatoms are largely unknown. Consequently, this work focuses on actin and actin-related proteins (ARPs) encoded in the diatom genomes of Thalassiosira pseudonana, Thalassiosira oceanica, Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries. Our comparative genomic study revealed that most diatoms possess only a single conventional actin and a small set of ARPs. Among these are the highly conserved cytoplasmic Arp1 protein and the nuclear Arp4 as well as Arp6. Diatom genomes contain genes coding for two structurally different homologues of Arp4 that might serve specific functions. All diatom species examined here lack ARP2 and ARP3 proteins, suggesting that diatoms are not capable of forming the Arp2/3 complex, which is essential in most eukaryotes for actin filament branching and plus-end dynamics. Interestingly, none of the sequenced representatives of the Bacillariophyta phylum code for profilin. Profilin is an essential actin-binding protein regulating the monomer actin pool and is involved in filament plus-end dynamics. This is the first report of organisms not containing profilin.

  3. Human muscle LIM protein dimerizes along the actin cytoskeleton and cross-links actin filaments.

    PubMed

    Hoffmann, Céline; Moreau, Flora; Moes, Michèle; Luthold, Carole; Dieterle, Monika; Goretti, Emeline; Neumann, Katrin; Steinmetz, André; Thomas, Clément

    2014-08-01

    The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

    PubMed Central

    Hoffmann, Céline; Moreau, Flora; Moes, Michèle; Luthold, Carole; Dieterle, Monika; Goretti, Emeline; Neumann, Katrin; Steinmetz, André

    2014-01-01

    The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. PMID:24934443

  5. The C-terminal dimerization motif of cyclase-associated protein is essential for actin monomer regulation.

    PubMed

    Iwase, Shohei; Ono, Shoichiro

    2016-12-01

    Cyclase-associated protein (CAP) is a conserved actin-regulatory protein that functions together with actin depolymerizing factor (ADF)/cofilin to enhance actin filament dynamics. CAP has multiple functional domains, and the function to regulate actin monomers is carried out by its C-terminal half containing a Wiskott-Aldrich Syndrome protein homology 2 (WH2) domain, a CAP and X-linked retinitis pigmentosa 2 (CARP) domain, and a dimerization motif. WH2 and CARP are implicated in binding to actin monomers and important for enhancing filament turnover. However, the role of the dimerization motif is unknown. Here, we investigated the function of the dimerization motif of CAS-2, a CAP isoform in the nematode Caenorhabditis elegans, in actin monomer regulation. CAS-2 promotes ATP-dependent recycling of ADF/cofilin-bound actin monomers for polymerization by enhancing exchange of actin-bound nucleotides. The C-terminal half of CAS-2 (CAS-2C) has nearly as strong activity as full-length CAS-2. Maltose-binding protein (MBP)-tagged CAS-2C is a dimer. However, MBP-CAS-2C with a truncation of either one or two C-terminal β-strands is monomeric. Truncations of the dimerization motif in MBP-CAS-2C nearly completely abolish its activity to sequester actin monomers from polymerization and enhance nucleotide exchange on actin monomers. As a result, these CAS-2C variants, also in the context of full-length CAS-2, fail to compete with ADF/cofilin to release actin monomers for polymerization. CAS-2C variants lacking the dimerization motif exhibit enhanced binding to actin filaments, which is mediated by WH2. Taken together, these results suggest that the evolutionarily conserved dimerization motif of CAP is essential for its C-terminal region to exert the actin monomer-specific regulatory function.

  6. Interaction of actin and the chloroplast protein import apparatus.

    PubMed

    Jouhet, Juliette; Gray, John C

    2009-07-10

    Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.

  7. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor.

    PubMed

    Ono, Shoichiro

    2013-08-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.

  8. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

  9. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  10. The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton

    PubMed Central

    1996-01-01

    Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2- H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament- associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis. PMID:8698808

  11. AKAP-independent localization of type-II protein kinase A to dynamic actin microspikes.

    PubMed

    Rivard, Robert L; Birger, Monique; Gaston, Kara J; Howe, Alan K

    2009-09-01

    Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RIIalpha subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RIIalpha and a GFP-RIIalpha fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RIIalpha-associated microspikes are highly dynamic and that the coupling of RIIalpha to actin is tight, as the movement of both actin and RIIalpha are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RIIalpha and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RIIalpha is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RIIalpha fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity.

  12. AKAP-Independent Localization of Type-II Protein Kinase A to Dynamic Actin Microspikes

    PubMed Central

    Rivard, Robert L.; Birger, Monique; Gaston, Kara J.; Howe, Alan K.

    2010-01-01

    Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RIIα subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RIIα and a GFP-RIIα fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RIIα-associated microspikes are highly dynamic and that the coupling of RIIα to actin is tight, as the movement of both actin and RIIα are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RIIα and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RIIα is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RIIα fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity. PMID:19536823

  13. A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.

    PubMed Central

    Eichinger, L; Bomblies, L; Vandekerckhove, J; Schleicher, M; Gettemans, J

    1996-01-01

    Actin-fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA-resistant 1:1 actin-fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of approximately 35 kDa each that are separated by a hinge-like proline/serine-rich segment of 50 residues. Whereas the N-terminal domain does not show any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C-terminal half of the molecule. To prove the intrinsic phosphorylation activity of AFK, full-length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli. In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N-terminal half. Interestingly, this region did not contain any of the known protein kinase consensus sequences. The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P-loop). However, introduction of two different point mutations into this putative P-loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer. Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non-muscle cells. Images PMID:8896448

  14. The ADF/cofilin family: actin-remodeling proteins.

    PubMed

    Maciver, Sutherland K; Hussey, Patrick J

    2002-01-01

    The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.

  15. Cooperativity and Frustration in Protein-Mediated Parallel Actin Bundles

    NASA Astrophysics Data System (ADS)

    Shin, Homin; Drew, Kirstin R. Purdy; Bartles, James R.; Wong, Gerard C. L.; Grason, Gregory M.

    2009-12-01

    We examine the mechanism of bundling of cytoskeletal actin filaments by two representative bundling proteins, fascin and espin. Small-angle x-ray studies show that increased binding from linkers drives a systematic overtwist of actin filaments from their native state, which occurs in a linker-dependent fashion. Fascin bundles actin into a continuous spectrum of intermediate twist states, while espin only allows for untwisted actin filaments and fully overtwisted bundles. Based on a coarse-grained, statistical model of protein binding, we show that the interplay between binding geometry and the intrinsic flexibility of linkers mediates cooperative binding in the bundle. We attribute the respective continuous (discontinuous) bundling mechanisms of fascin (espin) to difference in the stiffness of linker bonds themselves.

  16. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  17. Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

    PubMed

    Laine, R O; Phaneuf, K L; Cunningham, C C; Kwiatkowski, D; Azuma, T; Southwick, F S

    1998-08-01

    -end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.

  18. Gelsolin, a Protein That Caps the Barbed Ends and Severs Actin Filaments, Enhances the Actin-Based Motility of Listeria monocytogenes in Host Cells

    PubMed Central

    Laine, Roney O.; Phaneuf, Katherine L.; Cunningham, Casey C.; Kwiatkowski, David; Azuma, Toshi; Southwick, Frederick S.

    1998-01-01

    -end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system. PMID:9673261

  19. Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model.

    PubMed

    Walcott, Sam; Docken, Steffen; Harris, Samantha P

    2015-01-06

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin.

  20. Effects of Cardiac Myosin Binding Protein-C on Actin Motility Are Explained with a Drag-Activation-Competition Model

    PubMed Central

    Walcott, Sam; Docken, Steffen; Harris, Samantha P.

    2015-01-01

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin. PMID:25564844

  1. Conserved hydrophobic residues in the CARP/β-sheet domain of cyclase-associated protein are involved in actin monomer regulation.

    PubMed

    Iwase, Shohei; Ono, Shoichiro

    2017-09-01

    Cyclase-associated protein (CAP) is a multidomain protein that promotes actin filament dynamics. The C-terminal region of CAP contains a CAP and X-linked retinitis pigmentosa 2 protein (CARP) domain (or a β-sheet domain), which binds to actin monomer and is essential for enhancing exchange of actin-bound nucleotides. However, how the CARP domain binds to actin is not clearly understood. Here, we report that conserved hydrophobic residues in the CARP domain play important roles in the function of CAP to regulate actin dynamics. Single mutations of three conserved surface-exposed hydrophobic residues in the CARP domain of CAS-2, a Caenorhabditis elegans CAP, significantly reduce its binding to actin monomers and suppress its nucleotide exchange activity on actin. As a result, these mutants are weaker than wild-type to compete with ADF/cofilin to promote recycling of actin monomers for polymerization. A double mutation (V367A/I373A) eliminates these actin-regulatory functions of CAS-2. These hydrophobic residues and previously identified functional residues are scattered on a concave β-sheet of the CARP domain, suggesting that a wide area of the β-sheet is involved in binding to actin. These observations suggest that the CARP domain of CAP binds to actin in a distinct manner from other known actin-binding proteins. © 2017 Wiley Periodicals, Inc.

  2. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  3. How does the antagonism between capping and anti-capping proteins affect actin network dynamics?

    NASA Astrophysics Data System (ADS)

    Hu, Longhua; Papoian, Garegin A.

    2011-09-01

    Actin-based cell motility is essential to many biological processes. We built a simplified, three-dimensional computational model and subsequently performed stochastic simulations to study the growth dynamics of lamellipodia-like branched networks. In this work, we shed light on the antagonism between capping and anti-capping proteins in regulating actin dynamics in the filamentous network. We discuss detailed mechanisms by which capping and anti-capping proteins affect the protrusion speed of the actin network and the rate of nucleation of filaments. We computed a phase diagram showing the regimes of motility enhancement and inhibition by these proteins. Our work shows that the effects of capping and anti-capping proteins are mainly transmitted by modulation of the filamentous network density and local availability of monomeric actin. We discovered that the combination of the capping/anti-capping regulatory network with nucleation-promoting proteins introduces robustness and redundancy in cell motility machinery, allowing the cell to easily achieve maximal protrusion speeds under a broader set of conditions. Finally, we discuss distributions of filament lengths under various conditions and speculate on their potential implication for the emergence of filopodia from the lamellipodial network.

  4. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  5. Control of nuclear organization by F-actin binding proteins.

    PubMed

    Pfisterer, Karin; Jayo, Asier; Parsons, Maddy

    2017-03-04

    The regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

  6. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  7. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes.

    PubMed

    Chan, Maia M; Wooden, Jason M; Tsang, Mark; Gilligan, Diana M; Hirenallur-S, Dinesh K; Finney, Greg L; Rynes, Eric; Maccoss, Michael; Ramirez, Julita A; Park, Heon; Iritani, Brian M

    2013-01-01

    Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1⁻/⁻ erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1⁻/⁻ erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

  8. Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

    PubMed Central

    Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.

    2013-01-01

    Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621

  9. Multiple repeat elements within the FAM21 tail link the WASH actin regulatory complex to the retromer

    PubMed Central

    Jia, Da; Gomez, Timothy S.; Billadeau, Daniel D.; Rosen, Michael K.

    2012-01-01

    Wiskott–Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E]3-10-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation. PMID:22513087

  10. Actin Capping Protein is required for Dendritic Spine Development and Synapse Formation

    PubMed Central

    Fan, Yanjie; Tang, Xin; Vitriol, Eric; Chen, Gong; Zheng, James Q.

    2011-01-01

    Dendritic spines serve as the postsynaptic platform for most excitatory synapses in the mammalian brain and their shape and size are tightly correlated with synaptic strength. The actin cytoskeleton plays a crucial role in the spine structure and its modifications during synapse development and plasticity, but the underlying regulatory mechanisms remain to be elucidated. Here we report that actin capping protein (CP), a regulator of actin filament growth, plays an essential role for spine development and synapse formation. We found that CP expression in rat hippocampus is elevated at and after the stage of substantial synapse formation. CP knockdown in hippocampal cultures resulted in a marked decline in the spine density accompanied by increased filopodia-like protrusions. Moreover, the spines of CP knockdown neurons exhibited an altered morphology, highlighted by multiple thin filopodia-like protrusions emerging from the spine head. Finally, the number of functional synapses was reduced by CP knockdown as evidenced by a reduction in the density of paired pre- and postsynaptic markers and in the frequency of miniature excitatory postsynaptic currents. These findings indicate that capping of actin filaments by CP represents an essential step for the remodeling of the actin architecture underlying spine morphogenesis and synaptic formation during development. PMID:21752999

  11. Proteins of the ADF/cofilin family: essential regulators of actin dynamics.

    PubMed

    Bamburg, J R

    1999-01-01

    Ubiquitous among eukaryotes, the ADF/cofilins are essential proteins responsible for the high turnover rates of actin filaments in vivo. In vertebrates, ADF and cofilin are products of different genes. Both bind to F-actin cooperatively and induce a twist in the actin filament that results in the loss of the phalloidin-binding site. This conformational change may be responsible for the enhancement of the off rate of subunits at the minus end of ADF/cofilin-decorated filaments and for the weak filament-severing activity. Binding of ADF/cofilin is competitive with tropomyosin. Other regulatory mechanisms in animal cells include binding of phosphoinositides, phosphorylation by LIM kinases on a single serine, and changes in pH. Although vertebrate ADF/cofilins contain a nuclear localization sequence, they are usually concentrated in regions containing dynamic actin pools, such as the leading edge of migrating cells and neuronal growth cones. ADF/cofilins are essential for cytokinesis, phagocytosis, fluid phase endocytosis, and other cellular processes dependent upon actin dynamics.

  12. Interactions with Actin Monomers, Actin Filaments, and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks*

    PubMed Central

    Helgeson, Luke A.; Prendergast, Julianna G.; Wagner, Andrew R.; Rodnick-Smith, Max; Nolen, Brad J.

    2014-01-01

    Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks. PMID:25160634

  13. Actin-related proteins localized in the nucleus

    PubMed Central

    Oma, Yukako

    2011-01-01

    The actin family consists of conventional actin and actin-related proteins (ARPs), and the members show moderate similarity and share the same basal structure. Following the finding of various ARPs in the cytoplasm in the 1990s, multiple subfamilies that are localized predominantly in the nucleus were identified. Consistent with these cytological observations, subsequent biochemical analyses revealed the involvement of the nuclear ARPs in ATP-dependent chromatin-remodeling and histone acetyltransferase complexes. In addition to their contribution to chromatin remodeling, recent studies have shown that nuclear ARPs have roles in the organization of the nucleus that are independent of the activity of the above-mentioned complexes. Therefore, nuclear ARPs are recognized as novel key regulators of genome function, and affect not only the remodeling of chromatin but also the spatial arrangement and dynamics of chromatin within the nucleus. PMID:21647298

  14. Involvement of PCH family proteins in cytokinesis and actin distribution.

    PubMed

    Lippincott, J; Li, R

    2000-04-15

    Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.

  15. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor

    PubMed Central

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-01-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA. Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  16. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  17. Holding back the microfilament--structural insights into actin and the actin-monomer-binding proteins of apicomplexan parasites.

    PubMed

    Olshina, Maya A; Wong, Wilson; Baum, Jake

    2012-05-01

    Parasites from the phylum Apicomplexa are responsible for several major diseases of man, including malaria and toxoplasmosis. These highly motile protozoa use a conserved actomyosin-based mode of movement to power tissue traversal and host cell invasion. The mode termed as 'gliding motility' relies on the dynamic turnover of actin, whose polymerisation state is controlled by a markedly limited number of identifiable regulators when compared with other eukaryotic cells. Recent studies of apicomplexan actin regulator structure-in particular those of the core triad of monomer-binding proteins, actin-depolymerising factor/cofilin, cyclase-associated protein/Srv2, and profilin-have provided new insights into possible mechanisms of actin regulation in parasite cells, highlighting divergent structural features and functions to regulators from other cellular systems. Furthermore, the unusual nature of apicomplexan actin itself is increasingly coming into the spotlight. Here, we review recent advances in understanding of the structure and function of actin and its regulators in apicomplexan parasites. In particular we explore the paradox between there being an abundance of unpolymerised actin, its having a seemingly increased potential to form filaments relative to vertebrate actin, and the apparent lack of visible, stable filaments in parasite cells.

  18. Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

    PubMed Central

    Lin, Wei-Chi; Wang, Liang-Chen; Pang, Te-Ling; Chen, Mei-Yu

    2015-01-01

    Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG− cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG− cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG− cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion. PMID:25609090

  19. Mammalian CARMIL Inhibits Actin Filament Capping by Capping Protein

    PubMed Central

    Yang, Changsong; Pring, Martin; Wear, Martin A.; Huang, Minzhou; Cooper, John A.; Svitkina, Tatyana M.; Zigmond, Sally H.

    2009-01-01

    Summary Actin polymerization in cells occurs via filament elongation at the barbed end. Proteins that cap the barbed end terminate this elongation. Heterodimeric capping protein (CP) is an abundant and ubiquitous protein that caps the barbed end. We find that the mouse homolog of the adaptor protein CARMIL (mCARMIL) binds CP with high affinity and decreases its affinity for the barbed end. Addition of mCARMIL to cell extracts increases the rate and extent of Arp2/3 or spectrin-actin seed-induced polymerization. In cells, GFP-mCARMIL concentrates in lamellipodia and increases the fraction of cells with large lamellipodia. Decreasing mCARMIL levels by siRNA transfection lowers theF-actin level and slows cell migration through a mechanism that includes decreased lamellipodia protrusion. This phenotype is reversed by full-length mCARMIL but not mCARMIL lacking the domain that binds CP. Thus, mCARMIL is a key regulator of CP and has profound effects on cell behavior. PMID:16054028

  20. An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes.

    PubMed

    Wang, Huei-Jing; Wan, Ai-Ru; Jauh, Guang-Yuh

    2008-08-01

    Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

  1. Bacterial Actin-Like Proteins: Purification and Characterization of Self-Assembly Properties

    PubMed Central

    Petek, Natalie A.; Mullins, R. Dyche

    2014-01-01

    The sequence, structure, and assembly dynamics of eukaryotic actins are conserved across phyla. In contrast, actin-like proteins (ALPs) from eubacteria share little sequence homology, form polymers with different architectures, and assemble with different kinetics. The structural and functional diversity of the bacterial ALPs appears to arise from their (i) high degree of functional specialization and (ii) small number of regulatory factors. To understand the molecular mechanism by which a given ALP carries out its biological function, we must, therefore, understand its unique architecture and assembly dynamics. In this chapter, we provide a basic toolbox for studying the self-assembly of uncharacterized ALPs, including methods for characterizing the architecture and stability of polymers, specifying the mechanism of their nucleation, and measuring their rate of growth. Determining these basic properties provides a stable base for more complex reconstitutions of biological function. PMID:24630099

  2. The CPEB3 Protein Is a Functional Prion that Interacts with the Actin Cytoskeleton.

    PubMed

    Stephan, Joseph S; Fioriti, Luana; Lamba, Nayan; Colnaghi, Luca; Karl, Kevin; Derkatch, Irina L; Kandel, Eric R

    2015-06-23

    The mouse cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a translational regulator implicated in long-term memory maintenance. Invertebrate orthologs of CPEB3 in Aplysia and Drosophila are functional prions that are physiologically active in the aggregated state. To determine if this principle applies to the mammalian CPEB3, we expressed it in yeast and found that it forms heritable aggregates that are the hallmark of known prions. In addition, we confirm in the mouse the importance of CPEB3's prion formation for CPEB3 function. Interestingly, deletion analysis of the CPEB3 prion domain uncovered a tripartite organization: two aggregation-promoting domains surround a regulatory module that affects interaction with the actin cytoskeleton. In all, our data provide direct evidence that CPEB3 is a functional prion in the mammalian brain and underline the potential importance of an actin/CPEB3 feedback loop for the synaptic plasticity underlying the persistence of long-term memory.

  3. Nuclear actin-binding proteins as modulators of gene transcription.

    PubMed

    Gettemans, Jan; Van Impe, Katrien; Delanote, Veerle; Hubert, Thomas; Vandekerckhove, Joël; De Corte, Veerle

    2005-10-01

    Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

  4. A statistical model of protein binding in parallel actin bundles

    NASA Astrophysics Data System (ADS)

    Shin, Homin; Grason, Gregory; Purdy Drew, Kirstin; Wong, Gerard

    2010-03-01

    We propose a coarse-grained lattice model of cross-linking proteins in parallel actin bundles. Based on this model that captures the interplay between geometrical frustration of binding and the intrinsic flexibility of filaments and linkers, we predict a unique regular ground-state structure of fully cross-linked bundles. We also discuss the linker-dependent thermodynamic transition of actin filaments from their native state to the overtwisted state and map out the ``twist-state'' phase diagram in terms of linker flexibility as well as the chemical potential. A flexible linker regime exhibits a continuous spectrum of intermediate twist states, while a stiff linker regime only allows for untwisted actin filaments and fully overtwisted bundles. Our predictions compare well with small-angle scattering studies of bundles formed in the presence of two types of reconstituted cross-linking proteins, fascin and espin. Additionally, this study reveals how subtle differences in crosslinking agents themselves may be used by cells to achieve self-organized bundles with dramatically different properties.

  5. Engineered protein connectivity to actin mimics PDZ-dependent recycling of G protein-coupled receptors but not its regulation by Hrs.

    PubMed

    Lauffer, Benjamin E L; Chen, Stanford; Melero, Cristina; Kortemme, Tanja; von Zastrow, Mark; Vargas, Gabriel A

    2009-01-23

    Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the beta2-adrenergic receptor (beta2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the delta-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.

  6. Identification of obscure yet conserved actin-associated proteins in Giardia lamblia.

    PubMed

    Paredez, Alexander R; Nayeri, Arash; Xu, Jennifer W; Krtková, Jana; Cande, W Zacheus

    2014-06-01

    Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.

  7. Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

    PubMed Central

    Nayeri, Arash; Xu, Jennifer W.; Krtková, Jana; Cande, W. Zacheus

    2014-01-01

    Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton. PMID:24728194

  8. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

    PubMed Central

    Pestonjamasp, K; Amieva, M R; Strassel, C P; Nauseef, W M; Furthmayr, H; Luna, E J

    1995-01-01

    Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein. Images PMID:7612961

  9. Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

    PubMed

    Schneider, Gabriela; Nieznanski, Krzysztof; Jozwiak, Jolanta; Slomnicki, Lukasz P; Redowicz, Maria J; Filipek, Anna

    2010-11-01

    CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

  10. Cyclase-associated protein (CAP) acts directly on F-actin to accelerate cofilin-mediated actin severing across the range of physiological pH.

    PubMed

    Normoyle, Kieran P M; Brieher, William M

    2012-10-12

    Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.

  11. Cyclase-associated Protein (CAP) Acts Directly on F-actin to Accelerate Cofilin-mediated Actin Severing across the Range of Physiological pH*

    PubMed Central

    Normoyle, Kieran P. M.; Brieher, William M.

    2012-01-01

    Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization. PMID:22904322

  12. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  13. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells.

    PubMed

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnès; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J; Rider, Mark H; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca(2+)-dependent AMPK activation via calmodulin-dependent protein kinase kinase-beta(CaMKKbeta), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKbeta inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  14. Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter.

    PubMed Central

    Chung, Y T; Keller, E B

    1990-01-01

    The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the distal one of which controls synthesis of actin in a tissue- and developmental stage-specific manner. This very strong promoter has widely been used for expression of heterologous genes in cultured cells. To locate functional regulatory elements in this distal promoter, mutants of the promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. The results showed that the upstream end of the promoter extends to 522 bp from the transcription start site. In addition, there are two remote activating regions about 2 kb upstream. Between -522 and -379 are two regions that exert a strong negative effect. Downstream from these negative regions are at least six positive regions and a TATA element. The strongest positive determinant of the promoter was identified at -320 as AAAATGTG by footprinting and by a replacement experiment. When the relevant region was replaced by a synthetic sequence containing this element in a random context, the transient expression activity was restored. The sequence TGTATG located at -355 was also identified as a positive element by a similar replacement approach. Apparently the very high activity of this promoter is the result of the combined activities of multiple factors. Images PMID:2123290

  15. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    SciTech Connect

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  16. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis.

    PubMed

    Stossel, T P; Hartwig, J H

    1976-03-01

    Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.

  17. Nuclear actin-related proteins at the core of epigenetic control.

    PubMed

    Meagher, Richard B; Kandasamy, Muthugapatti K; Smith, Aaron P; McKinney, Elizabeth C

    2010-05-01

    Nuclear Actin-Related Proteins (ARPs) and actin combine as heterodimers to bind a large helicase subunit and form a core complex essential to the assembly and function of most chromatin remodeling and modifying machines. They are the most common shared subunits of these large and diverse assemblies in eukaryotes. We recently argued that most nuclear ARPs evolved directly from actin prior to the divergence of the eukaryotic kingdoms and did not evolve from pre-existing ARPs.2 Arabidopsis plants defective in nuclear ARP4, ARP5, ARP6, or ARP7 have extreme developmental phenotypes. Our recent publication demonstrates that ARP5-defective plants are not only dwarfed and have aberrant cell sizes, but are also hypersensitive to mutagenic agents that cause double strand DNA breaks.5 In Smith et al.6 we show that ARP6-defective plants, in addition to their extreme developmental phenotypes like small organs and early flowering, present an apparent "Phosphate Starvation Response" with strong morphological and molecular phenotypes. Herein, we interpret our latest data in the light of a hypothesis stating that in addition to their roles in overcoming DNA compaction that affects basal gene expression and silencing, nuclear ARP-containing chromatin complexes exert primary epigenetic control over high-level regulatory factors.

  18. Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia.

    PubMed

    Cohan, C S; Welnhofer, E A; Zhao, L; Matsumura, F; Yamashiro, S

    2001-02-01

    Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.

  19. Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

    PubMed Central

    Nakano, Kentaro; Satoh, Kazuomi; Morimatsu, Akeshi; Ohnuma, Masaaki; Mabuchi, Issei

    2001-01-01

    We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively. PMID:11694585

  20. The Nck family of adapter proteins: regulators of actin cytoskeleton.

    PubMed

    Buday, László; Wunderlich, Livius; Tamás, Peter

    2002-09-01

    SH2/SH3 domain-containing adapter proteins, such as the Nck family, play a major role in regulating tyrosine kinase signalling. They serve to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. Initially, it was not clear why cells from nematodes to vertebrates contain redundant and closely related SH2/SH3 adapters, such as Grb2, Crk and Nck. Recent evidence suggests that their biological roles are clearly different, whereas, for example, Grb2 connects activated receptor tyrosine kinases to Sos and Ras, leading to cell proliferation. The proteins of Nck family are implicated in organisation of actin cytoskeleton, cell movement or axon guidance in flies. In this review, the author attempts to summarise signalling pathways in which Nck plays a critical role.

  1. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

    PubMed

    Lila, T; Drubin, D G

    1997-02-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.

  2. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

    PubMed Central

    Lila, T; Drubin, D G

    1997-01-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions. Images PMID:9190214

  3. Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver.

    PubMed

    Poulter, Natalie S; Staiger, Christopher J; Rappoport, Joshua Z; Franklin-Tong, Vernonica E

    2010-03-01

    The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca(2+)-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.

  4. Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination

    PubMed Central

    Hao, Yi-Heng; Doyle, Jennifer M.; Ramanathan, Saumya; Gomez, Timothy S.; Jia, Da; Xu, Ming; Chen, Zhijian J.; Billadeau, Daniel D.; Rosen, Michael K.; Potts, Patrick Ryan

    2013-01-01

    SUMMARY Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a novel role for ubiquitination in this process. We find that the novel E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the Retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired Retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of Retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and Retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 and reveal novel aspects of retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex. PMID:23452853

  5. A new Tetrahymena actin-binding protein is localized in the division furrow.

    PubMed

    Watanabe, A; Kurasawa, Y; Watanabe, Y; Numata, O

    1998-04-01

    Using an F-actin affinity column, a 60 kDa fragment of a 71 kDa F-actin-binding protein was partially purified from Tetrahymena pyriformis. After digestion of the 60 kDa fragment with cyanogen bromide, the N-terminal 21-amino acid sequence of one of the resulting peptides was found to show sequence similarity to a region near the actin-binding site (amino acid residues 260-281) of yeast fimbrin. An antibody prepared against a synthesized 21-mer oligopeptide reacted with the 71 kDa proteins in T. pyriformis and T. thermophila cell extracts, suggesting that the 60 kDa fragment was produced from the 71 kDa protein through partial digestion occurring during isolation. The 60 kDa fragment bound to Tetrahymena F-actin as well as to rabbit skeletal muscle F-actin, and induced the bundling of Tetrahymena F-actin. Indirect immunofluorescence revealed colocalization of the 71 kDa protein and actin in the oral apparatus and the deep fiber bundles in T. pyriformis. On the other hand, in T. thermophila, the 71 kDa protein was localized in the oral apparatus and the contractile vacuole pores during the interphase. During cytokinesis, the 71 kDa protein was localized in the division furrow. Therefore, the 71 kDa protein seems to associate with the actin cytoskeleton, and to regulate the actin filament organization during phagocytosis and cytokinesis in Tetrahymena.

  6. Sequence and Comparative Genomic Analysis of Actin-related ProteinsD⃞

    PubMed Central

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-01-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of ∼700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4. PMID:16195354

  7. Mechanism for CARMIL protein inhibition of heterodimeric actin-capping protein.

    PubMed

    Kim, Taekyung; Ravilious, Geoffrey E; Sept, David; Cooper, John A

    2012-05-04

    Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP.

  8. Mechanism for CARMIL Protein Inhibition of Heterodimeric Actin-capping Protein*

    PubMed Central

    Kim, Taekyung; Ravilious, Geoffrey E.; Sept, David; Cooper, John A.

    2012-01-01

    Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP. PMID:22411988

  9. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    PubMed

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  10. A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells.

    PubMed

    Sheahan, Michael B; Staiger, Chris J; Rose, Ray J; McCurdy, David W

    2004-12-01

    The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.

  11. Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.

    PubMed

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.

  12. Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development.

    PubMed

    Lin, Zi Li; Cui, Xiang-Shun; Namgoong, Suk; Kim, Nam-Hyung

    2015-01-01

    Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.

  13. Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development

    PubMed Central

    LIN, Zi Li; CUI, Xiang-Shun; NAMGOONG, Suk; KIM, Nam-Hyung

    2015-01-01

    Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway. PMID:26052154

  14. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin.

    PubMed

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T; Rao, Madan; Mayor, Satyajit

    2015-11-05

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24-37 °C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface.

  15. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

    PubMed Central

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

    2015-01-01

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

  16. Rai14 (retinoic acid induced protein 14) is involved in regulating f-actin dynamics at the ectoplasmic specialization in the rat testis*.

    PubMed

    Qian, Xiaojing; Mruk, Dolores D; Cheng, C Yan

    2013-01-01

    Rai14 (retinoic acid induced protein 14) is an actin binding protein first identified in the liver, highly expressed in the placenta, the testis, and the eye. In the course of studying actin binding proteins that regulate the organization of actin filament bundles in the ectoplasmic specialization (ES), a testis-specific actin-rich adherens junction (AJ) type, Rai14 was shown to be one of the regulatory proteins at the ES. In the rat testis, Rai14 was found to be expressed by Sertoli and germ cells, structurally associated with actin and an actin cross-linking protein palladin. Its expression was the highest at the ES in the seminiferous epithelium of adult rat testes, most notably at the apical ES at the Sertoli-spermatid interface, and expressed stage-specifically during the epithelial cycle in stage VII-VIII tubules. However, Rai14 was also found at the basal ES near the basement membrane, associated with the blood-testis barrier (BTB) in stage VIII-IX tubules. A knockdown of Rai14 in Sertoli cells cultured in vitro by RNAi was found to perturb the Sertoli cell tight junction-permeability function in vitro, mediated by a disruption of F-actin, which in turn led to protein mis-localization at the Sertoli cell BTB. When Rai14 in the testis in vivo was knockdown by RNAi, defects in spermatid polarity and adhesion, as well as spermatid transport were noted mediated via changes in F-actin organization and mis-localization of proteins at the apical ES. In short, Rai14 is involved in the re-organization of actin filaments in Sertoli cells during the epithelial cycle, participating in conferring spermatid polarity and cell adhesion in the testis.

  17. Role of the Pi3k Regulatory Subunit in the Control of Actin Organization and Cell Migration

    PubMed Central

    Jiménez, Concepción; Portela, Rosario Armas; Mellado, Mario; Rodríguez-Frade, Jose Miguel; Collard, John; Serrano, Antonio; Martínez-A, Carlos; Avila, Jesus; Carrera, Ana C.

    2000-01-01

    Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85α regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85α in controlling PDGF receptor–induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics. PMID:11038173

  18. DNA binding properties of the actin-related protein Arp8 and its role in DNA repair.

    PubMed

    Osakabe, Akihisa; Takahashi, Yuichiro; Murakami, Hirokazu; Otawa, Kenji; Tachiwana, Hiroaki; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ich; Kurumizaka, Hitoshi; Harata, Masahiko

    2014-01-01

    Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair.

  19. Iron regulatory proteins in pathobiology.

    PubMed Central

    Cairo, G; Pietrangelo, A

    2000-01-01

    The capacity of readily exchanging electrons makes iron not only essential for fundamental cell functions, but also a potential catalyst for chemical reactions involving free-radical formation and subsequent oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated in order to maintain an adequate substrate while also minimizing the pool of potentially toxic 'free iron'. Iron homoeostasis is controlled through several genes, an increasing number of which have been found to contain non-coding sequences [i.e. the iron-responsive elements (IREs)] which are recognized at the mRNA level by two cytoplasmic iron-regulatory proteins (IRP-1 and IRP-2). The IRPs belong to the aconitase superfamily. By means of an Fe-S-cluster-dependent switch, IRP-1 can function as an mRNA-binding protein or as an enzyme that converts citrate into isocitrate. Although structurally and functionally similar to IRP-1, IRP-2 does not seem to assemble a cluster nor to possess aconitase activity; moreover, it has a distinct pattern of tissue expression and is modulated by means of proteasome-mediated degradation. In response to fluctuations in the level of the 'labile iron pool', IRPs act as key regulators of cellular iron homoeostasis as a result of the translational control of the expression of a number of iron metabolism-related genes. Conversely, various agents and conditions may affect IRP activity, thereby modulating iron and oxygen radical levels in different pathobiological settings. As the number of mRNAs regulated through IRE-IRP interactions keeps growing, the definition of IRPs as iron-regulatory proteins may in the near future become limiting as their role expands to other essential metabolic pathways. PMID:11085915

  20. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

    NASA Astrophysics Data System (ADS)

    Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

    1999-10-01

    Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

  1. Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection*

    PubMed Central

    Gordón-Alonso, Mónica; Rocha-Perugini, Vera; Álvarez, Susana; Ursa, Ángeles; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Muñoz-Fernández, María A.; Sánchez-Madrid, Francisco

    2013-01-01

    HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4+ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1. PMID:23926103

  2. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  3. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  4. Actin-crosslinking protein regulation of filament movement in motility assays: a theoretical model.

    PubMed Central

    Janson, L W; Taylor, D L

    1994-01-01

    The interaction of single actin filaments on a myosin-coated coverslip has been modeled by several authors. One model adds a component of "frictional drag" by myosin heads that oppose movement of the actin filaments. We have extended this concept by including the resistive drag from actin crosslinking proteins to understand better the relationship among crosslinking number, actin-myosin force generation, and motility. The validity of this model is supported by agreement with the experimental results from a previous study in which crosslinking proteins were added with myosin molecules under otherwise standard motility assay conditions. The theoretical relationship provides a means to determine many physical parameters that characterize the interaction between a single actin filament and a single actin-crosslinking molecule (various types). In particular, the force constant of a single filamin molecule is calculated as 1.105 pN, approximately 3 times less than a driving myosin head (3.4 pN). Knowledge of this parameter and others derived from this model allows a better understanding of the interaction between myosin and the actin/actin-binding protein cytoskeleton and the role of actin-binding proteins in the regulation and modulation of motility. PMID:7811954

  5. Regulation of osteoclast differentiation and actin ring formation by the cytolinker protein plectin.

    PubMed

    Matsubara, Takuma; Kinbara, Masayuki; Maeda, Toshihiro; Yoshizawa, Mitsuhiro; Kokabu, Shoichiro; Takano Yamamoto, Teruko

    2017-08-05

    Osteoclasts are cells that resorb the bone matrix and maintain bone and calcium homeostasis. An actin ring is a characteristic actin structure that is essential for bone resorption by osteoclasts. Tyrosine kinase Src deficient osteoclasts do not form actin rings; thus, Src is a key molecule for actin ring formation in osteoclasts. However, how Src regulates actin ring formation is not fully understood. We identified the cytolinker protein plectin as a Src-binding protein by immunoprecipitation and liquid chromatography tandem mass spectrometry. Plectin is a huge protein (>500 kDa) and regulates the cytoskeleton by binding to actin and tubulin. We assessed the expression and role of plectin in osteoclasts. Plectin was expressed and co-localized with Src close to the actin ring in osteoclasts. Moreover, plectin was tyrosine-phosphorylated by Src. Differentiation and actin ring formation were inhibited by downregulation of plectin. These results suggest an important role for plectin in osteoclast differentiation and actin ring formation through Src binding. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F- actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity. PMID:3972891

  7. Game of Zones: how actin-binding proteins organize muscle contraction

    PubMed Central

    Butkevich, Eugenia; Klopfenstein, Dieter R.; Schmidt, Christoph F.

    2016-01-01

    ABSTRACT Locomotion of C. elegans requires coordinated, efficient transmission of forces generated on the molecular scale by myosin and actin filaments in myocytes to dense bodies and the hypodermis and cuticle enveloping body wall muscles. The complex organization of the acto-myosin scaffold with its accessory proteins provides a fine-tuned machinery regulated by effectors that guarantees that sarcomere units undergo controlled, reversible cycles of contraction and relaxation. Actin filaments in sarcomeres dynamically undergo polymerization and depolymerization. In a recent study, the actin-binding protein DBN-1, the C. elegans ortholog of human drebrin and drebrin-like proteins, was discovered to stabilize actin in muscle cells. DBN-1 reversibly changes location between actin filaments and myosin-rich regions during muscle contraction. Mutations in DBN-1 result in mislocalization of other actin-binding proteins. Here we discuss implications of this finding for the regulation of sarcomere actin stability and the organization of other actin-binding proteins. PMID:27383012

  8. Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins.

    PubMed Central

    Leinweber, B D; Leavis, P C; Grabarek, Z; Wang, C L; Morgan, K G

    1999-01-01

    An interaction between extracellular regulated kinase 1 (ERK1) and calponin has previously been reported (Menice, Hulvershorn, Adam, Wang and Morgan (1997) J. Biol. Chem. 272 (40), 25157-25161) and has been suggested to reflect a function of calponin as a signalling molecule. We report in this study that calponin binds to both ERK1 and ERK2 under native conditions as well as in an overlay assay. Using chymotryptic fragments of calponin, the binding site of ERK on calponin was identified as the calponin homology (CH) domain, an N-terminal region of calponin found in other actin-binding proteins. ERK also bound, in a gel overlay assay, alpha-actinin, a protein with two tandem CH domains, as well as a 27 kDa thermolysin product of alpha-actinin containing the CH domains of alpha-actinin. The CH domain of calponin could compete with intact calponin or alpha-actinin for ERK binding. Titration of acrylodan-labelled calponin with ERK gave a K(a) of 6x10(6) M(-1) and titration of acrylodan-labelled calponin with a peptide from the alphaL16 helix of ERK gave a K(a) of 1x10(6) M(-1). Recombinant ERK was found to co-sediment with purified actin and induced a fluorescence change in pyrene-labelled F-actin (K(a)=5x10(6) M(-1)). The interaction of ERK with CH domains points to a new potential function for CH domains. The interaction of ERK with actin raises the possibility that actin may provide a scaffold for ERK signalling complexes in both muscle and non-muscle cells. PMID:10548541

  9. Actin-associated Proteins in the Pathogenesis of Podocyte Injury.

    PubMed

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-11-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies.

  10. Protein Kinase D Controls Actin Polymerization and Cell Motility through Phosphorylation of Cortactin*

    PubMed Central

    Eiseler, Tim; Hausser, Angelika; De Kimpe, Line; Van Lint, Johan; Pfizenmaier, Klaus

    2010-01-01

    We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility. PMID:20363754

  11. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division.

    PubMed

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-08-25

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

  12. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    PubMed Central

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-01-01

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

  13. Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

    PubMed

    Sato, Osamu; Komatsu, Satoshi; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Tanaka, Ryosuke; Mizutani, Takeomi; Watanabe, Tomonobu M; Ikebe, Reiko; Ikebe, Mitsuo

    2017-06-30

    Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s(-1) for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s(-1) We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Yamada, Noboru; Kagawa, Takatoshi; Yonekura, Hisashi; Uyeda, Taro Q P; Kadota, Akeo; Wada, Masamitsu

    2010-05-11

    Organelle movement is essential for efficient cellular function in eukaryotes. Chloroplast photorelocation movement is important for plant survival as well as for efficient photosynthesis. Chloroplast movement generally is actin dependent and mediated by blue light receptor phototropins. In Arabidopsis thaliana, phototropins mediate chloroplast movement by regulating short actin filaments on chloroplasts (cp-actin filaments), and the chloroplast outer envelope protein CHUP1 is necessary for cp-actin filament accumulation. However, other factors involved in cp-actin filament regulation during chloroplast movement remain to be determined. Here, we report that two kinesin-like proteins, KAC1 and KAC2, are essential for chloroplasts to move and anchor to the plasma membrane. A kac1 mutant showed severely impaired chloroplast accumulation and slow avoidance movement. A kac1kac2 double mutant completely lacked chloroplast photorelocation movement and showed detachment of chloroplasts from the plasma membrane. KAC motor domains are similar to those of the kinesin-14 subfamily (such as Ncd and Kar3) but do not have detectable microtubule-binding activity. The C-terminal domain of KAC1 could interact with F-actin in vitro. Instead of regulating microtubules, KAC proteins mediate chloroplast movement via cp-actin filaments. We conclude that plants have evolved a unique mechanism to regulate actin-based organelle movement using kinesin-like proteins.

  15. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

  16. Immunoinhibitory adapter protein Src homology domain 3 lymphocyte protein 2 (SLy2) regulates actin dynamics and B cell spreading.

    PubMed

    von Holleben, Max; Gohla, Antje; Janssen, Klaus-Peter; Iritani, Brian M; Beer-Hammer, Sandra

    2011-04-15

    Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.

  17. A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins

    PubMed Central

    Revenu, Céline; Ubelmann, Florent; Hurbain, Ilse; El-Marjou, Fatima; Dingli, Florent; Loew, Damarys; Delacour, Delphine; Gilet, Jules; Brot-Laroche, Edith; Rivero, Francisco; Louvard, Daniel; Robine, Sylvie

    2012-01-01

    Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology. PMID:22114352

  18. The actin binding protein profilin 2 is a novel regulator of iron homeostasis.

    PubMed

    Luscieti, Sara; Galy, Bruno; Gutierrez, Lucia; Reinke, Michael; Couso, Jorge; Shvartsman, Maya; Di Pascale, Antonio; Witke, Walter; Hentze, Matthias W; Pilo Boyl, Pietro; Sanchez, Mayka

    2017-08-03

    Cellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis-regulatory iron-responsive elements (IRE) on target mRNAs. We identified the profilin2 (Pfn2) mRNA, which encodes an actin binding protein involved in endocytosis and neurotransmitters release, as a novel IRP-interacting transcript and studied its role in iron metabolism. Combination of EMSA experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 3' untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal-IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Over-expression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus and midbrain) and reduction of the hepatic iron store without anaemia. In spite of low liver iron levels, hepatic hepcidin expression remained high, likely due to compensatory activation of Hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice. Copyright © 2017 American Society of Hematology.

  19. An antifungal protein from Ginkgo biloba binds actin and can trigger cell death.

    PubMed

    Gao, Ningning; Wadhwani, Parvesh; Mühlhäuser, Philipp; Liu, Qiong; Riemann, Michael; Ulrich, Anne S; Nick, Peter

    2016-07-01

    Ginkbilobin is a short antifungal protein that had been purified and cloned from the seeds of the living fossil Ginkgo biloba. Homologues of this protein can be detected in all seed plants and the heterosporic fern Selaginella and are conserved with respect to domain structures, peptide motifs, and specific cysteine signatures. To get insight into the cellular functions of these conserved motifs, we expressed green fluorescent protein fusions of full-length and truncated ginkbilobin in tobacco BY-2 cells. We show that the signal peptide confers efficient secretion of ginkbilobin. When this signal peptide is either cleaved or masked, ginkbilobin binds and visualizes the actin cytoskeleton. This actin-binding activity of ginkbilobin is mediated by a specific subdomain just downstream of the signal peptide, and this subdomain can also coassemble with actin in vitro. Upon stable overexpression of this domain, we observe a specific delay in premitotic nuclear positioning indicative of a reduced dynamicity of actin. To elucidate the cellular response to the binding of this subdomain to actin, we use chemical engineering based on synthetic peptides comprising different parts of the actin-binding subdomain conjugated with the cell-penetrating peptide BP100 and with rhodamine B as a fluorescent reporter. Binding of this synthetic construct to actin efficiently induces programmed cell death. We discuss these findings in terms of a working model, where ginkbilobin can activate actin-dependent cell death.

  20. Polycystin-2 (TRPP2) Regulation by Ca2+ Is Effected and Diversified by Actin-Binding Proteins

    PubMed Central

    Cantero, María del Rocío; Cantiello, Horacio F.

    2015-01-01

    Calcium regulation of Ca2+-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca2+ permeability. The molecular mechanisms associated with PC2 regulation by Ca2+ remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2hst) but not the in vitro translated protein (PC2iv), functionally responds to changes in intracellular (cis) Ca2+. In this study we determined the regulatory effect(s) of Ca2+-sensitive and -insensitive actin-binding proteins (ABPs) on PC2iv channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2iv channel function in the presence of cis Ca2+, although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2iv channel function in the presence, but no effect in the absence of cis Ca2+. Gelsolin stimulated PC2iv channel function in the presence, but not the absence of cis Ca2+. In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2iv channel function both in the presence and absence of Ca2+. The distinct effect(s) of the ABPs on PC2iv channel function demonstrate that Ca2+ regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca2+-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation. PMID:25954877

  1. CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle.

    PubMed

    Nomura, Kazumi; Ono, Kanako; Ono, Shoichiro

    2012-09-01

    Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

  2. The interaction between the adaptor protein APS and Enigma is involved in actin organisation.

    PubMed

    Barrès, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2005-08-15

    APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.

  3. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    NASA Astrophysics Data System (ADS)

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  4. CASK and protein 4.1 support F-actin nucleation on neurexins.

    PubMed

    Biederer, T; Sudhof, T C

    2001-12-21

    Rearrangements of the actin cytoskeleton are involved in a variety of cellular processes from locomotion of cells to morphological alterations of the cell surface. One important question is how local interactions of cells with the extracellular space are translated into alterations of their membrane organization. To address this problem, we studied CASK, a member of the membrane-associated guanylate kinase homologues family of adaptor proteins. CASK has been shown to bind the erythrocyte isoform of protein 4.1, a class of proteins that promote formation of actin/spectrin microfilaments. In neurons, CASK also interacts via its PDZ domain with the cytosolic C termini of neurexins, neuron-specific cell-surface proteins. We now show that CASK binds a brain-enriched isoform of protein 4.1, and nucleates local assembly of actin/spectrin filaments. These interactions can be reconstituted on the cytosolic tail of neurexins. Furthermore, CASK can be recovered with actin filaments prepared from rat brain extracts, and neurexins are recruited together with CASK and protein 4.1 into these actin filaments. Thus, analogous to the PDZ-domain protein p55 and glycophorin C at the erythrocyte membrane, a similar complex comprising CASK and neurexins exists in neurons. Our data suggest that intercellular junctions formed by neurexins, such as junctions initiated by beta-neurexins with neuroligins, are at least partially coupled to the actin cytoskeleton via an interaction with CASK and protein 4.1.

  5. Planar Cell Polarity (PCP) Protein Vangl2 Regulates Ectoplasmic Specialization Dynamics via Its Effects on Actin Microfilaments in the Testes of Male Rats.

    PubMed

    Chen, Haiqi; Mruk, Dolores D; Lee, Will M; Cheng, C Yan

    2016-05-01

    Planar cell polarity (PCP) proteins confer polarization of a field of cells (eg, elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (eg, Van Gogh-like 2 [Vangl2]), effectors, ligands, and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific, actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment and also Sertoli-Sertoli interface at the blood-testis barrier (BTB) and structurally interacted with actin, N-cadherin, and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNA interference in Sertoli cells cultured in vitro with an established tight junction-permeability barrier led to BTB tightening, whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin-regulatory proteins, epidermal growth factor receptor pathway substrate 8, actin-related protein 3, and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. Using Polyplus in vivo-jetPEI reagent as a transfection medium to silence Vangl2 in the testis in vivo by RNA interference with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and spermatid polarity, meiosis, and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, the PCP protein Vangl2 is an ES regulator through its effects on actin microfilaments in the testis.

  6. Planar Cell Polarity (PCP) Protein Vangl2 Regulates Ectoplasmic Specialization Dynamics via Its Effects on Actin Microfilaments in the Testes of Male Rats

    PubMed Central

    Chen, Haiqi; Mruk, Dolores D.; Lee, Will M.

    2016-01-01

    Planar cell polarity (PCP) proteins confer polarization of a field of cells (eg, elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (eg, Van Gogh-like 2 [Vangl2]), effectors, ligands, and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific, actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment and also Sertoli-Sertoli interface at the blood-testis barrier (BTB) and structurally interacted with actin, N-cadherin, and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNA interference in Sertoli cells cultured in vitro with an established tight junction-permeability barrier led to BTB tightening, whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin-regulatory proteins, epidermal growth factor receptor pathway substrate 8, actin-related protein 3, and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. Using Polyplus in vivo-jetPEI reagent as a transfection medium to silence Vangl2 in the testis in vivo by RNA interference with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and spermatid polarity, meiosis, and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, the PCP protein Vangl2 is an ES regulator through its effects on actin microfilaments in the testis. PMID:26990065

  7. Isolation and partial characterization of a 110-kD dimer actin-binding protein

    PubMed Central

    1986-01-01

    Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I. PMID:2942552

  8. THRUMIN1 is a light-regulated actin-bundling protein involved in chloroplast motility.

    PubMed

    Whippo, Craig W; Khurana, Parul; Davis, Phillip A; DeBlasio, Stacy L; DeSloover, Daniel; Staiger, Christopher J; Hangarter, Roger P

    2011-01-11

    Chloroplast movement in response to changing light conditions optimizes photosynthetic light absorption. This repositioning is stimulated by blue light perceived via the phototropin photoreceptors and is transduced to the actin cytoskeleton. Some actin-based motility systems use filament reorganizations rather than myosin-based translocations. Recent research favors the hypothesis that chloroplast movement is driven by actin reorganization at the plasma membrane, but no proteins affecting chloroplast movements have been shown to associate with both the plasma membrane and actin filaments in vivo. Here we identified THRUMIN1 as a critical link between phototropin photoreceptor activity at the plasma membrane and actin-dependent chloroplast movements. THRUMIN1 bundles filamentous actin in vitro, and it localizes to the plasma membrane and displays light- and phototropin-dependent localization to microfilaments in vivo. These results suggest that phototropin-induced actin bundling via THRUMIN1 is important for chloroplast movement. A mammalian homolog of THRUMIN1, GRXCR1, has been implicated in auditory responses and hair cell stereocilla development as a regulator of actin architecture. Studies of THRUMIN1 will help elucidate the function of this family of eukaryotic proteins.

  9. Inhibition of tobacco mosaic virus movement by expression of an actin-binding protein.

    PubMed

    Hofmann, Christina; Niehl, Annette; Sambade, Adrian; Steinmetz, André; Heinlein, Manfred

    2009-04-01

    The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.

  10. Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments.

    PubMed

    Klauschies, F; Gützkow, T; Hinkelmann, S; von Messling, V; Vaske, B; Herrler, G; Haas, L

    2010-09-01

    To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

  11. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite

    PubMed Central

    Ganter, Markus; Rizopoulos, Zaira; Schüler, Herwig; Matuschewski, Kai

    2015-01-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs. PMID:25565321

  12. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite.

    PubMed

    Ganter, Markus; Rizopoulos, Zaira; Schüler, Herwig; Matuschewski, Kai

    2015-04-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs.

  13. A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

    SciTech Connect

    Margarit,S.; Davidson, W.; Frego, L.; Stebbins, F.

    2006-01-01

    Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

  14. Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin.

    PubMed

    Pivovarova, Anastasia V; Chebotareva, Natalia A; Chernik, Ivan S; Gusev, Nikolai B; Levitsky, Dmitrii I

    2007-11-01

    Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.

  15. A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

    PubMed Central

    Hopmann, Roberta; Miller, Kathryn G.

    2003-01-01

    Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell. PMID:12529431

  16. Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly*

    PubMed Central

    Yu-Kemp, Hui-Chia; Brieher, William M.

    2016-01-01

    Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation. PMID:26598519

  17. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

    PubMed Central

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-01-01

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  18. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Regulation of blood-testis barrier by actin binding proteins and protein kinases

    PubMed Central

    Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

    2016-01-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  20. Regulation of blood-testis barrier by actin binding proteins and protein kinases.

    PubMed

    Li, Nan; Tang, Elizabeth I; Cheng, C Yan

    2016-03-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases.

  1. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

    PubMed

    Morita, Tsuyoshi; Hayashi, Ken'ichiro

    2013-08-02

    Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction.

  2. [The interactions of actin cell and membrane skeleton proteins with lipids].

    PubMed

    Bok, Ewa; Hryniewicz-Jankowska, Anita; Sikorski, Aleksander F

    2009-01-01

    The results of many studies indicate that many cytoskeletal proteins interact with lipids, or are regulated by phosphoinositides. Proteins may associate with membranes through specific domains, amphipathic helices and undefined motifs that interact through electrostatic or hydrophobic interactions. The interaction between specific proteins and certain lipids affect stabilization of lipid microdomains, which may provide an anchor for cytoskeletal proteins. In vitro, proteins that sever or depolymerize actin filaments, such as gelsolin, villin, cofilin and profilin, are inactivated by PI(4,5)P2. Conversely, proteins like e.g. vinculin, talin, alpha-actinin, ezrin, N-WASP, WAVE that promote actin polymerization and link actin filaments to each other, are activated by this lipid. The major components of red blood cell membrane skeleton: spectrin and protein 4.1, also their nonerythroid counterparts interact with lipids, and those interactions may be regulated by phosphoinositides.

  3. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.

    PubMed

    Hubberstey, Andrew V; Mottillo, Emilio P

    2002-04-01

    Many extracellular signals elicit changes in the actin cytoskeleton, which are mediated through an array of signaling proteins and pathways. One family of proteins that plays a role in regulating actin remodeling in response to cellular signals are the cyclase-associated proteins (CAPs). CAPs are highly conserved monomeric actin binding proteins present in a wide range of organisms including yeast, fly, plants, and mammals. The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding, and oligomerization. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multicellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity. This review will highlight the critical structural and functional domains of CAPs, describe recent studies that have implied important roles for these proteins in linking cell signaling with actin polymerization, and highlight their roles in vesicle trafficking and development.

  4. The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly

    PubMed Central

    1993-01-01

    Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F- actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization. PMID:8432731

  5. EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

    PubMed

    Prasath, Thiruketheeswaran; Greven, Hartmut; D'Haese, Jochen

    2012-06-01

    Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

  6. ZNF185, an actin-cytoskeleton-associated growth inhibitory LIM protein in prostate cancer.

    PubMed

    Zhang, J-S; Gong, A; Young, C Y F

    2007-01-04

    We have recently identified ZNF185 as a gene that is downregulated in prostate cancer (PCa), in part via epigenetic alteration, and maybe associated with disease progression. In this study, we cloned the ZNF185 cDNA from normal human prostate tissues and investigated its biological function. We show that ZNF185 is a novel actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein that localizes to F-actin structures, and is enriched at focal adhesions. We find that the NH(2)-terminal region, which we designate the actin-targeting domain, facilitates ZNF185 binding to actin in vitro and is both necessary and sufficient to mediate actin-cytoskeleton targeting of ZNF185, whereas the LIM domain, which is localized in the COOH-terminus is dispensable for this phenomenon. Interestingly, ectopic expression of full-length ZNF185, but not a mutant lacking the actin-targeting domain, could suppress proliferation and anchorage-independent growth of PCa cells. Together, our data suggest that ZNF185 may function as a tumor-suppressor protein by associating with the actin-cytoskeleton.

  7. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    PubMed Central

    Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

    2013-01-01

    The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

  8. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    PubMed Central

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  9. Differential effects of thin and thick filament disruption on zebrafish smooth muscle regulatory proteins

    PubMed Central

    Davuluri, G.; Seiler, C.; Abrams, J.; Soriano, A. J.; Pack, M.

    2013-01-01

    Background The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known. Methods Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae. Key Results Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae. Conclusions & Inferences These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism. PMID:20591105

  10. Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

    PubMed

    Jouhet, Juliette; Gray, John C

    2009-10-01

    Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

  11. F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse.

    PubMed

    Pérez-Martínez, Manuel; Gordón-Alonso, Mónica; Cabrero, José Román; Barrero-Villar, Marta; Rey, Mercedes; Mittelbrunn, María; Lamana, Amalia; Morlino, Giulia; Calabia, Carmen; Yamazaki, Hiroyuki; Shirao, Tomoaki; Vázquez, Jesús; González-Amaro, Roberto; Veiga, Esteban; Sánchez-Madrid, Francisco

    2010-04-01

    The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

  12. Identification of Arabidopsis cyclase-associated protein 1 as the first nucleotide exchange factor for plant actin.

    PubMed

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias; Blanchoin, Laurent; Staiger, Christopher J

    2007-08-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP-actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP- and ATP-monomeric actin (Kd approximately 1.3 microM). Binding of AtCAP1 to ATP-actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux

  13. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits

    PubMed Central

    Ferguson, Shawn M.; Raimondi, Andrea; Paradise, Summer; Shen, Hongying; Mesaki, Kumi; Ferguson, Agnes; Destaing, Olivier; Ko, Genevieve; Takasaki, Junko; Cremona, Ottavio; O’ Toole, Eileen; De Camilli, Pietro

    2010-01-01

    SUMMARY The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin nucleating proteins, actin and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wildtype cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin, and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin’s endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity. PMID:20059951

  14. How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

    NASA Technical Reports Server (NTRS)

    Tseng, Yiider; Kole, Thomas P.; Lee, Jerry S H.; Fedorov, Elena; Almo, Steven C.; Schafer, Benjamin W.; Wirtz, Denis

    2005-01-01

    Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.

  15. How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

    NASA Technical Reports Server (NTRS)

    Tseng, Yiider; Kole, Thomas P.; Lee, Jerry S H.; Fedorov, Elena; Almo, Steven C.; Schafer, Benjamin W.; Wirtz, Denis

    2005-01-01

    Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.

  16. An Actin-Binding Protein, LlLIM1, Mediates Calcium and Hydrogen Regulation of Actin Dynamics in Pollen Tubes1[C][W][OA

    PubMed Central

    Wang, Huei-Jing; Wan, Ai-Ru; Jauh, Guang-Yuh

    2008-01-01

    Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca2+: LlLIM1 showed a preference for F-actin binding under low pH and low Ca2+ concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed. PMID:18480376

  17. Protein Kinase D1 regulates Cofilin mediated F-actin reorganization and cell motility via Slingshot

    PubMed Central

    Eiseler, Tim; Döppler, Heike; Yan, Irene K.; Kitatani, Kanae; Mizuno, Kensaku; Storz, Peter

    2009-01-01

    Dynamic actin remodelling processes at the leading edge of migrating tumour cells are concerted events controlled by a fine-tuned temporal and spatial interplay of kinases and phosphatases. Actin severing is regulated by ADF/Cofilin which regulates stimulus-induced lamellipodia protrusion and directed cell motility. Cofilin is activated by dephosphorylation via phosphatases of the slingshot (SSH) family. SSH activity is strongly increased by its binding to filamentous actin (F-actin), however, other upstream regulators remain unknown. We show that in response to RhoA activation, Protein Kinase D1 (PKD1) phosphorylates the SSH enzyme SSH1L at a serine residue located in its actin binding motif. This generates a 14-3-3 binding motif, blocks the localization of SSH1L to F-actin-rich structures in the lamellipodium by sequestering it in the cytoplasm. Consequently, expression of constitutively-active PKD1 in invasive tumour cells enhanced phosphorylation of cofilin and effectively blocked the formation of free actin filament barbed ends and directed cell migration. PMID:19329994

  18. Characterization and regulation of an additional actin-filament-binding site in large isoforms of the stereocilia actin-bundling protein espin.

    PubMed

    Zheng, Lili; Beeler, Dina M; Bartles, James R

    2014-03-15

    The espin actin-bundling proteins, which are produced as isoforms of different sizes from a single gene, are required for the growth of hair cell stereocilia. We have characterized an additional actin-filament-binding site present in the extended amino-termini of large espin isoforms. Constitutively active in espin 2, the site increased the size of actin bundles formed in vitro and inhibited actin fluorescence recovery in microvilli. In espin 1, which has an N-terminal ankyrin repeat domain, the site was autoinhibited by binding between the ankyrin repeat domain and a peptide near the actin-binding site. Deletion of this peptide from espin 1 activated its actin-binding site. The peptide resembled tail homology domain I of myosin III, a ligand of the ankyrin repeat domain localized with espin 1 at the tip of stereocilia. A myosin III tail homology domain I peptide, but not scrambled control peptides, inhibited internal binding of the ankyrin repeat domain and released the espin 1 actin-binding site from autoinhibition. Thus, this regulation could result in local activation of the additional actin-binding site of espin 1 by myosin III in stereocilia.

  19. Danio rerio αE-catenin Is a Monomeric F-actin Binding Protein with Distinct Properties from Mus musculus αE-catenin*

    PubMed Central

    Miller, Phillip W.; Pokutta, Sabine; Ghosh, Agnidipta; Almo, Steven C.; Weis, William I.; Nelson, W. James; Kwiatkowski, Adam V.

    2013-01-01

    It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and β-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements. PMID:23788645

  20. Danio rerio αE-catenin is a monomeric F-actin binding protein with distinct properties from Mus musculus αE-catenin.

    PubMed

    Miller, Phillip W; Pokutta, Sabine; Ghosh, Agnidipta; Almo, Steven C; Weis, William I; Nelson, W James; Kwiatkowski, Adam V

    2013-08-02

    It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and β-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.

  1. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast.

    PubMed

    Gieselmann, R; Mann, K

    1992-02-24

    A new 56 kDa actin-binding protein (ASP-56) was isolated from pig platelet lysate. In falling ball viscosimetry it caused a reduction in viscosity that could be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD-labelled actin showed reduction of polymeric actin, too. These results could be explained by sequestering of actin in a non-polymerizable 1:1 ASP-56/actin complex. Sequencing of about 20 tryptic peptides of ASP-56 and comparison with known sequences revealed about 60% homology to the adenylate cyclase-associated protein (CAP) from yeast.

  2. Protein Disulfide Isomerase Directly Interacts with β-Actin Cys374 and Regulates Cytoskeleton Reorganization*

    PubMed Central

    Sobierajska, Katarzyna; Skurzynski, Szymon; Stasiak, Marta; Kryczka, Jakub; Cierniewski, Czeslaw S.; Swiatkowska, Maria

    2014-01-01

    Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the β-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with β-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and β-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbβ3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the β-actin Cys374 thiol. Formation of the β-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with β-actin when MEG-01 cells adhere via the αIIbβ3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in β-actin via a redox-dependent mechanism. PMID:24415753

  3. Modulation of Rho GTPases and the actin cytoskeleton by Yersinia outer proteins (Yops).

    PubMed

    Aepfelbacher, M; Heesemann, J

    2001-09-01

    Pathogenic species of the genus Yersinia employ a type III secretion apparatus to inject up to six effector proteins (Yersinia outer proteins; Yops) into host cells. Thereby yersiniae disarm the immune cell system of the host to proliferate extracellularly. At least four of the Yop effectors (YopE, YpkA/YopO, YopT and YopH) are involved in the rearrangement of the actin cytoskeleton: YopE, YopT and YpkA/YopO modulate the activity of actin-regulating Rho GTP-binding proteins, whereas YopH dephosphorylates phospho-tyrosine residues in focal adhesion proteins. In this review we will focus on recent evidence implicating Rho GTPases and the actin cytoskeleton as major targets of Yersinia Yops.

  4. Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum

    PubMed Central

    Ishikawa-Ankerhold, Hellen C.; Daszkiewicz, Wioleta; Schleicher, Michael; Müller-Taubenberger, Annette

    2017-01-01

    Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation. PMID:28074884

  5. Structure and mechanism of mouse cyclase-associated protein (CAP1) in regulating actin dynamics.

    PubMed

    Jansen, Silvia; Collins, Agnieszka; Golden, Leslie; Sokolova, Olga; Goode, Bruce L

    2014-10-31

    Srv2/CAP is a conserved actin-binding protein with important roles in driving cellular actin dynamics in diverse animal, fungal, and plant species. However, there have been conflicting reports about whether the activities of Srv2/CAP are conserved, particularly between yeast and mammalian homologs. Yeast Srv2 has two distinct functions in actin turnover: its hexameric N-terminal-half enhances cofilin-mediated severing of filaments, while its C-terminal-half catalyzes dissociation of cofilin from ADP-actin monomers and stimulates nucleotide exchange. Here, we dissected the structure and function of mouse CAP1 to better understand its mechanistic relationship to yeast Srv2. Although CAP1 has a shorter N-terminal oligomerization sequence compared with Srv2, we find that the N-terminal-half of CAP1 (N-CAP1) forms hexameric structures with six protrusions, similar to N-Srv2. Further, N-CAP1 autonomously binds to F-actin and decorates the sides and ends of filaments, altering F-actin structure and enhancing cofilin-mediated severing. These activities depend on conserved surface residues on the helical-folded domain. Moreover, N-CAP1 enhances yeast cofilin-mediated severing, and conversely, yeast N-Srv2 enhances human cofilin-mediated severing, highlighting the mechanistic conservation between yeast and mammals. Further, we demonstrate that the C-terminal actin-binding β-sheet domain of CAP1 is sufficient to catalyze nucleotide-exchange of ADP-actin monomers, while in the presence of cofilin this activity additionally requires the WH2 domain. Thus, the structures, activities, and mechanisms of mouse and yeast Srv2/CAP homologs are remarkably well conserved, suggesting that the same activities and mechanisms underlie many of the diverse actin-based functions ascribed to Srv2/CAP homologs in different organisms.

  6. Bacterial nucleators: actin' on actin

    PubMed Central

    Bugalhão, Joana N.; Mota, Luís Jaime; Franco, Irina S.

    2015-01-01

    The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways. PMID:26416078

  7. A posttranslational modification of beta-actin contributes to the slow dissociation of the spectrin-protein 4.1-actin complex of irreversibly sickled cells

    PubMed Central

    1995-01-01

    Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin. PMID:7876306

  8. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  9. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  10. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    PubMed

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

  11. [Some properties of complexes formed by small heat shock proteins with denatured actin].

    PubMed

    Pivovarova, A V; Chebotareva, N A; Guseev, N B; Levitskiĭ, D I

    2008-01-01

    We applied different methods to analyze the effects of the recombinant wild-type small heat shock protein with an apparent molecular mass of 27 kD (Hsp27-wt) and its S15,78,82D mutant (Hsp27-3D), which mimics the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin. It has been shown that, at the weight ratio of Hsp27/actin equal to 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal unfolding of F-actin but effectively prevent the aggregation of F-actin by forming soluble complexes with denatured actin. The formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. It is known that Hsp27-wt forms high-molecular-mass oligomers, whereas Hsp27-3D forms small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17-18 nm. On the other hand, the sedimentation coefficients of these complexes were distributed within the range 10-45 S in the case of Hsp27-3D and 18-60 S in the case of Hsp27-wt. Thus, the ability of Hsp27 to form soluble complexes with denatured actin does not significantly depend on the initial oligomeric state of Hsp27.

  12. FHOD proteins in actin dynamics--a formin' class of its own.

    PubMed

    Bechtold, Meike; Schultz, Jörg; Bogdan, Sven

    2014-01-01

    Eukaryotic cells have evolved a variety of actin-binding proteins to regulate the architecture and the dynamics of the actin cytoskeleton in time and space. The Diaphanous-related formins (DRF) represent a diverse group of Rho-GTPase-regulated actin regulators that control a range of actin structures composed of tightly-bundled, unbranched actin filaments as found in stress fibers and in filopodia. Under resting conditions, DRFs are auto-inhibited by an intra-molecular interaction between the C-terminal and the N-terminal domains. The auto-inhibition is thought to be released by binding of an activated RhoGTPase to the N-terminal GTPase-binding domain (GBD). However, there is growing evidence for more sophisticated variations from this simplified linear activation model. In this review we focus on the formin homology domain-containing proteins (FHOD), an unconventional group of DRFs. Recent findings on the molecular control and cellular functions of FHOD proteins in vivo are discussed in the light of the phylogeny of FHOD proteins.

  13. An immunodominant membrane protein (Imp) of 'Candidatus Phytoplasma mali' binds to plant actin.

    PubMed

    Boonrod, K; Munteanu, B; Jarausch, B; Jarausch, W; Krczal, G

    2012-07-01

    The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp▴Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp▴Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp▴Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized.

  14. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    SciTech Connect

    Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta; Hozak, Pavel; Goldberg, Ilya G.; Nakao, Mitsuyoshi; Saitoh, Noriko; Harata, Masahiko

    2015-08-21

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation.

  15. Actin capping protein alpha maintains vestigial-expressing cells within the Drosophila wing disc epithelium.

    PubMed

    Janody, Florence; Treisman, Jessica E

    2006-09-01

    Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.

  16. Biochemical evidence for the presence of an unconventional actin protein in a prokaryotic organism.

    PubMed

    Labbé, J P; Harricane, M C; Boyer, M; Derancourt, J; Roustan, C; Benyamin, Y

    1996-07-01

    The ubiquity of actin, like the functional diversity of many associated proteins, raises a question concerning diversification of motility mechanisms and thus the emergence of an elementary functional system. Our aim was to investigate, in particular, mobiles prokaryotics cells as Synechocystis lacking cilia and flagella, search for actin essential properties and then locate the molecular behaviours. Here we report the presence and purification of a 56-kDa (apparent molecular weight) prokaryotic protein that polymerizes to form filaments, activates myosin Mg(++)-ATPase activity, inhibits DNase-1 activity and affords close antigenic homology to skeletal actin. This protein was found to be associated with thylakoid membranes and extracted in the presence of Triton X-100.

  17. Creating biomolecular motors based on dynein and actin-binding proteins.

    PubMed

    Furuta, Akane; Amino, Misako; Yoshio, Maki; Oiwa, Kazuhiro; Kojima, Hiroaki; Furuta, Ken'ya

    2017-03-01

    Biomolecular motors such as myosin, kinesin and dynein are protein machines that can drive directional movement along cytoskeletal tracks and have the potential to be used as molecule-sized actuators. Although control of the velocity and directionality of biomolecular motors has been achieved, the design and construction of novel biomolecular motors remains a challenge. Here we show that naturally occurring protein building blocks from different cytoskeletal systems can be combined to create a new series of biomolecular motors. We show that the hybrid motors-combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins-robustly drive the sliding movement of an actin filament. Furthermore, the direction of actin movement can be reversed by simply changing the geometric arrangement of these building blocks. Our synthetic strategy provides an approach to fabricating biomolecular machines that work along artificial tracks at nanoscale dimensions.

  18. Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

    PubMed Central

    Bottazzi, Maria Elena; Buzzai, Monica; Zhu, Xiaoyun; Desdouets, Chantal; Bréchot, Christian; Assoian, Richard K.

    2001-01-01

    Soluble mitogens and adhesion-dependent organization of the actin cytoskeleton are required for cells to enter S phase in fibroblasts. The induction of cyclin A is also required for S-phase entry, and we now report that distinct effects of mitogens and the actin cytoskeleton on the phosphorylation of CREB and pocket proteins regulate the extent and timing of cyclin A promoter activity, respectively. First, we show that CREB phosphorylation and binding to the cyclic AMP response element (CRE) determines the extent, but not the timing, of cyclin A promoter activity. Second, we show that pocket protein inactivation regulates the timing, but not the extent, of cyclin A promoter activity. CREB phosphorylation and CRE occupancy are regulated by soluble mitogens alone, while the phosphorylation of pocket proteins requires both mitogens and the organized actin cytoskeleton. Mechanistically, cytoskeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our results lead to a model of cyclin A gene regulation in which mitogens play a permissive role by stimulating early G1-phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskeleton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation. PMID:11604497

  19. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    PubMed

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  20. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    PubMed Central

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  1. Actinous enigma or enigmatic actin

    PubMed Central

    Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

    2014-01-01

    Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins. PMID:28232879

  2. Yersinia protein kinase YopO is activated by a novel G-actin binding process.

    PubMed

    Trasak, Claudia; Zenner, Gerhardt; Vogel, Annette; Yüksekdag, Gülnihal; Rost, René; Haase, Ilka; Fischer, Markus; Israel, Lars; Imhof, Axel; Linder, Stefan; Schleicher, Michael; Aepfelbacher, Martin

    2007-01-26

    Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.

  3. Antibody against the actin-binding protein depactin attenuates Ca2+ signaling in starfish eggs.

    PubMed

    Chun, Jong T; Vasilev, Filip; Santella, Luigia

    2013-11-15

    Being present in starfish oocytes, the cofilin/ADF (actin-depolymerizing factor) family protein depactin severs actin filaments. Previously, we reported that exogenous cofilin microinjected into starfish eggs significantly augmented the Ca(2+) release in response to inositol 1,4,5-trisphosphate (InsP3) or fertilizing sperm, raising the possibility that intracellular Ca(2+) signaling could be modulated by the actin cytoskeleton. In this communication, we have targeted the endogenous depactin by use of the specific antibody that was raised against its actin-binding domain. The anti-depactin antibody microinjected into the starfish oocytes and eggs effectively altered the structure of the actin cytoskeleton, and significantly delayed the meiotic progression induced by 1-methyladenine. When microinjected into the mature eggs, the anti-depactin antibody markedly reduced the amplitude of the Ca(2+) response in a dose-dependent manner, corroborating the results of our previous study with cofilin. In addition, the eggs microinjected with the anti-depactin antibody displayed reduced rate of successful elevation of the fertilization envelope and an elevated tendency of polyspermic interaction. Taken together, our data suggest that the actin cytoskeleton is implicated not only in meiotic maturation and intracellular Ca(2+) signaling, but also in the fine regulation of gametes interaction and cortical granules exocytosis. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

    PubMed

    Schenk, Laura K; Möller-Kerutt, Annika; Klosowski, Rafael; Wolters, Dirk; Schaffner-Reckinger, Elisabeth; Weide, Thomas; Pavenstädt, Hermann; Vollenbröker, Beate

    2017-08-02

    Within the kidney, angiotensin II (AngII) targets different cell types in the vasculature, tubuli, and glomeruli. An important part of the renal filtration barrier is composed of podocytes with their actin-rich foot processes. In this study, we used stable isotope labeling with amino acids in cell culture coupled to mass spectrometry to characterize relative changes in the phosphoproteome of human podocytes in response to short-term treatment with AngII. In 4 replicates, we identified a total of 17,956 peptides that were traceable to 2081 distinct proteins. Bioinformatic analyses revealed that among the increasingly phosphorylated peptides are predominantly peptides that are related to actin filaments, cytoskeleton, lamellipodia, mammalian target of rapamycin, and MAPK signaling. Among others, this screening approach highlighted the increased phosphorylation of actin-bundling protein, l-plastin (LCP1). AngII-dependent phosphorylation of LCP1 in cultured podocytes was mediated by the kinases ERK, p90 ribosomal S6 kinase, PKA, or PKC. LCP1 phosphorylation increased filopodia formation. In addition, treatment with AngII led to LCP1 redistribution to the cell margins, membrane ruffling, and formation of lamellipodia. Our data highlight the importance of AngII-triggered actin cytoskeleton-associated signal transduction in podocytes.-Schenk, L. K., Möller-Kerutt, A., Klosowski, R., Wolters, D., Schaffner-Reckinger, E., Weide, T., Pavenstädt, H., Vollenbröker, B. Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes. © FASEB.

  5. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  6. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.

    PubMed

    Courtemanche, Naomi; Pollard, Thomas D; Chen, Qian

    2016-06-01

    When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP-LifeAct and LifeAct-mCherry bind actin filaments with Kd values of ∼10 μM. LifeAct-mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct-mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.

  7. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  8. Structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

    PubMed

    Kim, Min-Kyu; Kim, Ji-Hye; Kim, Ji-Sun; Kang, Sa-Ouk

    2015-09-01

    The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.

  9. Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly.

    PubMed

    Freeman, N L; Field, J

    2000-02-01

    Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.

  10. Creating biomolecular motors based on dynein and actin-binding proteins

    NASA Astrophysics Data System (ADS)

    Furuta, Akane; Amino, Misako; Yoshio, Maki; Oiwa, Kazuhiro; Kojima, Hiroaki; Furuta, Ken'ya

    2017-03-01

    Biomolecular motors such as myosin, kinesin and dynein are protein machines that can drive directional movement along cytoskeletal tracks and have the potential to be used as molecule-sized actuators. Although control of the velocity and directionality of biomolecular motors has been achieved, the design and construction of novel biomolecular motors remains a challenge. Here we show that naturally occurring protein building blocks from different cytoskeletal systems can be combined to create a new series of biomolecular motors. We show that the hybrid motors—combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins—robustly drive the sliding movement of an actin filament. Furthermore, the direction of actin movement can be reversed by simply changing the geometric arrangement of these building blocks. Our synthetic strategy provides an approach to fabricating biomolecular machines that work along artificial tracks at nanoscale dimensions.

  11. Creating biomolecular motors based on dynein and actin-binding proteins

    NASA Astrophysics Data System (ADS)

    Furuta, Akane; Amino, Misako; Yoshio, Maki; Oiwa, Kazuhiro; Kojima, Hiroaki; Furuta, Ken'ya

    2016-11-01

    Biomolecular motors such as myosin, kinesin and dynein are protein machines that can drive directional movement along cytoskeletal tracks and have the potential to be used as molecule-sized actuators. Although control of the velocity and directionality of biomolecular motors has been achieved, the design and construction of novel biomolecular motors remains a challenge. Here we show that naturally occurring protein building blocks from different cytoskeletal systems can be combined to create a new series of biomolecular motors. We show that the hybrid motors—combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins—robustly drive the sliding movement of an actin filament. Furthermore, the direction of actin movement can be reversed by simply changing the geometric arrangement of these building blocks. Our synthetic strategy provides an approach to fabricating biomolecular machines that work along artificial tracks at nanoscale dimensions.

  12. Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization.

    PubMed

    Pauker, Maor H; Reicher, Barak; Fried, Sophie; Perl, Orly; Barda-Saad, Mira

    2011-07-01

    T cell antigen receptor (TCR) activation triggers profound changes in the actin cytoskeleton. In addition to controlling cellular shape and polarity, this process regulates vital T cell responses, such as T cell adhesion, motility, and proliferation. These depend on the recruitment of the signaling proteins Nck and Wiskott-Aldrich syndrome protein (WASp) to the site of TCR activation and on the functional properties of the adapter proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). We now demonstrate that Nck is necessary but insufficient for the recruitment of WASp. We show that two pathways lead to SLP76-dependent actin rearrangement. One requires the SLP76 acidic domain, crucial to association with the Nck SH2 domain, and another requires the SLP76 SH2 domain, essential for interaction with the adhesion- and degranulation-promoting adapter protein ADAP. Functional cooperation between Nck and ADAP mediates SLP76-WASp interactions and actin rearrangement. We also reveal the molecular mechanism linking ADAP to actin reorganization.

  13. Activity-dependent spine morphogenesis: a role for the actin-capping protein Eps8

    PubMed Central

    Stamatakou, Eleanna; Marzo, Aude; Gibb, Alasdair; Salinas, Patricia C.

    2013-01-01

    Neuronal activity regulates the formation and morphology of dendritic spines through changes in the actin cytoskeleton. However, the molecular mechanisms that regulate this process remain poorly understood. Here we report that Eps8, an actin-capping protein, is required for spine morphogenesis. In rat hippocampal neurons gain and loss of function studies demonstrate that Eps8 promotes the formation of dendritic spines, whereas inhibits filopodium formation. Loss of function of Eps8 increases actin polymerization and induces fast actin turnover within dendritic spines, as revealed by free barbed-end and FRAP assays, consistent with a role for Eps8 as an actin-capping protein. Interestingly, Eps8 regulates the balance between excitatory synapses on spines and on the dendritic shaft, without affecting the total number of synapses or basal synaptic transmission. Importantly, Eps8 loss of function impairs the structural and functional plasticity of synapses induced by long-term potentiation. These findings demonstrate a novel role for Eps8 in spine formation and in activity-mediated synaptic plasticity. PMID:23392693

  14. Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

    PubMed Central

    Baranwal, Somesh

    2015-01-01

    Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

  15. Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN

    PubMed Central

    Cao, Lingyan

    2017-01-01

    Plants perceive microbe-associated molecular patterns and damage-associated molecular patterns to activate innate immune signaling events, such as bursts of reactive oxygen species (ROS). The actin cytoskeleton remodels during the first 5 min of innate immune signaling in Arabidopsis (Arabidopsis thaliana) epidermal cells; however, the immune signals that impinge on actin cytoskeleton and its response regulators remain largely unknown. Here, we demonstrate that rapid actin remodeling upon elicitation with diverse microbe-associated molecular patterns and damage-associated molecular patterns represent a conserved plant immune response. Actin remodeling requires ROS generated by the defense-associated NADPH oxidase, RBOHD. Moreover, perception of flg22 by its cognate receptor complex triggers actin remodeling through the activation of RBOHD-dependent ROS production. Our genetic studies reveal that the ubiquitous heterodimeric capping protein transduces ROS signaling to the actin cytoskeleton during innate immunity. Additionally, we uncover a negative feedback loop between actin remodeling and flg22-induced ROS production. PMID:27909046

  16. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  17. Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin

    PubMed Central

    Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ichi; Igarashi, Kazuhiko; Harata, Masahiko

    2017-01-01

    Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex. PMID:28270832

  18. Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

    PubMed

    Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-Ichi; Igarashi, Kazuhiko; Harata, Masahiko

    2017-01-01

    Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

  19. Biochemical interaction of an actin-capping protein, CapZ, with NAP-22.

    PubMed

    Odagaki, Sin-Ichi; Kumanogoh, Haruko; Nakamura, Shun; Maekawa, Shohei

    2009-07-01

    NAP-22 is a neuronal protein localized in the presynaptic membrane and synaptic vesicles and recovered in a Triton-insoluble low-density microdomain fraction after biochemical fractionation of the synaptic plasma membrane. NAP-22 organizes membrane microdomains through binding to membrane lipids such as cholesterol, phosphatidylethanolamine, and phosphatidylinositol 4,5-bisphosphate. In this study, NAP-22-binding proteins were screened through the pull-down assay using brain-derived NAP-22 bound to Sepharose 4B. An actin-capping protein, CapZ, was identified in the precipitate through mass spectrometry and Western blotting. CapZ was then expressed in E. coli and the purified protein-bound NAP-22 directly. Because bacterially expressed NAP-22 bound CapZ, it was determined that the N-terminal myristoyl moiety of NAP-22 is not necessary for the binding. The binding of NAP-22 showed no effect on the actin nucleation activity of CapZ measured with centrifugation and viscometric assays. Hence, the CapZ-NAP-22 complex could work as the nucleation site of actin polymerization or as the actin filament-anchoring site on the membrane microdomain.

  20. The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain.

    PubMed

    González-Jamett, Arlek M; Guerra, María J; Olivares, María J; Haro-Acuña, Valentina; Baéz-Matus, Ximena; Vásquez-Navarrete, Jacqueline; Momboisse, Fanny; Martinez-Quiles, Narcisa; Cárdenas, Ana M

    2017-01-01

    Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca(2+) signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca(2+)-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca(2+)-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells.

  1. The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain

    PubMed Central

    González-Jamett, Arlek M.; Guerra, María J.; Olivares, María J.; Haro-Acuña, Valentina; Baéz-Matus, Ximena; Vásquez-Navarrete, Jacqueline; Momboisse, Fanny; Martinez-Quiles, Narcisa; Cárdenas, Ana M.

    2017-01-01

    Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells. PMID:28522963

  2. A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor

    PubMed Central

    1990-01-01

    Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross- linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays. PMID:1698791

  3. An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein.

    PubMed

    Freeman, N L; Chen, Z; Horenstein, J; Weber, A; Field, J

    1995-03-10

    The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showing that CAP sequesters monomeric actin (Kd in the range of 0.5-5 microM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.

  4. Regulation of the Actin Cytoskeleton by an Interaction of IQGAP Related Protein GAPA with Filamin and Cortexillin I

    PubMed Central

    Rieger, Daniela; Müller, Rolf; Rivero, Francisco; Faix, Jan; Schleicher, Michael; Noegel, Angelika A.

    2010-01-01

    Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin. PMID:21085675

  5. Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis

    PubMed Central

    2011-01-01

    Background Neutrophils are abundant leukocytes that play a primary role in defence against pathogens. Neutrophils enter sites of infection where they eliminate pathogens via phagocytosis and the release of antimicrobial mediators via degranulation. Rho GTPases, particularly Rac2, play a key role in neutrophil degranulation. The purpose of this study was to identify Rac2-dependent changes in protein abundance in stimulated neutrophils. Methods We performed a proteomic analysis on secretagogue-stimulated bone marrow neutrophils that were isolated from wild-type and Rac2-/- mice. Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins. Results We identified 22 proteins that showed significant changes in abundance after secretagogue-stimulation of wild-type neutrophils, which did not occur in neutrophils isolated from Rac2-/- mice. As expected, the abundance of several granule proteins was reduced in wild-type cells; this did not occur in Rac2-/- neutrophils which confirms the requirement for Rac2 in degranulation. We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β). Coronin-1A showed elevated levels of several isoforms after stimulation of neutrophils from wild-type, but not from Rac2-/- mice. These isoforms were immunoreactive with anti-phospho-threonine antibodies, suggesting that neutrophil stimulation triggers a Rac2-dependent kinase cascade that results in the phosphorylation of coronin-1A. Conclusion The control of Rac2-mediated degranulation in neutrophils likely functions through actin remodelling via activation of several actin-binding proteins. We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation. PMID:22081935

  6. Actin-related proteins localized in the nucleus: from discovery to novel roles in nuclear organization.

    PubMed

    Oma, Yukako; Harata, Masahiko

    2011-01-01

    The actin family consists of conventional actin and actin-related proteins (ARPs), and the members show moderate similarity and share the same basal structure. Following the finding of various ARPs in the cytoplasm in the 1990s, multiple subfamilies that are localized predominantly in the nucleus were identified. Consistent with these cytological observations, subsequent biochemical analyses revealed the involvement of the nuclear ARPs in ATP-dependent chromatin-remodeling and histone acetyltransferase complexes. In addition to their contribution to chromatin remodeling, recent studies have shown that nuclear ARPs have roles in the organization of the nucleus that are independent of the activity of the above-mentioned complexes. Therefore, nuclear ARPs are recognized as novel key regulators of genome function, and affect not only the remodeling of chromatin but also the spatial arrangement and dynamics of chromatin within the nucleus.

  7. CIP4 coordinates with phospholipids and actin-associated proteins to localize to the protruding edge and produce actin ribs and veils.

    PubMed

    Saengsawang, Witchuda; Taylor, Kendra L; Lumbard, Derek C; Mitok, Kelly; Price, Amanda; Pietila, Lauren; Gomez, Timothy M; Dent, Erik W

    2013-06-01

    Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family of proteins, plays important roles in a variety of cellular events by regulating both membrane and actin dynamics. In many cell types, CIP4 functions in vesicle formation, endocytosis and membrane tubulation. However, recent data indicate that CIP4 is also involved in protrusion in some cell types, including cancer cells (lamellipodia and invadopodia) and neurons (ribbed lamellipodia and veils). In neurons, CIP4 localizes specifically to extending protrusions and functions to limit neurite outgrowth early in development. The mechanism by which CIP4 localizes to the protruding edge membrane and induces lamellipodial/veil protrusion and actin rib formation is not known. Here, we show that CIP4 localization to the protruding edge of neurons is dependent on both the phospholipid content of the plasma membrane and the underlying organization of actin filaments. Inhibiting phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production decreases CIP4 at the membrane. CIP4 localization to the protruding edge is also dependent on Rac1/WAVE1, rather than Cdc42/N-WASP. Capping actin filaments with low concentrations of cytochalasin D or by overexpressing capping protein dramatically decreases CIP4 at the protruding edge, whereas inactivating Arp2/3 drives CIP4 to the protruding edge. We also demonstrate that CIP4 dynamically colocalizes with Ena/VASP and DAAM1, two proteins known to induce unbranched actin filament arrays and play important roles in neuronal development. Together, this is the first study to show that the localization of an F-BAR protein depends on both actin filament architecture and phospholipids at the protruding edge of developing neurons.

  8. CIP4 coordinates with phospholipids and actin-associated proteins to localize to the protruding edge and produce actin ribs and veils

    PubMed Central

    Saengsawang, Witchuda; Taylor, Kendra L.; Lumbard, Derek C.; Mitok, Kelly; Price, Amanda; Pietila, Lauren; Gomez, Timothy M.; Dent, Erik W.

    2013-01-01

    Summary Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family of proteins, plays important roles in a variety of cellular events by regulating both membrane and actin dynamics. In many cell types, CIP4 functions in vesicle formation, endocytosis and membrane tubulation. However, recent data indicate that CIP4 is also involved in protrusion in some cell types, including cancer cells (lamellipodia and invadopodia) and neurons (ribbed lamellipodia and veils). In neurons, CIP4 localizes specifically to extending protrusions and functions to limit neurite outgrowth early in development. The mechanism by which CIP4 localizes to the protruding edge membrane and induces lamellipodial/veil protrusion and actin rib formation is not known. Here, we show that CIP4 localization to the protruding edge of neurons is dependent on both the phospholipid content of the plasma membrane and the underlying organization of actin filaments. Inhibiting phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production decreases CIP4 at the membrane. CIP4 localization to the protruding edge is also dependent on Rac1/WAVE1, rather than Cdc42/N-WASP. Capping actin filaments with low concentrations of cytochalasin D or by overexpressing capping protein dramatically decreases CIP4 at the protruding edge, whereas inactivating Arp2/3 drives CIP4 to the protruding edge. We also demonstrate that CIP4 dynamically colocalizes with Ena/VASP and DAAM1, two proteins known to induce unbranched actin filament arrays and play important roles in neuronal development. Together, this is the first study to show that the localization of an F-BAR protein depends on both actin filament architecture and phospholipids at the protruding edge of developing neurons. PMID:23572514

  9. Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation.

    PubMed

    Tsujita, Kazuya; Kondo, Akihiro; Kurisu, Shusaku; Hasegawa, Junya; Itoh, Toshiki; Takenawa, Tadaomi

    2013-05-15

    FBP17, an F-BAR domain protein, has emerged as a crucial factor linking the plasma membrane to WASP-mediated actin polymerization. Although it is well established that FBP17 has a powerful self-polymerizing ability that promotes actin nucleation on membranes in vitro, knowledge of inhibitory factors that counteract this activity in vivo is limited. Here, we demonstrate that the assembly of FBP17 on the plasma membranes is antagonized by PSTPIP2, another F-BAR protein implicated in auto-inflammatory disorder. Knockdown of PSTPIP2 in macrophage promotes the assembly of FBP17 as well as subsequent actin nucleation at podosomes, resulting in an enhancement of matrix degradation. This phenotype is rescued by expression of PSTPIP2 in a manner dependent on its F-BAR domain. Time-lapse total internal reflection fluorescence (TIRF) microscopy observations reveal that the self-assembly of FBP17 at the podosomal membrane initiates actin polymerization, whereas the clustering of PSTPIP2 has an opposite effect. Biochemical analysis and live-cell imaging show that PSTPIP2 inhibits actin polymerization by competing with FBP17 for assembly at artificial as well as the plasma membrane. Interestingly, the assembly of FBP17 is dependent on WASP, and its dissociation by WASP inhibition strongly induces a self-organization of PSTPIP2 at podosomes. Thus, our data uncover a previously unappreciated antagonism between different F-BAR domain assemblies that determines the threshold of actin polymerization for the formation of functional podosomes and may explain how the absence of PSTPIP2 causes auto-inflammatory disorder.

  10. Effect of cooling (4°C) and cryopreservation on cytoskeleton actin and protein tyrosine phosphorylation in buffalo spermatozoa.

    PubMed

    Naresh, Sai

    2016-02-01

    Semen cryopreservation is broadly utilized as a part of the bovine reproducing industry, a large portion of the spermatozoa does not survive and the majority of those that do survive experience various molecular and physiological changes that influence their fertilizing capacity. The main aim of this study is to determine the effect of cooling (4 °C) and cryopreservation on cytoskeleton actin, tyrosine phosphorylation and quality of buffalo spermatozoa, and to determine the similarity between in vitro capacitation and cryopreservation induced capacitation like changes. To achieve this, Western blot was used to examine the changes in actin expression and protein tyrosine phosphorylation, whereas changes in actin polymerization, localization of actin and protein tyrosine phosphorylation during capacitation and cryopreservation were evaluated by indirect immunofluorescence technique. Localization studies revealed that the actin localized to flagella and acrosome membrane regions and following, capacitation it migrated towards the acrosome region of sperm. Time dependent increase in actin polymerization and protein tyrosine phosphorylation was observed during in vitro capacitation. The cooling phase (4 °C) and cryopreservation processes resulted in the loss/damage of cytoskeleton actin. In addition, we performed the actin polymerization and protein tyrosine phosphorylation in cooled and cryopreserved buffalo spermatozoa. Interestingly, cooling and cryopreservation induces actin polymerization and protein tyrosine phosphorylation, which were similar to in vitro capacitation (cryo-capacitation). These changes showed 1.3 folds reduction in the sperm quality parameters which includes motility, viability and plasma membrane integrity. Furthermore, our findings indicate that cooling and cryopreservation damages the cytoskeleton actin and also induces capacitation like changes such as protein tyrosine phosphorylation and actin polymerization. This could be one of the

  11. Pearling instability of membrane tubes driven by curved proteins and actin polymerization

    NASA Astrophysics Data System (ADS)

    Jelerčič, U.; Gov, N. S.

    2015-12-01

    Membrane deformation inside living cells is crucial for the proper shaping of various intracellular organelles and is necessary during the fission/fusion processes that allow membrane recycling and transport (e.g. endocytosis). Proteins that induce membrane curvature play a key role in such processes, mostly by adsorbing to the membrane and forming a scaffold that deforms the membrane according to the curvature of the proteins. In this paper we explore the possibility of membrane tube destabilization through a pearling mechanism enabled by the combined effects of the adsorbed curved proteins and the actin polymerization that they recruit. The pearling instability can serve as the initiation for fission of the tube into vesicles. We find that adsorbed curved proteins are more likely to stabilize the tubes, while the actin polymerization can provide the additional constrictive force needed for the robust instability. We discuss the relevance of the theoretical results to in vivo and in vitro experiments.

  12. Characterization of the Yeast Actin Patch Protein App1p Phosphatidate Phosphatase*

    PubMed Central

    Chae, Minjung; Carman, George M.

    2013-01-01

    Yeast App1p is a phosphatidate phosphatase (PAP) that associates with endocytic proteins at cortical actin patches. App1p, which catalyzes the conversion of phosphatidate (PA) to diacylglycerol, is unique among Mg2+-dependent PAP enzymes in that its reaction is not involved with de novo lipid synthesis. Instead, App1p PAP is thought to play a role in endocytosis because its substrate and product facilitate membrane fission/fusion events and regulate enzymes that govern vesicular movement. App1p PAP was purified from yeast and characterized with respect to its enzymological, kinetic, and regulatory properties. Maximum PAP activity was dependent on Triton X-100 (20 mm), PA (2 mm), Mg2+ (0.5 mm), and 2-mercaptoethanol (10 mm) at pH 7.5 and 30 °C. Analysis of surface dilution kinetics with Triton X-100/PA-mixed micelles yielded constants for surface binding (KsA = 11 mm), interfacial PA binding (KmB = 4.2 mol %), and catalytic efficiency (Vmax = 557 μmol/min/mg). The activation energy, turnover number, and equilibrium constant were 16.5 kcal/mol, 406 s−1, and 16.2, respectively. PAP activity was stimulated by anionic lipids (cardiolipin, phosphatidylglycerol, phosphatidylserine, and CDP-diacylglycerol) and inhibited by zwitterionic (phosphatidylcholine and phosphatidylethanolamine) and cationic (sphinganine) lipids, nucleotides (ATP and CTP), N-ethylmaleimide, propranolol, phenylglyoxal, and divalent cations (Ca2+, Mn2+, and Zn2+). App1p also utilized diacylglycerol pyrophosphate and lyso-PA as substrates with specificity constants 4- and 7-fold lower, respectively, when compared with PA. PMID:23335564

  13. Comparative anatomy of a regulatory ribosomal protein.

    PubMed

    Worbs, Michael; Wahl, Markus C; Lindahl, Lasse; Zengel, Janice M

    2002-08-01

    Ribosomal protein L4 is a crucial folding mediator and an important architectural component of the large ribosomal subunit. Furthermore, Escherichia coli L4 produced in excess of its rRNA binding sites downregulates the transcription and translation of its own S10 operon, encoding 11 ribosomal proteins. Genetic experiments and the crystal structure of Thermotoga maritima L4 had implicated separable regions on L4 in ribosome association and expression control while RNA competition experiments and the regulatory capacity of heterologous L4 had suggested an overlap of the protein sequences involved in the two functions. We report herein that contrary to other foreign bacterial L4 proteins, L4 from T. maritima only weakly controlled expression of the S10 operon in E. coli. Also, wildtype T. maritima L4 was more weakly associated with E. coli ribosomes than with the E. coli analog. Rational mutageneses were performed to try to increase the regulatory competence of T. maritima L4. The ribosome incorporation of the mutant proteins was also investigated. Two different deletions removing T. maritima-specific sequences had little effects on regulation although one did improve ribosome association. Interestingly, a set of multiple mutations, which rendered the region around helices alpha4 and alpha5 in T. maritima L4 more E. coli-like, had no influence on the incorporation of the protein into the large ribosomal subunit but considerably improved its regulatory potential. Therefore, the area around helices alpha4 and alpha5, which is critical for the initial folding steps of the large subunit, is also a central element of autogenous control, presumably by contacting the S10 mRNA leader. Ribosome association is compounded at later stages of assembly by additional rRNA contacts through L4 areas which do not participate in regulation. Similarly, sequences outside the alpha4/alpha5 region aid expression control.

  14. Two Distinct Mechanisms for Actin Capping Protein Regulation—Steric and Allosteric Inhibition

    PubMed Central

    Takeda, Shuichi; Minakata, Shiho; Koike, Ryotaro; Kawahata, Ichiro; Narita, Akihiro; Kitazawa, Masashi; Ota, Motonori; Yamakuni, Tohru; Maéda, Yuichiro; Nitanai, Yasushi

    2010-01-01

    The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the α-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the

  15. Actin-binding cellular proteins inside human immunodeficiency virus type 1.

    PubMed

    Ott, D E; Coren, L V; Johnson, D G; Kane, B P; Sowder, R C; Kim, Y D; Fisher, R J; Zhou, X Z; Lu, K P; Henderson, L E

    2000-01-05

    Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding. Copyright 2000 Academic Press.

  16. Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics.

    PubMed

    Gay, Olivia; Gilquin, Benoît; Assard, Nicole; Stuelsatz, Pascal; Delphin, Christian; Lachuer, Joël; Gidrol, Xavier; Baudier, Jacques

    2016-10-15

    Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling.

  17. Tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton rearrangement and cancer cell invasion.

    PubMed

    Ohishi, Tomokazu; Yoshida, Haruka; Katori, Masamichi; Migita, Toshiro; Muramatsu, Yukiko; Miyake, Mao; Ishikawa, Yuichi; Saiura, Akio; Iemura, Shun-Ichiro; Natsume, Tohru; Seimiya, Hiroyuki

    2017-02-15

    Tankyrase, a poly(ADP-ribose) polymerase (PARP) that promotes telomere elongation and Wnt/β-catenin signaling, has various binding partners, suggesting that it has as-yet unidentified functions. Here we report that the tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton and cancer cell invasion, which is closely associated with cancer progression. TNKS1BP1 colocalized with actin filaments and negatively regulated cell invasion. In TNKS1BP1-depleted cells, actin filament dynamics, focal adhesion, and lamellipodia ruffling were increased with activation of the ROCK-LIMK-cofilin pathway. TNKS1BP1 bound the actin capping protein CapZA2. TNKS1BP1 depletion dissociated CapZA2 from the cytoskeleton, leading to cofilin phosphorylation and enhanced cell invasion. Tankyrase overexpression increased cofilin phosphorylation, dissociated CapZA2 from cytoskeleton, and enhanced cell invasion in a PARP activity-dependent manner. In clinical samples of pancreatic cancer, TNKS1BP1 expression was reduced in invasive regions. We propose that the tankyrase-TNKS1BP1 axis constitutes a post-translational modulator of cell invasion whose aberration promotes cancer malignancy.

  18. Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics

    PubMed Central

    Gay, Olivia; Gilquin, Benoît; Assard, Nicole; Stuelsatz, Pascal; Delphin, Christian; Lachuer, Joël; Gidrol, Xavier; Baudier, Jacques

    2016-01-01

    ABSTRACT Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling. PMID:27744291

  19. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

    PubMed

    Nomura, Kazumi; Ono, Shoichiro

    2013-07-15

    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  20. Differential effects of an expected actin-tropomyosin binding region of heat shock protein 20 on the relaxation in skinned carotid artery and taenia cecum from guinea pig.

    PubMed

    Hashimoto, Ryo; Yumoto, Masatoshi; Watanabe, Masaru; Konishi, Masato; Haraoka, Jo; Miki, Tamotsu

    2009-02-01

    To explore the possible role of heat shock protein 20 (HSP20) -linked regulation of actin-myosin interaction in living vascular smooth muscle contraction, we studied the effects of HSP20p and TnIp, synthetic peptides originating from an actin tropomyosin binding region of human heat shock protein 20 [residues 110-121; GFVAREFHRRYR] and that of rabbit cardiac troponin I [residues 136-147; GKFKRPTLRRVR], respectively, on the active stress and phosphorylation level of myosin regulatory light chain (MLC(20)) during relaxation of skinned (cell membrane permeabilized) preparations from "tonic" carotid artery and "phasic" taenia cecum from guinea pig. Active stress of the skinned preparations, resulting from actin-myosin interaction, biphasically decayed following Ca(2+) removal (relaxation). Decay of MLC(20) phosphorylation level by Ca(2+) removal was much faster than active stress in an exponential manner. In skinned carotid artery, HSP20p did neither affect relaxation time course nor MLC(20) dephosphorylation, whereas, in skinned taenia cecum, the peptide slowed relaxation time course through inhibition of MLC(20) dephosphorylation and slowing "latch"-bridge dissociation. On the other hand, TnIp accelerated relaxation time course without affecting MLC(20) dephosphorylation in both skinned carotid artery and skinned taenia cecum. Our present results suggest that, HSP20p slows the relaxation processes through intracellular regulatory mechanisms such as Rho A/Rho-kinase mediated pathways, which are known to be dominant in "phasic" smooth muscles but to be recessive in "tonic" smooth muscles.

  1. Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins

    PubMed Central

    1994-01-01

    Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells. PMID:8051203

  2. What We Know and Do Not Know About Actin.

    PubMed

    Pollard, Thomas D

    2017-01-01

    Seven decades of research have revealed much about actin structure, assembly, regulatory proteins, and cellular functions. However, some key information is still missing, so we do not understand the mechanisms of most processes that depend on actin. This chapter summarizes our current knowledge and explains some examples of work that will be required to fill these gaps and arrive at a mechanistic understanding of actin biology.

  3. Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein1[W][OPEN

    PubMed Central

    Jimenez-Lopez, Jose C.; Wang, Xia; Kotchoni, Simeon O.; Huang, Shanjin; Szymanski, Daniel B.; Staiger, Christopher J.

    2014-01-01

    The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction. PMID:25201878

  4. Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

    PubMed Central

    Loo, Chin-San; Chen, Cheng-Wei; Wang, Po-Jen; Chen, Pei-Yu; Lin, Shu-Yu; Khoo, Kay-Hooi; Fenton, Robert A.; Knepper, Mark A.; Yu, Ming-Jiun

    2013-01-01

    In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressin-induced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase. PMID:24085853

  5. Stain-Free total protein staining is a superior loading control to β-actin for Western blots.

    PubMed

    Gilda, Jennifer E; Gomes, Aldrin V

    2013-09-15

    Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots.

  6. Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization.

    PubMed

    Miehe, Ulrich; Neumaier-Wagner, Peruka; Kadyrov, Mamed; Goyal, Pankaj; Alfer, Joachim; Rath, Werner; Huppertz, Berthold

    2005-01-01

    The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.

  7. Crystal structure of the actin binding domain of the cyclase-associated protein.

    PubMed

    Dodatko, Tetyana; Fedorov, Alexander A; Grynberg, Marcin; Patskovsky, Yury; Rozwarski, Denise A; Jaroszewski, Lukasz; Aronoff-Spencer, Eliah; Kondraskina, Elena; Irving, Tom; Godzik, Adam; Almo, Steven C

    2004-08-24

    Cyclase-associated protein (CAP or Srv2p) is a modular actin monomer binding protein that directly regulates filament dynamics and has been implicated in a number of complex developmental and morphological processes, including mRNA localization and the establishment of cell polarity. The crystal structure of the C-terminal dimerization and actin monomer binding domain (C-CAP) reveals a highly unusual dimer, composed of monomers possessing six coils of right-handed beta-helix flanked by antiparallel beta-strands. Domain swapping, involving the last two strands of each monomer, results in the formation of an extended dimer with an extensive interface. This structural and biochemical characterization provides new insights into the organization and potential mechanistic properties of the multiprotein assemblies that integrate dynamic actin processes into the overall physiology of the cell. An unanticipated finding is that the unique tertiary structure of the C-CAP monomer provides a structural model for a wide range of molecules, including RP2 and cofactor C, proteins involved in X-linked retinitis pigmentosa and tubulin maturation, respectively, as well as several uncharacterized proteins that exhibit very diverse domain organizations. Thus, the unusual right-handed beta-helical fold present in C-CAP appears to support a wide range of biological functions.

  8. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    PubMed

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase.

  9. Bacterial actin MreB assembles in complex with cell shape protein RodZ

    PubMed Central

    van den Ent, Fusinita; Johnson, Christopher M; Persons, Logan; de Boer, Piet; Löwe, Jan

    2010-01-01

    Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB–RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm. PMID:20168300

  10. Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin

    SciTech Connect

    Lee, Y.-J. . E-mail: lee_yi_jang@hotmail.com; Sheu, T.-J.; Keng, Peter C.

    2005-09-23

    Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.

  11. Charge effects modulate actin assembly by classic myelin basic protein isoforms

    SciTech Connect

    Hill, Christopher M.D.; Harauz, George . E-mail: gharauz@uoguelph.ca

    2005-04-01

    Myelin basic protein (MBP), a highly cationic structural protein of the myelin sheath, is believed to be associated with the cytoskeleton in vivo and interacts with actin in vitro, but little is known about the regulation of this interaction. The rate and extent of actin polymerization induced by 18.5 kDa MBP charge isomers were correlated to charge reduction by post-translational modifications. Increased ionic strength attenuated the initial rate but not the final extent of polymerization achieved. Reduced pH enhanced the rate and extent of polymerization, presumably via partial protonation of intrinsic histidyl residues. The polymerizing activities of the 21.5, 17, and 14 kDa MBP splice variants were not proportionate to their net charges or charge densities. The presence of at least one region derived from exon II or VI of the 'classic' MBP gene was required for effective bundling as assessed by light scattering and transmission electron microscopy.

  12. The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

    PubMed

    Douglas, Lois M; Wang, Hong X; Konopka, James B

    2013-11-26

    Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis. The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane

  13. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity.

    PubMed

    Vander Broek, Charles W; Zainal Abidin, Nurhamimah; Stevens, Joanne M

    2017-01-01

    Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin) and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins.

  14. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity

    PubMed Central

    Vander Broek, Charles W.; Zainal Abidin, Nurhamimah; Stevens, Joanne M.

    2017-01-01

    Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin) and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins. PMID:28770177

  15. Whirlin interacts with espin and modulates its actin-regulatory function: an insight into the mechanism of Usher syndrome type II.

    PubMed

    Wang, Le; Zou, Junhuang; Shen, Zuolian; Song, E; Yang, Jun

    2012-02-01

    Whirlin mutations cause retinal degeneration and hearing loss in Usher syndrome type II (USH2) and non-syndromic deafness, DFNB31. Its protein recruits other USH2 causative proteins to form a complex at the periciliary membrane complex in photoreceptors and the ankle link of the stereocilia in hair cells. However, the biological function of this USH2 protein complex is largely unknown. Using a yeast two-hybrid screen, we identified espin, an actin-binding/bundling protein involved in human deafness when defective, as a whirlin-interacting protein. The interaction between these two proteins was confirmed by their coimmunoprecipitation and colocalization in cultured cells. This interaction involves multiple domains of both proteins and only occurs when espin does not bind to actin. Espin was partially colocalized with whirlin in the retina and the inner ear. In whirlin knockout mice, espin expression changed significantly in these two tissues. Further studies found that whirlin increased the mobility of espin and actin at the actin bundles cross-linked by espin and, eventually, affected the dimension of these actin bundles. In whirlin knockout mice, the stereocilia were thickened in inner hair cells. We conclude that the interaction between whirlin and espin and the balance between their expressions are required to maintain the actin bundle network in photoreceptors and hair cells. Disruption of this actin bundle network contributes to the pathogenic mechanism of hearing loss and retinal degeneration caused by whirlin and espin mutations. Espin is a component of the USH2 protein complex and could be a candidate gene for Usher syndrome.

  16. Mammalian and malaria parasite cyclase-associated proteins catalyze nucleotide exchange on G-actin through a conserved mechanism.

    PubMed

    Makkonen, Maarit; Bertling, Enni; Chebotareva, Natalia A; Baum, Jake; Lappalainen, Pekka

    2013-01-11

    Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.

  17. Actin and ubiquitin protein sequences support a cercozoan/foraminiferan ancestry for the plasmodiophorid plant pathogens.

    PubMed

    Archibald, John M; Keeling, Patrick J

    2004-01-01

    The plasmodiophorids are a group of eukaryotic intracellular parasites that cause disease in a variety of economically significant crops. Plasmodiophorids have traditionally been considered fungi but have more recently been suggested to be members of the Cercozoa, a morphologically diverse group of amoeboid, flagellate, and amoeboflagellate protists. The recognition that Cercozoa constitute a monophyletic lineage has come from phylogenetic analyses of small subunit ribosomal RNA genes. Protein sequence data have suggested that the closest relatives of Cercozoa are the Foraminifera. To further test a cercozoan origin for the plasmodiophorids, we isolated actin genes from Plasmodiophora brassicae, Sorosphaera veronicae, and Spongospora subterranea, and polyubiquitin gene fragments from P. brassicae and S. subterranea. We also isolated actin genes from the chlorarachniophyte Lotharella globosa. In protein phylogenies of actin, the plasmodiophorid sequences consistently branch with Cercozoa and Foraminifera, and weakly branch as the sister group to the foraminiferans. The plasmodiophorid polyubiquitin sequences contain a single amino acid residue insertion at the functionally important processing point between ubiquitin monomers, the same place in which an otherwise unique insertion exists in the cercozoan and foraminiferan proteins. Taken together, these results indicate that plasmodiophorids are indeed related to Cercozoa and Foraminifera, although the relationships amongst these groups remain unresolved.

  18. Cooperation of Cdc42 small G protein-activating and actin filament-binding activities of frabin in microspike formation.

    PubMed

    Ikeda, W; Nakanishi, H; Tanaka, Y; Tachibana, K; Takai, Y

    2001-06-14

    Frabin is a GDP/GTP exchange protein for Cdc42 with actin filament (F-actin)-binding activity. Cdc42 is a small GTP-binding protein that forms filopodia-like microspikes in a variety of cells. Expression of frabin indeed forms microspikes through at least activation of Cdc42 in MDCK cells and fibroblasts such as COS7, L, and NIH3T3 cells. However, the role of the F-actin-binding activity of frabin in the microspike formation remains unknown. We have examined here this role of frabin by expressing various frabin mutants, which have lost Cdc42-activating or F-actin-binding activity, with or without a dominant active mutant of Cdc42 in MDCK and COS7 cells. We show here that for the microspike formation, either of the Cdc42-activating and F- actin-binding activities of frabin alone is not sufficient and both the activities are necessary and that both the activities play a cooperative role in the microspike formation. The present results, together with the earlier finding that Cdc42 reorganizes the actin cytoskeleton at least through the N-WASP-Arp2/3 complex, suggest that frabin directly and indirectly reorganizes the actin cytoskeleton through its F-actin-binding and Cdc42-activating activities, respectively, in a cooperative manner, eventually leading to microspike formation.

  19. Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex.

    PubMed

    Eisenmann, Kathryn M; Harris, Elizabeth S; Kitchen, Susan M; Holman, Holly A; Higgs, Henry N; Alberts, Arthur S

    2007-04-03

    Mammalian Diaphanous (mDia)-related formins and the N-WASP-activated Arp2/3 complex initiate the assembly of filamentous actin. Dia-interacting protein (DIP) binds via its amino-terminal SH3 domain to the proline-rich formin homology 1 (FH1) domain of mDia1 and mDia2 and to the N-WASp proline-rich region. Here, we investigated an interaction between a conserved leucine-rich region (LRR) in DIP and the mDia FH2 domain that nucleates, processively elongates, and bundles actin filaments. DIP binding to mDia2 was regulated by the same Rho-GTPase-controlled autoinhibitory mechanism modulating formin-mediated actin assembly. DIP was previously shown to interact with and stimulate N-WASp-dependent branched filament assembly via Arp2/3. Despite direct binding to both mDia1 and mDia2 FH2 domains, DIP LRR inhibited only mDia2-dependent filament assembly and bundling in vitro. DIP expression interfered with filopodia formation, consistent with a role for mDia2 in assembly of these structures. After filopodia retraction into the cell body, DIP expression induced excessive nonapoptotic membrane blebbing, a physiological process involved in both cytokinesis and amoeboid cell movement. DIP-induced blebbing was dependent on mDia2 but did not require the activities of either mDia1 or Arp2/3. These observations point to a pivotal role for DIP in the control of nonbranched and branched actin-filament assembly that is mediated by Diaphanous-related formins and activators of Arp2/3, respectively. The ability of DIP to trigger blebbing also suggests a role for mDia2 in the assembly of cortical actin necessary for maintaining plasma-membrane integrity.

  20. Export of virulence proteins by malaria-infected erythrocytes involves remodeling of host actin cytoskeleton.

    PubMed

    Rug, Melanie; Cyrklaff, Marek; Mikkonen, Antti; Lemgruber, Leandro; Kuelzer, Simone; Sanchez, Cecilia P; Thompson, Jennifer; Hanssen, Eric; O'Neill, Matthew; Langer, Christine; Lanzer, Michael; Frischknecht, Friedrich; Maier, Alexander G; Cowman, Alan F

    2014-11-27

    Following invasion of human red blood cells (RBCs) by the malaria parasite, Plasmodium falciparum, a remarkable process of remodeling occurs in the host cell mediated by trafficking of several hundred effector proteins to the RBC compartment. The exported virulence protein, P falciparum erythrocyte membrane protein 1 (PfEMP1), is responsible for cytoadherence of infected cells to host endothelial receptors. Maurer clefts are organelles essential for protein trafficking, sorting, and assembly of protein complexes. Here we demonstrate that disruption of PfEMP1 trafficking protein 1 (PfPTP1) function leads to severe alterations in the architecture of Maurer's clefts. Furthermore, 2 major surface antigen families, PfEMP1 and STEVOR, are no longer displayed on the host cell surface leading to ablation of cytoadherence to host receptors. PfPTP1 functions in a large complex of proteins and is required for linking of Maurer's clefts to the host actin cytoskeleton.

  1. Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein1[W][OA

    PubMed Central

    Hofmann, Christina; Niehl, Annette; Sambade, Adrian; Steinmetz, André; Heinlein, Manfred

    2009-01-01

    The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane. PMID:19218363

  2. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells

    PubMed Central

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830–840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250–1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and

  3. Mechanics and multiple-particle tracking microheterogeneity of alpha-actinin-cross-linked actin filament networks.

    PubMed Central

    Tseng, Y; Wirtz, D

    2001-01-01

    Cell morphology is controlled by the actin cytoskeleton organization and mechanical properties, which are regulated by the available contents in actin and actin regulatory proteins. Using rheometry and the recently developed multiple-particle tracking method, we compare the mechanical properties and microheterogeneity of actin filament networks containing the F-actin cross-linking protein alpha-actinin. The elasticity of F-actin/alpha-actinin networks increases with actin concentration more rapidly for a fixed molar ratio of actin to alpha-actinin than in the absence of alpha-actinin, for networks of fixed alpha-actinin concentration and of fixed actin concentration, but more slowly than theoretically predicted for a homogeneous cross-linked semiflexible polymer network. These rheological measurements are complemented by multiple-particle tracking of fluorescent microspheres imbedded in the networks. The distribution of the mean squared displacements of these microspheres becomes progressively more asymmetric and wider for increasing concentration in alpha-actinin and, to a lesser extent, for increasing actin concentration, which suggests that F-actin networks become progressively heterogeneous for increasing protein content. This may explain the slower-than-predicted rise in elasticity of F-actin/alpha-actinin networks. Together these in vitro results suggest that actin and alpha-actinin provides the cell with an unsuspected range of regulatory pathways to modulate its cytoskeleton's micromechanics and local organization in vivo. PMID:11509377

  4. Binding of a C-terminal fragment (residues 369 to 435) of vitamin D-binding protein to actin.

    PubMed

    Buch, Stefan; Gremm, Dagmar; Wegner, Albrecht; Mannherz, Hans Georg

    2002-10-01

    The vitamin D-binding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The full-length protein and the N- and C-terminal halves of DBP remained insoluble probably because the protein did not fold to its native three-dimensional structure due to formation of accidental intra- and inter-molecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a C-terminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the C-terminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than full-length DBP. This suggests that in addition to the C-terminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The C-terminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentration-dependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the C-terminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.

  5. The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

    PubMed Central

    Miller, Rita K.; Matheos, Dina; Rose, Mark D.

    1999-01-01

    In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)– Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Δ, pea2Δ, bud6Δ, and bni1Δ, required for normal polarization and actin cytoskeleton functions and, of these, bni1Δ affected Kar9p localization most severely. Like kar9Δ, bni1Δ mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Δ, the bni1Δ mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Δ bni1Δ double mutants suggested that Kar9p retained some function in bni1Δ mitotic cells. Unlike the polarization mutants, kar9Δ shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Δ. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks. PMID:10085294

  6. Structural reorganization of parallel actin bundles by crosslinking proteins: Incommensurate states of twist

    NASA Astrophysics Data System (ADS)

    Shin, Homin; Grason, Gregory M.

    2010-11-01

    We construct a coarse-grained model of parallel actin bundles crosslinked by compact globular bundling proteins, such as fascin and espin, necessary components of filopodial and mechanosensory bundles. Consistent with structural observations of bundles, we find that the optimal geometry for crosslinking is overtwisted, requiring a coherent structural change of the helical geometry of the filaments. We study the linker-dependent thermodynamic transition of bundled actin filaments from their native state to the overtwisted state and map out the “twist-state” phase diagram in terms of the availability as well as the flexibility of crosslinker proteins. We predict that the transition from the uncrosslinked to fully crosslinked state is highly sensitive to linker flexibility: flexible crosslinking smoothly distorts the twist state of bundled filaments, while rigidly crosslinked bundles undergo a phase transition, rapidly overtwisting filaments over a narrow range of free crosslinker concentrations. Additionally, we predict a rich spectrum of intermediate structures, composed of alternating domains of sparsely bound (untwisted) and strongly bound (overtwisted) filaments. This model reveals that subtle differences in crosslinking agents themselves modify not only the detailed structure of parallel actin bundles, but also the thermodynamic pathway by which they form.

  7. Polycomb group protein ezh2 controls actin polymerization and cell signaling.

    PubMed

    Su, I-hsin; Dobenecker, Marc-Werner; Dickinson, Ephraim; Oser, Matthew; Basavaraj, Ashwin; Marqueron, Raphael; Viale, Agnes; Reinberg, Danny; Wülfing, Christoph; Tarakhovsky, Alexander

    2005-05-06

    Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

  8. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    PubMed Central

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  9. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    PubMed

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins.

    PubMed

    Wegner, Waja; Ilgen, Peter; Gregor, Carola; van Dort, Joris; Mott, Alexander C; Steffens, Heinz; Willig, Katrin I

    2017-09-18

    The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.

  11. Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flexneri.

    PubMed Central

    Suzuki, T; Miki, H; Takenawa, T; Sasakawa, C

    1998-01-01

    Shigella, the causative agent of bacillary dysentery, is capable of directing its own movement in the cytoplasm of infected epithelial cells. The bacterial surface protein VirG recruits host components mediating actin polymerization, which is thought to serve as the propulsive force. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP), which is a critical target for filopodium formation downstream of Cdc42, is required for assembly of the actin tail generated by intracellular S.flexneri. N-WASP accumulates at the front of the actin tail and is capable of interacting with VirG in vitro and in vivo, a phenomenon that is not observed in intracellular Listeria monocytogenes. The verprolin-homology region in N-WASP was required for binding to the glycine-rich repeats domain of VirG, an essential domain for recruitment of F-actin on intracellular S.flexneri. Overexpression of a dominant-negative N-WASP mutant greatly inhibited formation of the actin tail by intracellular S.flexneri. Furthermore, depletion of N-WASP from Xenopus egg extracts shut off Shigella actin tail assembly, and this was restored upon addition of N-WASP protein, suggesting that N-WASP is a critical host factor for the assembly of the actin tail by intracellular Shigella. PMID:9582270

  12. A high-affinity interaction with ADP-actin monomers underlies the mechanism and in vivo function of Srv2/cyclase-associated protein.

    PubMed

    Mattila, Pieta K; Quintero-Monzon, Omar; Kugler, Jamie; Moseley, James B; Almo, Steven C; Lappalainen, Pekka; Goode, Bruce L

    2004-11-01

    Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

  13. Correlation between the structure and biochemical activities of FtsA, an essential cell division protein of the actin family.

    PubMed

    Sánchez, M; Valencia, A; Ferrándiz, M J; Sander, C; Vicente, M

    1994-10-17

    Cell division protein FtsA, predicted to belong to the actin family, is present in different cell compartments depending on its phosphorylation state. The FtsA fraction isolated from the cytoplasm is phosphorylated and capable of binding ATP, while the membrane-bound form is unphosphorylated and does not bind ATP. A variant of the protein FtsA102, in which the nucleotide binding site was destroyed by mutagenesis of a highly conserved residue predicted to be needed for the binding, does not bind ATP. Another variant, FtsA104, cannot be phosphorylated because the predicted phosphorylatable residue has been replaced by a non-phosphorylatable one. This protein although unable to bind ATP in vitro, is able to rescue the reversible ftsA2, the irreversible ftsA3 and, almost with the same efficiency, the ftsA16 amber alleles. Consequently, phosphorylation and ATP binding may not be essential for the function of FtsA. Alternatively they may have a regulatory role on the action of FtsA in the septator.

  14. Correlation between the structure and biochemical activities of FtsA, an essential cell division protein of the actin family.

    PubMed Central

    Sánchez, M; Valencia, A; Ferrándiz, M J; Sander, C; Vicente, M

    1994-01-01

    Cell division protein FtsA, predicted to belong to the actin family, is present in different cell compartments depending on its phosphorylation state. The FtsA fraction isolated from the cytoplasm is phosphorylated and capable of binding ATP, while the membrane-bound form is unphosphorylated and does not bind ATP. A variant of the protein FtsA102, in which the nucleotide binding site was destroyed by mutagenesis of a highly conserved residue predicted to be needed for the binding, does not bind ATP. Another variant, FtsA104, cannot be phosphorylated because the predicted phosphorylatable residue has been replaced by a non-phosphorylatable one. This protein although unable to bind ATP in vitro, is able to rescue the reversible ftsA2, the irreversible ftsA3 and, almost with the same efficiency, the ftsA16 amber alleles. Consequently, phosphorylation and ATP binding may not be essential for the function of FtsA. Alternatively they may have a regulatory role on the action of FtsA in the septator. Images PMID:7957059

  15. Actin mRNA levels and actin synthesis during the encystation of Entamoeba invadens.

    PubMed

    Manning-Cela, R; Meraz, M A; Hernandez, J M; Meza, I

    1994-01-01

    Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate the encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeletion occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.

  16. The novel, actin-like protein Tact3 is expressed in rodent testicular haploid germ cells.

    PubMed

    Oh, Sung-Dug; Park, Soo-Yun; Park, Jae-Il; Chun, Sang-Young; Ryu, Tae-hun; Soh, Jaemog

    2013-12-01

    Mouse testis actin-like proteins 1 and 2 (mTact1 and mTact2), which are expressed in murine haploid germ cells, have been described previously. Here, we report the cloning and characterization of a third actin-like protein from rat, rat testis actin-like protein 3 (rTact3). The complete cDNA of the rTact3 gene was approximately 3.7 kb in length, and its corresponding amino acid sequence consisted of 1219 amino acids. The rTact3 gene lacks introns, similar to mTact1 and mTact2. The 356 C-terminal amino acids of rTact3 showed 43% homology with mTact1, whereas the 863 N-terminal amino acids did not show any significant homology. Northern blot analysis revealed that rTact3 mRNA was expressed only in adult rat testes and not during the prepubescent stage. In situ hybridization revealed that rTact3 was expressed exclusively during round and elongated spermatids maturation stages in rat testes. Immunohistochemical experiments using antibodies raised against a synthetic peptide showed that the expression of the rTact3 protein was also restricted in round and elongated spermatids, specifically in the head and acrosome of mature rat sperm. The 5′-flanking region of the mTact3 gene was found to contain a TATA-box motif as well as two putative CREB/c-Jun and five C/EBP motifs. mTact3 promoter activity was enhanced in a dose-dependent manner by the transfection of CREB, c-Jun, or C/EBP in NIH3T3 cells. These results suggest that Tact3 proteins might play an important role in rodent germ-cell development. © 2013 Wiley Periodicals, Inc.

  17. Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin function, the actin cytoskeleton, and cell adhesion.

    PubMed

    Zhang, Haitao; Ghai, Pooja; Wu, Huhehasi; Wang, Changhui; Field, Jeffrey; Zhou, Guo-Lei

    2013-07-19

    CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

  18. A Novel Monoclonal Antibody Against a Synthetic Peptide from β-Actin can React with its Corresponding Protein.

    PubMed

    Amini, Nazila; Bayat, Ali-Ahmad; Zarei, Omid; Hadavi, Reza; Mahmoudian, Jafar; Mahmoudi, Ahmad R; Darzi, Maryam; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2015-01-01

    Actin is one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells with important roles in many cell functions. Antibodies against β-actin and other housekeeping gene-encoded proteins are used as internal loading controls in Western blot analyses. The aim of this study was to produce a monoclonal antibody (mAb) against a synthetic peptide derived from N-terminal region of β-actin and to study its reactivity with different organisms. A synthetic peptide, derived from β-actin, was designed and used to produce a mAb by hybridoma technology. The produced antibody (clone 4E5- A10) was purified by an affinity chromatography column followed by characterization of purified mAb using SDS-PAGE, ELISA and Western blot. Our results showed that 4E5-A10 was an IgM and had desired purity and excellent reactivity with the immunizing peptide with an affinity constant of 2.7x10(8) M(-1)>. It could detect a band of about 45 kDa, corresponding to β-actin, in Western blot. Furthermore, it could react in a more sensitive manner and with a wider range of organisms than a known commercial anti β-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of β-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.

  19. FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions

    PubMed Central

    Brieher, William M.

    2013-01-01

    By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell–cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell–cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity. PMID:24322428

  20. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    PubMed

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-07-06

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

  1. FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions.

    PubMed

    Tang, Vivian W; Brieher, William M

    2013-12-09

    By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell-cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell-cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity.

  2. Mitochondrial function and actin regulate dynamin-related protein 1-dependent mitochondrial fission.

    PubMed

    De Vos, Kurt J; Allan, Victoria J; Grierson, Andrew J; Sheetz, Michael P

    2005-04-12

    Mitochondria display a variety of shapes, ranging from small and spherical or the classical tubular shape to extended networks. Shape transitions occur frequently and include fusion, fission, and branching. It was reported that some mitochondrial shape transitions are developmentally regulated, whereas others were linked to disease or apoptosis. However, if and how mitochondrial function controls mitochondrial shape through regulation of mitochondrial fission and fusion is unclear. Here, we show that inhibitors of electron transport, ATP synthase, or the permeability transition pore (mtPTP) induced reversible mitochondrial fission. Mitochondrial fission depended on dynamin-related protein 1 (DRP1) and F-actin: Disruption of F-actin attenuated fission and recruitment of DRP1 to mitochondria. In contrast, uncoupling of electron transport and oxidative phosphorylation caused mitochondria to adopt a distinct disk shape. This shape change was independent of the cytoskeleton and DRP1 and was most likely caused by swelling. Thus, disruption of mitochondrial function rapidly and reversibly altered mitochondrial shape either by activation of DRP1-dependent fission or by swelling, indicating a close relationship between mitochondrial fission, shape, and function. Furthermore, our results suggest that the actin cytoskeleton is involved in mitochondrial fission by facilitating mitochondrial recruitment of DRP1.

  3. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  4. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  5. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP.

    PubMed

    Hussain, N K; Jenna, S; Glogauer, M; Quinn, C C; Wasiak, S; Guipponi, M; Antonarakis, S E; Kay, B K; Stossel, T P; Lamarche-Vane, N; McPherson, P S

    2001-10-01

    Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

  6. Prediction and integration of regulatory and protein-protein interactions

    SciTech Connect

    Wichadakul, Duangdao; McDermott, Jason E.; Samudrala, Ram

    2009-04-20

    Knowledge of transcriptional regulatory interactions (TRIs) is essential for exploring functional genomics and systems biology in any organism. While several results from genome-wide analysis of transcriptional regulatory networks are available, they are limited to model organisms such as yeast [1] and worm [2]. Beyond these networks, experiments on TRIs study only individual genes and proteins of specific interest. In this chapter, we present a method for the integration of various data sets to predict TRIs for 54 organisms in the Bioverse [3]. We describe how to compile and handle various formats and identifiers of data sets from different sources, and how to predict the TRIs using a homology-based approach, utilizing the compiled data sets. Integrated data sets include experimentally verified TRIs, binding sites of transcription factors, promoter sequences, protein sub-cellular localization, and protein families. Predicted TRIs expand the networks of gene regulation for a large number of organisms. The integration of experimentally verified and predicted TRIs with other known protein-protein interactions (PPIs) gives insight into specific pathways, network motifs, and the topological dynamics of an integrated network with gene expression under different conditions, essential for exploring functional genomics and systems biology.

  7. Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

    PubMed Central

    1995-01-01

    The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin. PMID:7822422

  8. Disruption of actin-binding domain-containing Dystonin protein causes dystonia musculorum in mice.

    PubMed

    Horie, Masao; Watanabe, Keisuke; Bepari, Asim K; Nashimoto, Jun-Ichiro; Araki, Kimi; Sano, Hiromi; Chiken, Satomi; Nambu, Atsushi; Ono, Katsuhiko; Ikenaka, Kazuhiro; Kakita, Akiyoshi; Yamamura, Ken-Ichi; Takebayashi, Hirohide

    2014-11-01

    The Dystonin gene (Dst) is responsible for dystonia musculorum (dt), an inherited mouse model of hereditary neuropathy accompanied by progressive motor symptoms such as dystonia and cerebellar ataxia. Dst-a isoforms, which contain actin-binding domains, are predominantly expressed in the nervous system. Although sensory neuron degeneration in the peripheral nervous system during the early postnatal stage is a well-recognised phenotype in dt, the histological characteristics and neuronal circuits in the central nervous system responsible for motor symptoms remain unclear. To analyse the causative neuronal networks and roles of Dst isoforms, we generated novel multipurpose Dst gene trap mice, in which actin-binding domain-containing isoforms are disrupted. Homozygous mice showed typical dt phenotypes with sensory degeneration and progressive motor symptoms. The gene trap allele (Dst(Gt) ) encodes a mutant Dystonin-LacZ fusion protein, which is detectable by X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) staining. We observed wide expression of the actin-binding domain-containing Dystonin isoforms in the central nervous system (CNS) and peripheral nervous system. This raised the possibility that not only secondary neuronal defects in the CNS subsequent to peripheral sensory degeneration but also cell-autonomous defects in the CNS contribute to the motor symptoms. Expression analysis of immediate early genes revealed decreased neuronal activity in the cerebellar-thalamo-striatal pathway in the homozygous brain, implying the involvement of this pathway in the dt phenotype. These novel Dst(Gt) mice showed that a loss-of-function mutation in the actin-binding domain-containing Dystonin isoforms led to typical dt phenotypes. Furthermore, this novel multipurpose Dst(Gt) allele offers a unique tool for analysing the causative neuronal networks involved in the dt phenotype.

  9. Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones.

    PubMed

    Piper, Michael; Lee, Aih Cheun; van Horck, Francisca P G; McNeilly, Heather; Lu, Trina Bo; Harris, William A; Holt, Christine E

    2015-02-25

    Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis. Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2. Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.

  10. A microtubule-facilitated nuclear import pathway for cancer regulatory proteins.

    PubMed

    Roth, Daniela Martino; Moseley, Gregory W; Glover, Dominic; Pouton, Colin W; Jans, David A

    2007-06-01

    Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormone-related protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110(Rb) tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.

  11. Arf1 and Arf6 Promote Ventral Actin Structures formed by acute Activation of Protein Kinase C and Src

    PubMed Central

    Caviston, Juliane P.; Cohen, Lee Ann; Donaldson, Julie G.

    2016-01-01

    Arf proteins regulate membrane traffic and organelle structure. Although Arf6 is known to initiate actin-based changes in cell surface architecture, Arf1 may also function at the plasma membrane. Here we show that acute activation of protein kinase C (PKC) induced by the phorbol ester PMA led to the formation of motile actin structures on the ventral surface of Beas-2b cells, a lung bronchial epithelial cell line. Ventral actin structures also formed in PMA-treated HeLa cells that had elevated levels of Arf activation. For both cell types, formation of the ventral actin structures was enhanced by expression of active forms of either Arf1 or Arf6, and by the expression of guanine nucleotide exchange factors that activate these Arfs. By contrast, formation of these structures was blocked by inhibitors of PKC and Src, and required phosphatidylinositol 4, 5-bisphosphate, Rac, Arf6 and Arf1. Furthermore, expression of ASAP1, an Arf1 GTPase activating protein (GAP) was more effective at inhibiting the ventral actin structures than was ACAP1, an Arf6 GAP. This study adds to the expanding role for Arf1 in the periphery and identifies a requirement for Arf1, a “Golgi Arf”, in the reorganization of the cortical actin cytoskeleton on ventral surfaces, against the substratum. PMID:24916416

  12. Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins.

    PubMed

    de Arruda, M V; Watson, S; Lin, C S; Leavitt, J; Matsudaira, P

    1990-09-01

    Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

  13. Villin-Like Actin-Binding Proteins Are Expressed Ubiquitously in Arabidopsis1

    PubMed Central

    Klahre, Ulrich; Friederich, Evelyne; Kost, Benedikt; Louvard, Daniel; Chua, Nam-Hai

    2000-01-01

    In an attempt to elucidate the biological function of villin-like actin-binding proteins in plants we have cloned several genes encoding Arabidopsis proteins with high homology to animal villin. We found that Arabidopsis contains at least four villin-like genes (AtVLNs) encoding four different VLN isoforms. Two AtVLN isoforms are more closely related to mammalian villin in their primary structure and are also antigenically related, whereas the other two contain significant changes in the C-terminal headpiece domain. RNA and promoter/β-glucuronidase expression studies demonstrated that AtVLN genes are expressed in all organs, with elevated expression levels in certain types of cells. These results suggest that AtVLNs have less-specialized functions than mammalian villin, which is found only in the microvilli of brush border cells. Immunoblot experiments using a monoclonal antibody against pig villin showed that AtVLNs are widely distributed in a variety of plant tissues. Green fluorescent protein fused to full-length AtVLN and individual AtVLN headpiece domains can bind to both animal and plant actin filaments in vivo. PMID:10631247

  14. Muscle lim protein isoform negatively regulates striated muscle actin dynamics and differentiation.

    PubMed

    Vafiadaki, Elizabeth; Arvanitis, Demetrios A; Papalouka, Vasiliki; Terzis, Gerasimos; Roumeliotis, Theodoros I; Spengos, Konstantinos; Garbis, Spiros D; Manta, Panagiota; Kranias, Evangelia G; Sanoudou, Despina

    2014-07-01

    Muscle lim protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, whereas aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically, it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/cofilin-2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components, including α-actinin, T-cap and MLP. The findings of the present study unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, as well as in differentiated striated muscles as a contributor to sarcomeric integrity. © 2014 FEBS.

  15. Muscle Lim Protein isoform negatively regulates striated muscle actin dynamics and differentiation

    PubMed Central

    Vafiadaki, Elizabeth; Arvanitis, Demetrios A.; Papalouka, Vasiliki; Terzis, Gerasimos; Roumeliotis, Theodoros I.; Spengos, Konstantinos; Garbis, Spiros D.; Manta, Panagiota; Kranias, Evangelia G.; Sanoudou, Despina

    2015-01-01

    Muscle Lim Protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, while aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/CFL2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components including α-actinin, T-cap and MLP. Our findings unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, and in differentiated striated muscles as a contributor to sarcomeric integrity. PMID:24860983

  16. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

    PubMed

    Gottwald, U; Brokamp, R; Karakesisoglou, I; Schleicher, M; Noegel, A A

    1996-02-01

    In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.

  17. Effect of Metals on β-Actin and Total Protein Synthesis in Cultured Human Intestinal Epithelial Cells

    PubMed Central

    Calabro, Anthony R.; Gazarian, Dmitry I.; Barile, Frank A.

    2010-01-01

    As an important structural protein, β-actin is associated with anchoring of tight junctions (TJs) to the cell scaffold. Caco-2 cells, an immortal intestinal epithelial cell line, rely on β-actin to form intact monolayers with high transepithelial electrical resistance in cell culture inserts. We examined the effect of six metals on expression of β-actin mRNA and β-actin synthesis, on total and net production of newly synthesized proteins, on paracellular transport of TJ markers, and on cell viability in confluent monolayers. [3H]-glycine and [3H]-tyrosine were used as indicators of newly synthesized proteins in the absence or presence of increasing concentrations of arsenic, cadmium, copper, manganese, mercury and nickel. The monolayers were exposed to 24-hr single exposures as well as continuous daily repeated doses of metals for 48-hr and 96-hr. Results suggest that decreases in newly synthesized proteins, in which β-actin represents about 10%, correlated with 2- to 5-fold higher expression of β-actin mRNA for the higher concentrations of metals. Interestingly, IC50s calculated for each chemical for 24-hr acute and 48- and 96-hr repeated dosing experiments, using the MTT viability assay and paracellular permeability markers, decreased newly synthesized and total proteins to 10% and 40% of control, respectively. Overall, the results indicate that, at equivalent concentrations, the metals affect β-actin mRNA and newly synthesized proteins before cell viability and paracellular permeability are compromised. Consequently the results help in elucidating mechanisms of metal cytotoxicity that lead to understanding the relationship between tight junction integrity, paracellular transport, and cell viability. PMID:20452446

  18. The putative pocket protein binding site of Autographa californica nucleopolyhedrovirus BV/ODV-C42 is required for virus-induced nuclear actin polymerization.

    PubMed

    Li, Kun; Wang, Yun; Bai, Huimin; Wang, Qian; Song, Jianhua; Zhou, Yuan; Wu, Chunchen; Chen, Xinwen

    2010-08-01

    Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAc(p78/83nls-gfp), with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAc(p78/83nls-gfp)-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.

  19. Role of Proteins of the Ena/VASP Family in Actin-based Motility of Listeria monocytogenes

    PubMed Central

    Laurent, Valérie; Loisel, Thomas P.; Harbeck, Birgit; Wehman, Ann; Gröbe, Lothar; Jockusch, Brigitte M.; Wehland, Jürgen; Gertler, Frank B.; Carlier, Marie-France

    1999-01-01

    Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (−/−) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association–dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 × 108 M−1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment–detachment of VASP to F-actin allows its sliding along the growing filament. PMID:10087267

  20. Actin filament curvature biases branching direction

    PubMed Central

    Risca, Viviana I.; Wang, Evan B.; Chaudhuri, Ovijit; Chia, Jia Jun; Geissler, Phillip L.; Fletcher, Daniel A.

    2012-01-01

    Mechanical cues affect many important biological processes in metazoan cells, such as migration, proliferation, and differentiation. Such cues are thought to be detected by specialized mechanosensing molecules linked to the cytoskeleton, an intracellular network of protein filaments that provide mechanical rigidity to the cell and drive cellular shape change. The most abundant such filament, actin, forms branched networks nucleated by the actin-related protein (Arp) 2/3 complex that support or induce membrane protrusions and display adaptive behavior in response to compressive forces. Here we show that filamentous actin serves in a mechanosensitive capacity itself, by biasing the location of actin branch nucleation in response to filament bending. Using an in vitro assay to measure branching from curved sections of immobilized actin filaments, we observed preferential branch formation by the Arp2/3 complex on the convex face of the curved filament. To explain this behavior, we propose a fluctuation gating model in which filament binding or branch nucleation by Arp2/3 occur only when a sufficiently large, transient, local curvature fluctuation causes a favorable conformational change in the filament, and we show with Monte Carlo simulations that this model can quantitatively account for our experimental data. We also show how the branching bias can reinforce actin networks in response to compressive forces. These results demonstrate how filament curvature can alter the interaction of cytoskeletal filaments with regulatory proteins, suggesting that direct mechanotransduction by actin may serve as a general mechanism for organizing the cytoskeleton in response to force. PMID:22308368

  1. The Actin-Binding Proteins Eps8 and Gelsolin Have Complementary Roles in Regulating the Growth and Stability of Mechanosensory Hair Bundles of Mammalian Cochlear Outer Hair Cells

    PubMed Central

    Johnson, Stuart L.; Parker, Andy; Kuhn, Stephanie; Bowl, Mike; Marcotti, Walter; Brown, Steve D. M.

    2014-01-01

    Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing. PMID:24475274

  2. The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells.

    PubMed

    Olt, Jennifer; Mburu, Philomena; Johnson, Stuart L; Parker, Andy; Kuhn, Stephanie; Bowl, Mike; Marcotti, Walter; Brown, Steve D M

    2014-01-01

    Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.

  3. The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility

    PubMed Central

    Fang, Zhiyou; Takizawa, Norio; Wilson, Korey A.; Smith, Tara C.; Delprato, Anna; Davidson, Michael W.; Lambright, David G.; Luna, Elizabeth J.

    2010-01-01

    In migrating cells, the cytoskeleton coordinates signal transduction and re-distributions of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F-actin- and myosin II-binding protein that tightly associates with signaling proteins in cholesterol-rich, “lipid raft” membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin-knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed-end inhibitor, cytochalasin D, suggests that both treatments affect actin-dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal-regulated kinases 1 and 2 (ERK) and increases the velocity of cell translocation. These results suggest that supervillin, F-actin, and associated proteins may coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin-based cell motility. PMID:20331534

  4. The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells.

    PubMed

    Yasunaga, Takayuki; Hoff, Sylvia; Schell, Christoph; Helmstädter, Martin; Kretz, Oliver; Kuechlin, Sebastian; Yakulov, Toma A; Engel, Christina; Müller, Barbara; Bensch, Robert; Ronneberger, Olaf; Huber, Tobias B; Lienkamp, Soeren S; Walz, Gerd

    2015-12-07

    Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.

  5. The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

    PubMed Central

    Yasunaga, Takayuki; Hoff, Sylvia; Schell, Christoph; Helmstädter, Martin; Kretz, Oliver; Kuechlin, Sebastian; Yakulov, Toma A.; Engel, Christina; Müller, Barbara; Bensch, Robert; Ronneberger, Olaf; Huber, Tobias B.; Lienkamp, Soeren S.

    2015-01-01

    Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton. PMID:26644512

  6. The membrane-associated protein, supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility.

    PubMed

    Fang, Zhiyou; Takizawa, Norio; Wilson, Korey A; Smith, Tara C; Delprato, Anna; Davidson, Michael W; Lambright, David G; Luna, Elizabeth J

    2010-06-01

    In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F-actin- and myosin II-binding protein that tightly associates with signaling proteins in cholesterol-rich, 'lipid raft' membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin-knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed-end inhibitor, cytochalasin D, suggests that both treatments affect actin-dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal-regulated kinases (ERKs) 1 and 2 and increases the velocity of cell translocation. These results suggest that supervillin, F-actin and associated proteins coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin-based cell motility.

  7. Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

    PubMed Central

    Bencsik, Norbert; Szíber, Zsófia; Liliom, Hanna; Tárnok, Krisztián; Borbély, Sándor; Gulyás, Márton; Rátkai, Anikó; Szűcs, Attila; Hazai-Novák, Diána; Ellwanger, Kornelia; Rácz, Bence; Pfizenmaier, Klaus; Hausser, Angelika

    2015-01-01

    Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines. PMID:26304723

  8. Mcp4, a Meiotic Coiled-Coil Protein, Plays a Role in F-Actin Positioning during Schizosaccharomyces pombe Meiosis▿

    PubMed Central

    Ohtaka, Ayami; Okuzaki, Daisuke; Saito, Takamune T.; Nojima, Hiroshi

    2007-01-01

    Some meiosis-specific proteins of Schizosaccharomyces pombe harbor coiled-coil motifs and play essential roles in meiotic progression. Here we describe Mcp4, a novel meiosis-specific protein whose expression is abruptly induced at the horsetail phase and which remains expressed until sporulation is finished. Fluorescence microscopic analysis revealed that Mcp4 alters its subcellular localization during meiosis in a manner that partially resembles the movement of F-actin during meiosis. Mcp4 and F-actin never colocalize; rather, they are located in a side-by-side manner. When forespore membrane formation begins at metaphase II, the Mcp4 signals assemble at the lagging face of the dividing nuclei. At this stage, they are sandwiched between F-actin and the nucleus. Mcp4, in turn, appears to sandwich F-actin with Meu14. In mcp4Δ cells at anaphase II, the F-actin, which is normally dumbbell-shaped, adopts an abnormal balloon shape. Spores of mcp4Δ cells were sensitive to NaCl, although their shape and viability were normal. Taken together, we conclude that Mcp4 plays a role in the accurate positioning of F-actin during S. pombe meiosis. PMID:17435009

  9. Tyrosine phosphorylation of the chlamydial effector protein Tarp is species specific and not required for recruitment of actin.

    PubMed

    Clifton, Dawn R; Dooley, Cheryl A; Grieshaber, Scott S; Carabeo, Reynaldo A; Fields, Kenneth A; Hackstadt, Ted

    2005-07-01

    Chlamydiae are obligate intracellular pathogens that efficiently induce their endocytosis by susceptible eukaryotic host cells. Recently, a Chlamydia trachomatis type III secreted effector protein, Tarp, was found to be translocated and tyrosine phosphorylated at the site of entry and associated with the recruitment of actin that coincides with endocytosis. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. Here we have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrated that tyrosine phosphorylation occurs primarily within the repeat region, while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp, although Chlamydia muridarum, Chlamydophila caviae, and Chlamydophila pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an antiphosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae, although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis.

  10. Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

    NASA Astrophysics Data System (ADS)

    Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

    2006-03-01

    Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

  11. Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.

    PubMed

    Matthews, Paul R; Eastwood, Sharon L; Harrison, Paul J

    2012-01-01

    Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17) from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]). Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.

  12. F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells

    PubMed Central

    Feygin, Alex; Ivanov, Andrei I.

    2015-01-01

    Tight junctions (TJ) and adherens junctions (AJ) are key morphological features of differentiated epithelial cells that regulate the integrity and permeability of tissue barriers. Structure and remodeling of epithelial junctions depends on their association with the underlying actomyosin cytoskeleton. Anillin is a unique scaffolding protein interacting with different cytoskeletal components, including actin filaments and myosin motors. Its role in the regulation of mammalian epithelial junctions remains unexplored. Downregulation of anillin expression in human prostate, colonic, and lung epithelial cells triggered AJ and TJ disassembly without altering the expression of junctional proteins. This junctional disassembly was accompanied by dramatic disorganization of the perijunctional actomyosin belt; while the general architecture of the actin cytoskeleton, and activation status of non-muscle myosin II, remained unchanged. Furthermore, loss of anillin disrupted the adducin-spectrin membrane skeleton at the areas of cell-cell contact, selectively decreased γ-adducin expression, and induced cytoplasmic aggregation of αII-spectrin. Anillin knockdown activated c-Jun N-terminal kinase (JNK), and JNK inhibition restored AJ and TJ integrity and cytoskeletal organization in anillin-depleted cells. These findings suggest a novel role for anillin in regulating intercellular adhesion in model human epithelia by mechanisms involving the suppression of JNK activity and controlling the assembly of the perijunctional cytoskeleton. PMID:25809162

  13. Membrane trafficking in protozoa SNARE proteins, H+-ATPase, actin, and other key players in ciliates.

    PubMed

    Plattner, Helmut

    2010-01-01

    Due to their well-defined pathways of vesicle trafficking and manyfold mutants ciliates have served as good model systems. Further studies required the development of databases, now available for Paramecium and Tetrahymena. A variety of key players have been identified and characterized based on BLAST search, domain analysis, localization, and gene-silencing studies. They include NSF (N-ethylmaleimide sensitive factor), SNAREs (soluble NSF attachment protein [SNAP] receptors), the H(+)-ATPase (V-ATPase) and actin, while Arf (ADP-ribosylation factor) and Rab-type small GTPases, COPs (coatamer proteins) and many others remain to be elucidated. The number of SNAREs, H(+)-ATPase subunits, and actins ever found within one cell type are unexpectedly high and most of the manifold vesicle types seem to be endowed with specific molecular components pertinent to trafficking. As in higher eukaryotes, multifactorial targeting likely occurs. It appears that, in parallel to higher organisms, ciliates have evolved a similar structural and molecular complexity of vesicle trafficking.

  14. Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

    PubMed Central

    Yamashiro, Shigeko; Yamakita, Yoshihiko; Ono, Shoichiro; Matsumura, Fumio

    1998-01-01

    Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery. PMID:9571235

  15. Pattern formation in actin gels: A study in the mechanics of gels formed by the important cytoskeletal protein actin, especially as applied to cellular motility

    NASA Astrophysics Data System (ADS)

    Balter, Ariel

    We have studied pattern formation in actin gels to better understand how they function in biological systems, especially in the motility mechanism used by some pathogenic bacteria such as Listeria. By coating themselves with certain enzymes, these bacteria appropriate actin (a protein) from the surrounding host cell's cytoplasm and cause a network or "gel" of actin filaments to grow on their outer surface. As the resulting "comet tail" shaped protrusion grows, it pushes the bacterium away. In experiments, polystyrene beads coated with the same enzymes will also generate comet tails and swim in a very similar manner. However, these bead experiments have also generated anomalous results such as the formation of many comet tails. In some experiments, when two comet tails formed they systematically grew into regular, oppositely handed helices. The formation of any comet tails on a bead poses a physical conundrum. The bacterial enzyme coating is asymmetrical so the comet tail forms in a particular place. But the beads are symmetrical, so comet tails formation constitutes symmetry breaking and spontaneous pattern formation. We have modeled this process as a competition between elastic energy (which favors many tails) and chemical energy (which favors few tails). Our analytical model explains the factors that experimentally determine the number of tails, and numerical simulations confirm these predictions. To understand the helical tails, we did extensive data analysis involving image processing, statistical analysis and mathematical modeling of images of the helical tails. We identified some important features of how the twin tails form. For instance, the tail growth rate is independent of drag force, and bead rotation must accompany helical tail formation. We also created a physical model for helical growth. Numerical simulations of our model show that at very low Reynolds number, a cylindrical object growing under the conditions of an actin comet tail can spontaneously

  16. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells.

    PubMed

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

  17. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.

    PubMed

    Blanchoin, L; Amann, K J; Higgs, H N; Marchand, J B; Kaiser, D A; Pollard, T D

    2000-04-27

    Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

  18. Zyxin antagonizes the FERM protein expanded to couple F-actin and Yorkie-dependent organ growth.

    PubMed

    Gaspar, Pedro; Holder, Maxine V; Aerne, Birgit L; Janody, Florence; Tapon, Nicolas

    2015-03-16

    Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. We generated a zyx mutant in Drosophila using TALEN endonucleases and used this to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, we show that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). Our results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

    PubMed Central

    Gay, Olivia; Gilquin, Benoît; Nakamura, Fumihiko; Jenkins, Zandra A.; McCartney, Rosannah; Krakow, Deborah; Deshiere, Alexandre; Assard, Nicole; Hartwig, John H.; Robertson, Stephen P.; Baudier, Jacques

    2011-01-01

    The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial–mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. PMID:21709252

  20. The Role of the Actin Cytoskeleton in Regulating Drosophila Behavior

    PubMed Central

    Ojelade, Shamsideen A.; Acevedo, Summer F.; Rothenfluh, Adrian

    2014-01-01

    Over the past decade, the function of the cytoskeleton has been extensively studied in developing and in mature neurons. Actin, a major cytoskeletal protein, is indispensable for the structural integrity and plasticity of neurons and their synapses. Disruption of actin dynamics has significant consequence for neurons, neuronal circuits, and the functions they govern. In particular, cell adhesion molecules (CAMs), members of the Rho family of GTPases, and actin binding proteins (ABPs) are important modulators of actin dynamics and neuronal as well as behavioral plasticity. In this review, we discuss recent advances in Drosophila that highlight the importance of actin regulatory proteins in mediating fly behaviors such as circadian rhythm, courtship behavior, learning and memory, and the development of drug addiction. PMID:24077615

  1. REM sleep deprivation attenuates actin-binding protein cortactin: a link between sleep and hippocampal plasticity.

    PubMed

    Davis, Christopher J; Meighan, Peter C; Taishi, Ping; Krueger, James M; Harding, Joseph W; Wright, John W

    2006-06-12

    Rapid eye-movement sleep (REMS) is thought to affect synaptic plasticity. Cortactin is a cytoskeletal protein critically involved in the regulation of actin branching and stabilization including the actin backbone of dendritic spines. Hippocampal cortactin levels, phosphorylation, and processing appear to be altered during learning and long-term potentiation (LTP); consistent with a role for cortactin in the dendritic restructuring that accompanies synaptic plasticity. In this study juvenile male Sprague-Dawley rats were selectively REMS-deprived (RD) for 48 h by the flowerpot method. Cage control (CC) and large pedestal control (PC) animals were used for comparison. Animals were euthanized immediately, or 12 h, after removal from the pedestal. The hippocampus was dissected, flash-frozen, and stored for subsequent Western blot or quantitative RT-PCR analysis of cortactin. Cortactin mRNA/cDNA levels initially rose in PC and RD rats but returned to CC levels by 12 h after removal from the pedestal. Predictably cortactin protein levels were initially unchanged but were up-regulated after 12 h. The PC group had more total and tyrosine-phosphorylated cortactin protein expression than the RD and CC groups. This increase in cortactin was likely due to the exposure of the rats to the novel environment of the deprivation chambers thus triggering plasticity events. The lack of REMS, however, severely hampered cortactin protein up-regulation and phosphorylation observed in the PC group suggesting an attenuation of plasticity-related events. Thus, these data support a functional link between REMS and cytoskeletal reorganization in the hippocampus, a process that is essential for synaptic plasticity.

  2. Differential regulation of cortactin and N-WASP-mediated actin polymerization by missing in metastasis (MIM) protein.

    PubMed

    Lin, Jinxiu; Liu, Jiali; Wang, Ying; Zhu, Jianwei; Zhou, Kang; Smith, Nicole; Zhan, Xi

    2005-03-17

    Missing in metastasis (MIM) gene encodes an actin binding protein that is expressed at low levels in a subset of malignant cell lines. MIM protein tagged by green fluorescent protein (GFP) colocalizes with cortactin, an Arp2/3 complex activator, and interacts directly with the SH3 domain of cortactin. Recombinant full-length MIM promotes markedly cortactin and Arp2/3 complex-mediated actin polymerization in an SH3 dependent manner. In contrast, MIM-CT, a short splicing variant of MIM, binds poorly to cortactin in vitro and is unable to enhance actin polymerization. Full-length MIM binds to G-actin with a similar affinity as N-WASP-VCA, a constitutively active form of N-WASP, and inhibits N-WASP-VCA-mediated actin polymerization as analysed in vitro. The significance of the association of MIM with cortactin and G-actin was evaluated in NIH3T3 cells expressing several MIM constructs. Overexpression of full-length wild-type MIM-GFP inhibited markedly the motility of NIH3T3 cells induced by PDGF and that of human vein umbilical endothelial cells induced by sphingosine 1 phosphate. However, an MIM mutant with deletion of the WH2 domain, which is responsible for G-actin binding, enhanced cell motility. The motility inhibition imposed by MIM was compromised in the cells overexpressing N-WASP. In contrast, deletion of an MIM proline-rich domain, which is required for an optimal binding to cortactin, substantiated the MIM-mediated inhibition of cell motility. These data imply that MIM regulates cell motility by modulating different Arp2/3 activators in a distinguished manner.

  3. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

    PubMed Central

    Gao, Ying; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  4. Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein

    PubMed Central

    Kim, Kyoungtae; McCully, Michelle E.; Bhattacharya, Nandini; Butler, Boyd; Sept, David; Cooper, John A.

    2008-01-01

    The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP2). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP2 binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP2 binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP2 rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can “wobble” when bound to the barbed end solely by the C-terminal “tentacle” of its β-subunit. PMID:17182619

  5. Mutational Analysis Reveals a Noncontractile but Interactive Role of Actin and Profilin in Viral RNA-Dependent RNA Synthesis▿

    PubMed Central

    Harpen, Mary; Barik, Tiasha; Musiyenko, Alla; Barik, Sailen

    2009-01-01

    As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role. PMID:19710142

  6. A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction

    PubMed Central

    Van Itallie, Christina M.; Tietgens, Amber Jean; Krystofiak, Evan; Kachar, Bechara; Anderson, James M.

    2015-01-01

    Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR–domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation–promoting factor N-WASP to tight junctions. CRISPR-Cas9–mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1–knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin. PMID:26063734

  7. The kinesin-like proteins, KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens.

    PubMed

    Shen, Zhiyuan; Liu, Yen-Chen; Bibeau, Jeffrey P; Lemoi, Kyle P; Tüzel, Erkan; Vidali, Luis

    2015-01-01

    In plants, light determines chloroplast position; these organelles show avoidance and accumulation responses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thaliana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins (called KACs) are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene-specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specific cytoskeletal elements responsible for motility, this loss-of-function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament-dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.

  8. Cytoskeleton alterations in melanoma: aberrant expression of cortactin, an actin-binding adapter protein, correlates with melanocytic tumor progression

    PubMed Central

    Xu, Xu-Zhi; Garcia, Marileila Varella; Li, Tian-yu; Khor, Li-Yan; Gajapathy, R Sujatha; Spittle, Cindy; Weed, Scott; Lessin, Stuart R; Wu, Hong

    2010-01-01

    Cortactin is a multidomain actin-binding protein important for the functions of cytoskeleton by regulating cortical actin dynamics. It is involved in a diverse array of basic cellular functions. Tumorigenesis and tumor progression involves alterations in actin cytoskeleton proteins. We sought to study the role of cortactin in melanocytic tumor progression using immunohistochemistry on human tissues. The results reveal quantitative differences between benign and malignant lesions. Significantly higher cortactin expression is found in melanomas than in nevi (P<0.0001), with levels greater in metastatic than in invasive melanomas (P<0.05). Qualitatively, tumor tissues often show aberrant cortactin localization at the cell periphery, corresponding to its colocalization with filamentous actin in cell cortex of cultured melanoma cells. This suggests an additional level of protein dysregulation. Furthermore, in patients with metastatic disease, high-level cortactin expression correlates with poor disease-specific survival. Our data, in conjunction with outcome data on several other types of human cancers and experimental data from melanoma cell lines, supports a potential role of aberrant cortactin expression in melanoma tumor progression and a rational for targeting key elements of actin-signaling pathway for developmental therapeutics in melanomas. PMID:19898426

  9. Regulation of actin dynamics and protein trafficking during spermatogenesis – insights into a complex process*

    PubMed Central

    Su, Wenhui; Mruk, Dolores; Cheng, C Yan

    2013-01-01

    In the mammalian testis, extensive restructuring takes place across the seminiferous epithelium at the Sertoli-Sertoli and Sertoli-germ cell interface during the epithelial cycle of spermatogenesis, which is important to facilitate changes in the cell shape and morphology of developing germ cells. However, precise communications also take place at the cell junctions to coordinate the discrete events pertinent to spermatogenesis, namely spermatogonial renewal via mitosis, cell cycle progression and meiosis, spermiogenesis, and spermiation. It is obvious that these cellular events are intimately related to the underlying actin-based cytoskeleton which is being used by different cell junctions for their attachment. However, little is known on the biology and regulation of this cytoskeleton, in particular its possible involvement in endocytic vesicle-mediated trafficking during spermatogenesis, which in turn affects cell adhesive function and communication at the cell-cell interface. Studies in other epithelia in recent years have shed insightful information on the intimate involvement of actin dynamics and protein trafficking in regulating cell adhesion and communications. The goal of this critical review is to provide an updated assessment of the latest findings in the field on how these complex processes regulate spermatogenesis. We also provide a working model based on the latest findings in the field to provide our thoughts on an apparent complicated subject, which also serves as the framework for investigators in the field. It is obvious that this model will be rapidly updated when more data are available in future years. PMID:23339542

  10. The Disruption of the Cytoskeleton during Semaphorin 3A induced Growth Cone Collapse Correlates with Differences in Actin Organization and Associated Binding Proteins

    PubMed Central

    Brown, Jacquelyn A; Bridgman, Paul C

    2010-01-01

    Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp-2 and cortactin decreases relative to total protein, while in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f-actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. PMID:19513995

  11. [The contractile, regulatory and structural proteins of myocardium].

    PubMed

    Adamcová, Michaela; Pelouch, Václav

    2003-01-01

    The myocardium consists of three basic categories of proteins. The myofibrillar proteins trasform the chemical energy of ATP to the mechanical work of the heart. The metabolic proteins located both in the cytosol and in the mitochondrial compartments provide energy for the cardiac contraction. The interstitial space between myocytes is occupied by the extracellular proteins (collagens, glycoproteins, glycosaminoglycans, elastins). By far the greater percentage of myofibrillar proteins (about 80%) is that concerned with contraction (actin and myosin), with about 10% concerned with its regulation (troponin, tropomyosin and tropomodulin) and another 10% concerned with maintenance of the structure of myofibril (C, M-, H-proteins, myomesin, nebulette, alpha-actinin, titin, CapZ protein). Most collagenous and non-collagenous proteins exist in many isoforms that originate from the same genom but are the product of alternative splicing of a primary RNA transcript.

  12. Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

    PubMed Central

    Visser, Michelle B.; Koh, Adeline; Glogauer, Michael; Ellen, Richard P.

    2011-01-01

    The major outer sheath protein (Msp) of Treponema denticola perturbs actin dynamics in fibroblasts by inducing actin reorganization, including subcortical actin filament assembly, leading to defective calcium flux, diminished integrin engagement of collagen, and retarded cell migration. Yet, its mechanisms of action are unknown. We challenged Rat-2 fibroblasts with enriched native Msp. Msp activated the small GTPases Rac1, RhoA and Ras, but not Cdc42, yet only Rac1 localized to areas of actin rearrangement. We used Rac1 dominant negative transfection and chemical inhibition of phosphatidylinositol-3 kinase (PI3K) to show that even though Rac1 activation was PI3K-dependent, neither was required for Msp-induced actin rearrangement. Actin free barbed end formation (FBE) by Msp was also PI3K-independent. Immunoblotting experiments showed that gelsolin and CapZ were released from actin filaments, whereas cofilin remained in an inactive state. Msp induced phosphatidylinositol (4,5)-bisphosphate (PIP2) formation through activation of a phosphoinositide 3-phosphatase and its recruitment to areas of actin assembly at the plasma membrane. Using a PIP2 binding peptide or lipid phosphatase inhibitor, PIP2 was shown to be required for Msp-mediated actin uncapping and FBE formation. Evidently, Msp induces actin assembly in fibroblasts by production and recruitment of PIP2 and release of the capping proteins CapZ and gelsolin from actin barbed ends. PMID:21901132

  13. Ras GTPase-Activating Protein Regulation of Actin Cytoskeleton and Hyphal Polarity in Aspergillus nidulans▿ †

    PubMed Central

    Harispe, Laura; Portela, Cecilia; Scazzocchio, Claudio; Peñalva, Miguel A.; Gorfinkiel, Lisette

    2008-01-01

    Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more “true” Ras proteins. A gapAΔ strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapAΔ strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapAΔ disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapAΔ conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapAΔ cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapAΔ cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis. PMID:18039943

  14. beta-Catenin associates with the actin-bundling protein fascin in a noncadherin complex

    PubMed Central

    1996-01-01

    Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesivity, catenins have more recently been indicated to participate in cell and developmental signaling pathways. beta-Catenin, for example, associates directly with at least two receptor tyrosine kinases and transduces developmental signals within the Wnt pathway. Catenins also complex with the tumor suppressor protein adenomatous polyposis coli (APC), which appears to have a role in regulating cell proliferation. We have used the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to beta-catenin's central Armadillo repeat domain. Western blotting of immunoprecipitates from cell line and mouse and rat brain extracts indicate that this interaction exists in vivo. Fascin and beta-catenin's association was further substantiated in vitro using purified proteins isolated from recombinant bacterial and baculoviral sources. Immunoprecipitation analysis indicates that fascin additionally binds to plakoglobin, which is highly homologous to beta-catenin but not to p120cas, a newly described catenin which contains a more divergent Armadillo-repeat domain. Immunoprecipitation, in vitro competition, and domain-mapping experiments demonstrate that fascin and E-cadherin utilize a similar binding site within beta-catenin, such that they form mutually exclusive complexes with beta-catenin. Immunofluorescence microscopy reveals that fascin and beta-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins were unexpectedly observed to colocalize with fascin and beta-catenin at cell leading edges. It is conceivable that beta-catenin participates in modulating cytoskeletal dynamics in association with the microfilament-bundling protein fascin, perhaps in a

  15. CLIC proteins, ezrin, radixin, moesin and the coupling of membranes to the actin cytoskeleton: a smoking gun?

    PubMed

    Jiang, Lele; Phang, Juanita M; Yu, Jiang; Harrop, Stephen J; Sokolova, Anna V; Duff, Anthony P; Wilk, Krystyna E; Alkhamici, Heba; Breit, Samuel N; Valenzuela, Stella M; Brown, Louise J; Curmi, Paul M G

    2014-02-01

    The CLIC proteins are a highly conserved family of metazoan proteins with the unusual ability to adopt both soluble and integral membrane forms. The physiological functions of CLIC proteins may include enzymatic activity in the soluble form and anion channel activity in the integral membrane form. CLIC proteins are associated with the ERM proteins: ezrin, radixin and moesin. ERM proteins act as cross-linkers between membranes and the cortical actin cytoskeleton. Both CLIC and ERM proteins are controlled by Rho family small GTPases. CLIC proteins, ERM and Rho GTPases act in a concerted manner to control active membrane processes including the maintenance of microvillar structures, phagocytosis and vesicle trafficking. All of these processes involve the interaction of membranes with the underlying cortical actin cytoskeleton. The relationships between Rho GTPases, CLIC proteins, ERM proteins and the membrane:actin cytoskeleton interface are reviewed. Speculative models are proposed involving the formation of localised multi-protein complexes on the membrane surface that assemble via multiple weak interactions. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

  16. Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1.

    PubMed

    Discher, D; Parra, M; Conboy, J G; Mohandas, N

    1993-04-05

    Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the previously identified spectrin-actin binding domain (10 kDa molecular mass) of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, denoted 10k21 and 10k19,21, were able to bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette, 10k19 and 10k0, did not function in these assays. A bacterially expressed fusion protein with glutathione-S-transferase, designated GST-10k21, congealed spectrin-actin into a network in vitro as found with purified protein 4.1. Additionally, incorporation of GST-10k21 into mechanically weak, 4.1-deficient membranes increased mechanical strength of these membranes to normal. GST-10k19 did not function in these assays. These results show that the 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 are suggested to have different, yet to be defined functions.

  17. Differential expression of smooth muscle regulatory proteins in the uterosacral ligaments of women with uterine prolapse.

    PubMed

    Takacs, Peter; Gualtieri, Marc; Nassiri, Mehdi; Candiotti, Keith; Fornoni, Alessia; Medina, Carlos A

    2010-06-01

    To compare smooth muscle regulatory protein expression in the uterosacral ligament (USL) of women with and without uterine prolapse. USLs ligament were sampled in women with (n = 9) or without (n = 9) uterine prolapse. Caldesmon, smooth muscle actin (SMA), myosin heavy chain, and zinc finger protein messenger RNA expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry and digital image analysis were used to determine protein expression. Caldesmon messenger RNA expression and the ratio of caldesmon-SMA messenger RNA expression was significantly increased in the USL from women with uterine prolapse compared with women without prolapse (caldesmon mean +/- standard deviation messenger RNA, 0.81 +/- 0.46 vs 0.39 +/- 0.16; P = .01 and caldesmon-SMA messenger RNA ratio, mean +/- standard deviation, 0.11 +/- 0.04 vs 0.07 +/- 0.02; P = .01). In addition, the ratio of caldesmon-SMA staining was significantly increased in women with uterine prolapse compared with women without prolapse (mean +/- standard deviation, 0.44 +/- 0.28 vs 0.28 +/- 0.16; P = .03). Uterine prolapse is associated with an increased ratio of caldesmon-SMA actin expression. Copyright 2010 Mosby, Inc. All rights reserved.

  18. Costars, a Dictyostelium protein similar to the C-terminal domain of STARS, regulates the actin cytoskeleton and motility.

    PubMed

    Pang, Te-Ling; Chen, Fung-Chi; Weng, Yi-Lan; Liao, Hsien-Ching; Yi, Yung-Hsiang; Ho, Chia-Lin; Lin, Chi-Hung; Chen, Mei-Yu

    2010-11-01

    Through analysis of a chemotaxis mutant obtained from a genetic screen in Dictyostelium discoideum, we have identified a new gene involved in regulating cell migration and have named it costars (cosA). The 82 amino acid Costars protein sequence appears highly conserved among diverse species, and significantly resembles the C-terminal region of the striated muscle activator of Rho signaling (STARS), a mammalian protein that regulates the serum response factor transcriptional activity through actin binding and Rho GTPase activation. The cosA-null (cosA(-)) cells formed smooth plaques on bacterial lawns, produced abnormally small fruiting bodies when developed on the non-nutrient agar and displayed reduced migration towards the cAMP source in chemotactic assays. Analysis of cell motion in cAMP gradients revealed decreased speed but wild-type-like directional persistence of cosA(-) cells, suggesting a defect in the cellular machinery for motility rather than for chemotactic orientation. Consistent with this notion, cosA(-) cells exhibited changes in the actin cytoskeleton, showing aberrant distribution of F-actin in fluorescence cell staining and an increased amount of cytoskeleton-associated actin. Excessive pseudopod formation was also noted in cosA(-) cells facing chemoattractant gradients. Expressing cosA or its human counterpart mCostars eliminated abnormalities of cosA(-) cells. Together, our results highlight a role for Costars in modulating actin dynamics and cell motility.

  19. The actin-related protein hArp8 accumulates on the mitotic chromosomes and functions in chromosome alignment

    SciTech Connect

    Aoyama, Naoki; Oka, Asako; Kitayama, Kumiko; Kurumizaka, Hitoshi; Harata, Masahiko

    2008-02-15

    The actin family consists of conventional actin and various actin-related proteins (Arps). Some of these Arps are localized in the nucleus, and a fraction of each of these nuclear Arps is functionally involved in chromatin remodeling and histone acetyltransferase complexes. On the other hand, in mitotic cells, the localization and function of the nuclear Arps are largely unknown. Human Arp8 (hArp8), an ortholog of yeast nuclear Arp8, was recently found to be associated with the hINO80-chromatin remodeling complex along with hArp5. Here we report that hArp8, but not hArp5, accumulates on mitotic chromosomes. This is the first example where a member of the actin family is found to be associated with mitotic chromosomes. Expression of truncated hArp8 proteins and depletion of endogenous hArp8 by RNA interference caused misalignment of mitotic chromosomes, suggesting that chromosome-associated hArp8 has a role in chromosome behavior. In contrast, depletion of hIno80 and hArp5 did not cause misalignment of chromosomes, suggesting that the role of hArp8 at mitotic chromosomes is independent of the activity of hINO80 complexes. These findings provide the first insight into a novel function of actin family members in mitosis.

  20. Bacterial Actins.

    PubMed

    Izoré, Thierry; van den Ent, Fusinita

    2017-01-01

    A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.

  1. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    NASA Technical Reports Server (NTRS)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  2. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    NASA Technical Reports Server (NTRS)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  3. Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement.

    PubMed

    Disanza, A; Steffen, A; Hertzog, M; Frittoli, E; Rottner, K; Scita, G

    2005-05-01

    Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.

  4. Actin cytoskeleton remodelling in the anterior pituitary folliculostellate cell line TtT/GF: participation of the actin-binding protein cortactin.

    PubMed

    Zheng, Guifu; Solinet, Sara; Pelletier, R-Marc; Vitale, María Leiza

    2005-10-01

    We have previously shown that the folliculostellate (FS) cells of the anterior pituitary change their shape from stellate (type I) to polygonal (type II) coincidently with variations in the secretory activity of the pituitary. To elucidate the mechanisms involved in this switch in phenotypes, here we studied the impact of serum factors on the morphology of the FS cell line TtT/GF. TtT/GF cells cultured in serum-containing medium displayed elongated shapes and membrane ruffles similarly to type I cells. Serum deprivation caused the loss of plasma membrane activity and the acquisition by the cells of a sedentary phenotype and of a polygonal shape typical of type II FS cells. Addition of serum to the starved cells induced the reappearance of membrane raffles and lamellipodia. The switch in phenotypes and the maintenance of a motile phenotype depended on tyrosine kinase but not on Erk activity. Because the transition between phenotypes involved the tyrosine kinase-dependent reorganization of cortical actin filaments, we studied the participation of the actin-binding protein, cortactin, a tyrosine kinase substrate. Cortactin and its tyrosine-phosphorylated form, pY421-cortactin, localized to membrane ruffles and lamellipodia in serum-cultured TtT/GF cells, while they were evenly distributed over the whole cell cortex in serum-starved cells. Serum treatment of starved cells induced a transient increase in pY421-cortactin levels and the clustering of pY421-cortactin in membrane regions where protrusions were developing. Both serum responses were blocked by a tyrosine kinase inhibitor. Together, the results indicate that the transition from a polygonal to an elongated shape entails the acquisition of a dynamic cortical actin cytoskeleton that involves the tyrosine kinase-dependent phosphorylation of cortactin and the translocation of cortical pY421-cortactin to sites of ruffle formation at the plasma membrane.

  5. Functional characterization of protein 4.1 homolog in amphioxus: defining a cryptic spectrin-actin-binding site.

    PubMed

    Wang, Lixia; Wang, Yuan; Li, Zhaohe; Gao, Zhan; Zhang, Shicui

    2013-10-07

    Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a "cryptic" SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins.

  6. Autographa californica Multiple Nucleopolyhedrovirus Ac34 Protein Retains Cellular Actin-Related Protein 2/3 Complex in the Nucleus by Subversion of CRM1-Dependent Nuclear Export

    PubMed Central

    Mu, Jingfang; Zhang, Yongli; Hu, Yangyang; Hu, Xue; Zhou, Yuan; Pei, Rongjuan; Wu, Chunchen; Chen, Jizheng; van Oers, Monique M.; Chen, Xinwen; Wang, Yun

    2016-01-01

    Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs, determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin. A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3, which induces actin polymerization in the nucleus to assist in virus replication. We found that Ac34, a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is involved in Arp2/3 nuclear accumulation during virus infection. Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 (CRM1)-dependent nuclear export. Upon AcMNPV infection, Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus. PMID:27802336

  7. [Nucleotide receptors and actin cytoskeleton dynamics].

    PubMed

    Kłopocka, Wanda; Korczyński, Jarosław

    2014-01-01

    Signaling cascades evoked by P2Y2 receptor plays an important role in the phenomena dependent on the actin cytoskeleton dynamics endocy-tosis, cell division, adhesion, intracellular transport and migration. P2Y2R coupled with G proteins, in response to ATP or UTP activates Rac1 and RhoA proteins important factors in actin cytoskeletal reorganization and regulates the level of phosphatidylinositol-4,5-bisphosphate (PIP2) that binds directly to a variety of actin regulatory proteins and modulates their function. The P2Y2 nucleotide receptor contains the integrin-binding domain enables it to interact selectively with α(v)β3 and α(v)β5 integrins and is required for G0-mediated Rac1 activation. Interaction with α(v)β5 is necessary for coupling the P2Y2 receptor to G12 and subsequent activation of RhoA.

  8. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells

    SciTech Connect

    Weisinger, Gary . E-mail: gary_w@tasmc.health.gov.il; Limor, Rona; Marcus-Perlman, Yonit; Knoll, Esther; Kohen, Fortune; Schinder, Vera; Firer, Michael; Stern, Naftali

    2007-05-11

    The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

  9. 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

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

    SciTech Connect

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

    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 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.

  11. Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein.

    PubMed

    Ge, Jiachun; Dong, Zhangji; Li, Jingyun; Xu, Zhiqiang; Song, Wei; Bao, Jie; Liang, Dong; Li, Junbo; Li, Kui; Jia, Wenshuang; Zhao, Muzi; Cai, Yongxiang; Yang, Jiaxin; Pan, Jianlin; Zhao, Qingshun

    2012-10-01

    Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of β-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

  12. Species and tissue distribution of the regulatory protein of glucokinase.

    PubMed

    Vandercammen, A; Van Schaftingen, E

    1993-09-01

    Rat liver is known to contain a regulatory protein that inhibits glucokinase (hexokinase IV or D) competitively versus glucose. This inhibition is greatly reinforced by the presence of fructose 6-phosphate and antagonized by fructose 1-phosphate and by KCl. This protein was now measured in various rat tissues and in the livers of various species by the inhibition it exerts on rat liver glucokinase. Rat, mouse, rabbit, guinea-pig and pig liver, all of which contain glucokinase, also contained between 60 and 200 units/g of tissue of a regulatory protein displaying the properties mentioned above. By contrast, this protein could not be detected in cat, goat, chicken or trout liver, or in rat brain, heart, skeletal muscle, kidney and spleen, all tissues from which glucokinase is missing. Fructose 1-phosphate stimulated glucokinase in extracts of human liver, indicating the presence of regulatory protein. In addition, antibodies raised against rat regulatory protein allowed the detection of an approximately 60 kDa polypeptide in rat, guinea pig, rabbit and human liver. The livers of the toad Bufo marinus, of Xenopus laevis and of the turtle Pseudemys scripta elegans contained a regulatory protein similar to that of the rat, with, however, the major difference that it was not sensitive to fructose 6-phosphate or fructose 1-phosphate. In rat liver, the regulatory protein was detectable 4 days before birth. Its concentration increased afterwards to reach the adult level at day 30 of extrauterine life, whereas glucokinase only appeared after day 15. In the liver of the adult rat, starvation and streptozotocin-diabetes caused a 50-60% decrease in the concentration of regulatory protein after 7 days, whereas glucokinase activity fell to about 20% of its initial level. When 4-day-starved rats were refed, or when diabetic rats were treated with insulin, the concentration of regulatory protein slowly increased to reach about 85% of the control level after 3 days, whereas the

  13. Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex.

    PubMed Central

    Millard, Thomas H; Sharp, Stewart J; Machesky, Laura M

    2004-01-01

    The assembly of a branched network of actin filaments provides the mechanical propulsion that drives a range of dynamic cellular processes, including cell motility. The Arp2/3 complex is a crucial component of such filament networks. Arp2/3 nucleates new actin filaments while bound to existing filaments, thus creating a branched network. In recent years, a number of proteins that activate the filament nucleation activity of Arp2/3 have been identified, most notably the WASP (Wiskott-Aldrich syndrome protein) family. WASP-family proteins activate the Arp2/3 complex, and consequently stimulate actin assembly, in response to extracellular signals. Structural studies have provided a significant refinement in our understanding of the molecular detail of how the Arp2/3 complex nucleates actin filaments. There has also been much progress towards an understanding of the complicated signalling processes that regulate WASP-family proteins. In addition, the use of gene disruption in a number of organisms has led to new insights into the specific functions of individual WASP-family members. The present review will discuss the Arp2/3 complex and its regulators, in particular the WASP-family proteins. Emphasis will be placed on recent developments in the field that have furthered our understanding of actin dynamics and cell motility. PMID:15040784

  14. Gas7b (growth arrest specific protein 7b) regulates neuronal cell morphology by enhancing microtubule and actin filament assembly.

    PubMed

    Gotoh, Aina; Hidaka, Masafumi; Hirose, Keiko; Uchida, Takafumi

    2013-11-29

    Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.

  15. The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

    PubMed Central

    Graziano, Brian R.; Yu, Hoi-Ying E.; Alioto, Salvatore L.; Eskin, Julian A.; Ydenberg, Casey A.; Waterman, David P.; Garabedian, Mikael; Goode, Bruce L.

    2014-01-01

    Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network. PMID:24719456

  16. Following the Viterbi Path to Deduce Flagellar Actin-Interacting Proteins of Leishmania spp.: Report on Cofilins and Twinfilins

    NASA Astrophysics Data System (ADS)

    Pacheco, Ana Carolina L.; Araújo, Fabiana F.; Kamimura, Michel T.; Medeiros, Sarah R.; Viana, Daniel A.; Oliveira, Fátima de Cássia E.; Filho, Raimundo Araújo; Costa, Marcília P.; Oliveira, Diana M.

    2007-11-01

    For performing vital cellular processes, such as motility, eukaryotic cells rely on the actin cytoskeleton, whose structure and dynamics are tightly controlled by a large number of actin-interacting (AIP) or actin-related/regulating (ARP) proteins. Trypanosomatid protozoa, such as Leishmania, rely on their flagellum for motility and sensory reception, which are believed to allow parasite migration, adhesion, invasion and even persistence on mammalian host tissues to cause disease. Actin can determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes, while the identification and analyses of AIPs can help to improve understanding of their mechanical properties on physiological architectures, such as the present case regarding Leishmania flagellar apparatus. This work conveniently apply bioinformatics tools in some refined pattern recognition techniques (such as hidden Markov models (HMMs) through the Viterbi algorithm/path) in order to improve the recognition of actin-binding/interacting activity through identification of AIPs in genomes, transcriptomes and proteomes of Leishmania species. We here report cofilin and twinfilin as putative components of the flagellar apparatus, a direct bioinformatics contribution in the secondary annotation of Leishmania and trypanosomatid genomes.

  17. A family of ROP proteins that suppresses actin dynamics, and is essential for polarized growth and cell adhesion.

    PubMed

    Burkart, Graham M; Baskin, Tobias I; Bezanilla, Magdalena

    2015-07-15

    In plants, the ROP family of small GTPases has been implicated in the polarized growth of tip-growing cells, such as root hairs and pollen tubes; however, most of the data derive from overexpressing ROP genes or constitutively active and dominant-negative isoforms, whereas confirmation by using loss-of-function studies has generally been lacking. Here, in the model moss Physcomitrella patens, we study ROP signaling during tip growth by using a loss-of-function approach based on RNA interference (RNAi) to silence the entire moss ROP family. We find that plants with reduced expression of ROP genes, in addition to failing to initiate tip growth, have perturbed cell wall staining, reduced cell adhesion and have increased actin-filament dynamics. Although plants subjected to RNAi against the ROP family also have reduced microtubule dynamics, this reduction is not specific to loss of ROP genes, as it occurs when actin function is compromised chemically or genetically. Our data suggest that ROP proteins polarize the actin cytoskeleton by suppressing actin-filament dynamics, leading to an increase in actin filaments at the site of polarized secretion. © 2015. Published by The Company of Biologists Ltd.

  18. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  19. On the association of glycoprotein Ib and actin-binding protein in human platelets

    PubMed Central

    1985-01-01

    Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets. PMID:3155520

  20. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

    PubMed Central

    Gottwald, U; Brokamp, R; Karakesisoglou, I; Schleicher, M; Noegel, A A

    1996-01-01

    In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner. Images PMID

  1. A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin

    PubMed Central

    1984-01-01

    A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization. PMID:6540784

  2. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

    PubMed

    Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

    2016-04-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

  3. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

    PubMed Central

    Fujiwara, Takahiro K.; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A.; Watanabe, Yusuke; Umemura, Yasuhiro M.; Murakoshi, Hideji; Suzuki, Kenichi G. N.; Nemoto, Yuri L.; Morone, Nobuhiro; Kusumi, Akihiro

    2016-01-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed “hop diffusion”) for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

  4. NAB-1 instructs synapse assembly by linking adhesion molecules and F-actin to active zone proteins.

    PubMed

    Chia, Poh Hui; Patel, Maulik R; Shen, Kang

    2012-01-08

    During synaptogenesis, macromolecular protein complexes assemble at the pre- and postsynaptic membrane. Extensive literature identifies many transmembrane molecules sufficient to induce synapse formation and several intracellular scaffolding molecules responsible for assembling active zones and recruiting synaptic vesicles. However, little is known about the molecular mechanisms coupling membrane receptors to active zone molecules during development. Using Caenorhabditis elegans, we identify an F-actin network present at nascent presynaptic terminals and required for presynaptic assembly. We unravel a sequence of events whereby specificity-determining adhesion molecules define the location of developing synapses and locally assemble F-actin. Next, the adaptor protein NAB-1 (neurabin) binds to F-actin and recruits active zone proteins SYD-1 and SYD-2 (liprin-α) by forming a tripartite complex. NAB-1 localizes transiently to synapses during development and is required for presynaptic assembly. Altogether, we identify a role for the actin cytoskeleton during presynaptic development and characterize a molecular pathway whereby NAB-1 links synaptic partner recognition to active zone assembly.

  5. The ALP-Enigma protein ALP-1 functions in actin filament organization to promote muscle structural integrity in Caenorhabditis elegans.

    PubMed

    Han, Hsiao-Fen; Beckerle, Mary C

    2009-05-01

    Mutations that affect the Z-disk-associated ALP-Enigma proteins have been linked to human muscular and cardiac diseases. Despite their clear physiological significance for human health, the mechanism of action of ALP-Enigma proteins is largely unknown. In Caenorhabditis elegans, the ALP-Enigma protein family is encoded by a single gene, alp-1; thus C. elegans provides an excellent model to study ALP-Enigma function. Here we present a molecular and genetic analysis of ALP-Enigma function in C. elegans. We show that ALP-1 and alpha-actinin colocalize at dense bodies where actin filaments are anchored and that the proper localization of ALP-1 at dense bodies is dependent on alpha-actinin. Our analysis of alp-1 mutants demonstrates that ALP-1 functions to maintain actin filament organization and participates in muscle stabilization during contraction. Reducing alpha-actinin activity enhances the actin filament phenotype of the alp-1 mutants, suggesting that ALP-1 and alpha-actinin function in the same cellular process. Like alpha-actinin, alp-1 also interacts genetically with a connectin/titin family member, ketn-1, to provide mechanical stability for supporting body wall muscle contraction. Taken together, our data demonstrate that ALP-1 and alpha-actinin function together to stabilize actin filaments and promote muscle structural integrity.

  6. A 27,000-D core of the Dictyostelium 34,000-D protein retains Ca(2+)- regulated actin cross-linking but lacks bundling activity

    PubMed Central

    1993-01-01

    Actin cross-linking proteins are important for formation of isotropic F- actin networks and anisotropic bundles of filaments in the cytoplasm of eucaryotic cells. A 34,000-D protein from the cellular slime mold Dictyostelium discoideum mediates formation of actin bundles in vitro, and is specifically incorporated into filopodia. The actin cross- linking activity of this protein is inhibited by the presence of micromolar calcium. A 27,000-D fragment obtained by digestion with alpha-chymotrypsin lacks the amino-terminal six amino acids and the carboxyl-terminal 7,000 D of the intact polypeptide. The 27,000-D fragment retains F-actin binding activity assessed by cosedimentation assays and by 125I-[F-actin] blot overlay technique, F-actin cross- linking activity as assessed by viscometry, and calcium binding activity. Ultrastructural analyses indicate that the 27,000-D fragment is deficient in the bundling activity characteristic of the intact 34,000-D protein. Actin filaments are aggregated into microdomains but not bundle in the presence of the 27,000-D fragment. A polarized light scattering assay was used to demonstrate that the 34,000-D protein increases the orientational correlation among F-actin filaments. The 27,000-D fragment does not increase the orientation of the actin filaments as assessed by this technique. A terminal segment(s) of the 34,000-D protein, lacking in the 27,000-D fragment, contributes significantly to the ability to cross-link actin filaments into bundles. PMID:8436589

  7. Control of actin filament dynamics at barbed ends by WH2 domains: from capping to permissive and processive assembly.

    PubMed

    Carlier, Marie-France; Pernier, Julien; Avvaru, Balendu Sankara

    2013-10-01

    WH2 domains are multifunctional regulators of actin assembly that can either sequester G-actin or allow polarized barbed end growth. They all bind similarly to a hydrophobic pocket at the barbed face of actin. Depending on their electrostatic environment, WH2 domains can nucleate actin assembly by facilitating the formation of prenuclei dimers along the canonical spontaneous assembly pathway. They also modulate filament barbed end dynamics in a versatile fashion, acting either as barbed end cappers or assisting barbed end growth like profilin or uncapping barbed ends and potentially mediating processive elongation like formins when they are dimerized. Tandem repeats of WH2 domains can sever filaments and either remain bound to created barbed ends like gelsolin, or strip off an ADP-actin subunit from the severed polymer end, depending on their relative affinity for terminal ADP-F-actin or ADP-G-actin. In summary, WH2 domains recapitulate all known elementary regulatory functions so far found in individual actin-binding proteins. By combining different discrete sets of these multifunctional properties, they acquire specific functions in various actin-based processes, and participate in activities as diverse as filament branching, filopodia extension, or actin remodeling in ciliogenesis and asymmetric meiotic division. They also integrate these functions with other actin-binding motifs present either in the same protein or in a complex with another protein, expanding the range of complexity in actin regulation. The details of their molecular mechanisms and the underlying structural basis provide exciting avenues in actin research.

  8. The actin crosslinking protein palladin modulates force generation and mechanosensitivity of tumor associated fibroblasts

    PubMed Central

    Azatov, Mikheil; Goicoechea, Silvia M.; Otey, Carol A.; Upadhyaya, Arpita

    2016-01-01

    Cells organize actin filaments into higher-order structures by regulating the composition, distribution and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin network and stress fibers, which are critical for mechanosensing of the environment. Palladin also serves as a molecular scaffold for α-actinin, another key actin crosslinker. By virtue of its close interactions with actomyosin structures in the cell, palladin may play an important role in cell mechanics. However, the role of palladin in cellular force generation and mechanosensing has not been studied. Here, we investigate the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. Traction force microscopy revealed that tumor-associated fibroblasts generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells and inhibited their ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in actin organization, adhesion dynamics and altered myosin organization in palladin knock-down cells. Our results suggest that actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis. PMID:27353427

  9. Nuclear membrane protein emerin: roles in gene regulation, actin dynamics and human disease.

    PubMed

    Wilson, Katherine L; Holaska, James M; Montes de Oca, Rocio; Tifft, Kathryn; Zastrow, Michael; Segura-Totten, Miriam; Mansharamani, Malini; Bengtsson, Luiza

    2005-01-01

    Loss of emerin, a nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy (EDMD), characterized by muscle weakening, contractures of major tendons and potentially lethal cardiac conduction system defects. Emerin has a LEM-domain and therefore binds barrier-to-autointegration factor (BAF), a conserved chromatin protein essential for cell division. BAF recruits emerin to chromatin and regulates higher-order chromatin structure during nuclear assembly. Emerin also binds filaments formed by A-type lamins, mutations in which also cause EDMD. Other partners for emerin include nesprin-1alpha and transcriptional regulators such as germ cell-less (GCL). The binding affinities of these partners range from 4nM (nesprin-1alpha) to 200 nM (BAF), and are physiologically significant. Biochemical studies therefore provide a valid means to predict the properties of emerin-lamin complexes in vivo. Emerin and lamin A together form stable complexes with either BAF or GCL in vitro. BAF, however, competes with GCL for binding to emerin in vitro. These and additional partners, notably actin and nuclear myosin II, suggest disease-relevant roles for emerin in gene regulation and the mechanical interity of the nucleus.

  10. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.

    PubMed

    Ren, L; Hong, S H; Cassavaugh, J; Osborne, T; Chou, A J; Kim, S Y; Gorlick, R; Hewitt, S M; Khanna, C

    2009-02-12

    Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCalpha, PKCiota and PKCgamma. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

  11. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

    The intracellular protein actin, by reversibly polymerizing into filaments, generates forces for motion and shape changes of many types of biological cells. Fluorescence imaging studies show that actin often occurs in the form of localized patches of size roughly one micrometer at the cell membrane. Patch formation is most prevalent when the free-actin concentration is low. I investigate possible mechanisms for the formation of actin patches by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations include filament growth, capping, branching, severing, and debranching. The attachment of membrane-bound activators to actin filaments, and subsequent membrane diffusion of unattached activators, are also included. It is found that as the actin concentration increases from zero, the actin occurs in patches at lower actin concentrations, and the size of the patches increases with increasing actin concentration. At a critical value of the actin concentration, the system undergoes a transition to complete coverage. The results are interpreted within the framework of reaction-diffusion equations in two dimensions.

  12. Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin-Dendra2 fusion proteins.

    PubMed

    Dovas, Athanassios; Gligorijevic, Bojana; Chen, Xiaoming; Entenberg, David; Condeelis, John; Cox, Dianne

    2011-02-14

    Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin-Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.

  13. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

    PubMed

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  14. The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.

    PubMed

    Zhang, Bing; Yu, Qilin; Jia, Chang; Wang, Yuzhou; Xiao, Chenpeng; Dong, Yijie; Xu, Ning; Wang, Lei; Li, Mingchun

    2015-08-01

    Candida albicans is a common pathogenic fungus and has aroused widespread attention recently. Actin cytoskeleton, an important player in polarized growth, protein secretion and organization of cell shape, displays irreplaceable role in hyphal development and cell integrity. In this study, we demonstrated a homologue of Saccharomyces cerevisiae Sac1, in C. albicans. It is a potential PIP phosphatase with Sac domain which is related to actin organization, hyphal development, biofilm formation and cell wall integrity. Deletion of SAC1 did not lead to insitiol-auxotroph phenotype in C. albicans, but this gene rescued the growth defect of S. cerevisiae sac1Δ in the insitiol-free medium. Hyphal induction further revealed the deficiency of sac1Δ/Δ in hyphal development and biofilm formation. Fluorescence observation and real time PCR (RT-PCR) analysis suggested both actin and the hyphal cell wall protein Hwp1 were overexpressed and mislocated in this mutant. Furthermore, cell wall integrity (CWI) was largely affected by deletion of SAC1, due to the hypersensitivity to cell wall stress, changed content and distribution of chitin in the mutant. As a result, the virulence of sac1Δ/Δ was seriously attenuated. Taken together, this study provides evidence that Sac1, as a potential PIP phosphatase, is essential for actin organization, hyphal development, CWI and pathogenicity in C. albicans.

  15. Vital role for the Plasmodium actin capping protein (CP) beta-subunit in motility of malaria sporozoites

    PubMed Central

    Ganter, Markus; Schüler, Herwig; Matuschewski, Kai

    2009-01-01

    Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F-actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein (PbCP), an orthologue of the heterodimeric regulator of filament barbed end growth, by reverse genetics. Parasites containing a deletion of the CP beta-subunit developed normally during the pathogenic erythrocytic cycle. However, due to reduced ookinete motility, mutant parasites form fewer oocysts and sporozoites in the Anopheles vector. These sporozoites display a vital deficiency in forward gliding motility and fail to colonize the mosquito salivary glands, resulting in complete attenuation of life cycle progression. Together, our results show that the CP beta-subunit exerts an essential role in the insect vector before malaria transmission to the mammalian host. The vital role is restricted to fast locomotion, as displayed by Plasmodium sporozoites. PMID:19682250

  16. Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear.

    PubMed

    Li, Huawei; Liu, Hong; Balt, Steve; Mann, Sabine; Corrales, C Eduardo; Heller, Stefan

    2004-01-01

    The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.

  17. Identification and Characterization of a Ca2+-Dependent Actin Filament-Severing Protein from Lily Pollen1

    PubMed Central

    Fan, Xiaoxue; Hou, Jian; Chen, Xiaoliang; Chaudhry, Faisal; Staiger, Christopher J.; Ren, Haiyun

    2004-01-01

    It is well known that a tip-focused intracellular Ca2+ gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca2+-dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with >98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca2+-sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca2+ gradient. PMID:15557101

  18. Diversification of caldesmon-linked actin cytoskeleton in cell motility

    PubMed Central

    Mayanagi, Taira

    2011-01-01

    The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions. PMID:21350330

  19. Structures of actin-bound Wiskott-Aldrich syndrome protein homology 2 (WH2) domains of Spire and the implication for filament nucleation

    PubMed Central

    Ducka, Anna M.; Joel, Peteranne; Popowicz, Grzegorz M.; Trybus, Kathleen M.; Schleicher, Michael; Noegel, Angelika A.; Huber, Robert; Holak, Tad A.; Sitar, Tomasz

    2010-01-01

    Three classes of proteins are known to nucleate new filaments: the Arp2/3 complex, formins, and the third group of proteins that contain ca. 25 amino acid long actin-binding Wiskott-Aldrich syndrome protein homology 2 domains, called the WH2 repeats. Crystal structures of the complexes between the actin-binding WH2 repeats of the Spire protein and actin were determined for the Spire single WH2 domain D, the double (SpirCD), triple (SpirBCD), quadruple (SpirABCD) domains, and an artificial Spire WH2 construct comprising three identical D repeats (SpirDDD). SpirCD represents the minimal functional core of Spire that can nucleate actin filaments. Packing in the crystals of the actin complexes with SpirCD, SpirBCD, SpirABCD, and SpirDDD shows the presence of two types of assemblies, “side-to-side” and “straight-longitudinal,” which can serve as actin filament nuclei. The principal feature of these structures is their loose, open conformations, in which the sides of actins that normally constitute the inner interface core of a filament are flipped inside out. These Spire structures are distant from those seen in the filamentous nuclei of Arp2/3, formins, and in the F-actin filament. PMID:20538977

  20. Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo.

    PubMed Central

    Prendergast, G C; Ziff, E B

    1991-01-01

    We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin. Images PMID:1849072

  1. Correlation between cell cycle proteins and hMSH2 in actinic cheilitis and lip cancer.

    PubMed

    Lopes, Maria Luiza Diniz de Sousa; de Oliveira, Denise Hélen Imaculada Pereira; Sarmento, Dmitry José de Santana; Queiroz, Lélia Maria Guedes; Miguel, Márcia Cristina da Costa; da Silveira, Éricka Janine Dantas

    2016-04-01

    This study aims to evaluate and verify the relationship between the immunoexpression of hMSH2, p53 and p21 in actinic cheilitis (AC) and lower lip squamous cell carcinoma (SCC) cases. Forty AC and 40 SCC cases were submitted to immunoperoxidase method and quantitatively analyzed. Expression was compared by Mann-Whitney test, Student t test or one-way ANOVA. To correlate the variables, Pearson's correlation coefficient was calculated. The expression of p53 and p21 showed no significant differences between histopathological grades of AC or lower lip SCC (p > 0.05). Immunoexpression of p53 was higher in SCC than in AC (p < 0.001), while p21 expression was more observed in AC when compared to SCC group (p = 0.006). The AC group revealed an inverse correlation between p53 and hMSH2 expression (r = -0.30, p = 0.006). Alterations in p53 and p21 expression suggest that these proteins are involved in lower lip carcinogenesis. Moreover, p53 and hMSH2 seem to be interrelated in early events of this process.

  2. Expression of drebrin, an actin binding protein, in basal cell carcinoma, trichoblastoma and trichoepithelioma.

    PubMed

    Mizutani, Yoko; Iwamoto, Ikuko; Kanoh, Hiroyuki; Seishima, Mariko; Nagata, Koh-ichi

    2014-06-01

    Drebrin, an F-actin binding protein, is known to play important roles in cell migration, synaptogenesis and neural plasticity. Although drebrin was long thought to be specific for neuronal cells, its expression has recently been reported in non-neuronal cells. As for skin-derived cells, drebrin was shown to be enriched at adhering junctions (AJs) in cultured primary keratinocytes and also be highly expressed in basal cell carcinoma (BCC) cells. Since BCC and two types of benign neoplasm, trichoblastoma and trichoepithelioma, are considered to derive from the same origin, follicular germinative cells, it is sometimes difficult to morphologically distinguish BCC from trichoblastoma and trichoepithelioma. In this study, we performed immunohistochemical staining of drebrin in BCC, trichoblastoma and trichoepithelioma, to examine whether drebrin could serve as a biomarker for BCC diagnosis. In western blotting, drebrin was detected highly and moderately in the lysates from a squamous cell carcinoma cell line, DJM-1, and normal human epidermis, respectively. In immunofluorescence analyses, drebrin was colocalized with markers of AJs and tight junctions in DJM-1 cells and detected at cell-cell junction areas of human normal epidermis tissue. We then examined the distribution patterns of drebrin in BCC, trichoblastoma and trichoepithelioma. In BCC tissues, intense and homogeneous drebrin expression was observed mainly at tumor cell-cell boundaries. In contrast, drebrin was stained only weakly and non-homogeneously in trichoblastoma and trichoepthelioma tissue samples. For differential diagnosis of BCC, drebrin may be a novel and useful marker.

  3. The Role of CKIP-1 in Cell Morphology Depends on Its Interaction with Actin-capping Protein*

    PubMed Central

    Canton, David A.; Olsten, Mary Ellen K.; Niederstrasser, Hanspeter; Cooper, John A.; Litchfield, David W.

    2008-01-01

    CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide “walking arrays” and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects. PMID:16987810

  4. Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo.

    PubMed

    Chen, Chih-Ying; Brodsky, Frances M

    2005-02-18

    Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of clathrin light chains.

  5. How VASP enhances actin-based motility

    PubMed Central

    Samarin, Stanislav; Romero, Stéphane; Kocks, Christine; Didry, Dominique; Pantaloni, Dominique; Carlier, Marie-France

    2003-01-01

    The function of vasodilator-stimulated phosphoprotein (VASP) in motility is analyzed using a biomimetic motility assay in which ActA-coated microspheres propel themselves in a medium containing actin, the Arp2/3 complex, and three regulatory proteins in the absence or presence of VASP. Propulsion is linked to cycles of filament barbed end attachment-branching-detachment-growth in which the ActA-activated Arp2/3 complex incorporates at the junctions of branched filaments. VASP increases the velocity of beads. VASP increases branch spacing of filaments in the actin tail, as it does in lamellipodia in living cells. The effect of VASP on branch spacing of Arp2/3-induced branched actin arrays is opposed to the effect of capping proteins. However, VASP does not compete with capping proteins for binding barbed ends of actin filaments. VASP enhances branched actin polymerization only when ActA is immobilized on beads or on Listeria. VASP increases the rate of dissociation of the branch junction from immobilized ActA, which is the rate-limiting step in the catalytic cycle of site-directed filament branching. PMID:14557252

  6. Axonal actin in action: Imaging actin dynamics in neurons.

    PubMed

    Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit

    2016-01-01

    Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

  7. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    SciTech Connect

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  8. Functional Classification of Immune Regulatory Proteins

    SciTech Connect

    Rubinstein, Rotem; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.; Fiser, Andras

    2013-05-01

    Members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. We were guided by the Brotherhood approach and present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

  9. Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane

    PubMed Central

    Dason, Jeffrey S; Smith, Alex J; Marin, Leo; Charlton, Milton P

    2014-01-01

    AbstractSynaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins. PMID:24297851

  10. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    PubMed

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  11. Single-molecule studies of actin assembly and disassembly factors.

    PubMed

    Smith, Benjamin A; Gelles, Jeff; Goode, Bruce L

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.

  12. Histones bundle F-actin filaments and affect actin structure.

    PubMed

    Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

    2017-01-01

    Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  13. Sumoylation: a regulatory protein modification in health and disease.

    PubMed

    Flotho, Annette; Melchior, Frauke

    2013-01-01

    Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

  14. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

    EPA Science Inventory

    IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

  15. Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

    PubMed Central

    Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

    2016-01-01

    The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

  16. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

    EPA Science Inventory

    IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

  17. NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.

    PubMed

    Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi

    2016-02-01

    The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.

  18. The actin-bundling protein L-plastin: a novel local inflammatory marker associated with periodontitis.

    PubMed

    Öztürk, V Ö; Emingil, G; Osterwalder, V; Bostanci, N

    2015-06-01

    L-plastin, an actin-bundling protein, is exclusively expressed in leukocytes and plays a crucial role in immune-mediated events. Periodontitis is a common infectious inflammatory disease that destroys the tooth-supporting tissues. Recent findings using proteomic technologies have demonstrated that L-plastin is one of the few molecules consistently present in the inflammatory exudate of the gingiva in periodontal disease, but not in health. Therefore, this study aimed to investigate in detail the local and systemic role of this molecule in different forms of periodontitis. A total of 61 subjects who met the inclusion/exclusion criteria were recruited, including 21 with chronic periodontitis, 20 generalized aggressive periodontitis and 20 nonperiodontitis control subjects. Gingival tissue biopsies, gingival crevicular fluid, as well as serum and saliva, were obtained. Immunohistochemistry and quantitative real-time PCR were employed to evaluate the localization and mRNA expression, respectively, of L-plastin. L-plastin levels in gingival crevicular fluid, saliva and serum were measured using ELISA. Statistical analysis was performed using nonparametric methods. Subjects with chronic periodontitis and generalized aggressive periodontitis exhibited significantly higher tissue L-plastin gene expression and gingival crevicular fluid levels than did subjects in the control group but there was no significant difference between the two forms of periodontitis. Within gingival tissue, L-plastin was confined to the inflammatory infiltrate. There was no statistically significant difference between serum and salivary L-plastin levels among the three study groups. The elevated gingival tissue expression and gingival crevicular fluid levels of L-plastin in both forms of periodontitis may denote the localized involvement of this novel molecule in the pathogenesis of the disease. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Microarray phenotyping places cyclase associated protein CAP at the crossroad of signaling pathways reorganizing the actin cytoskeleton in Dictyostelium.

    PubMed

    Sultana, Hameeda; Neelakanta, Girish; Eichinger, Ludwig; Rivero, Francisco; Noegel, Angelika A

    2009-01-15

    Large-scale gene expression analysis has been applied recently to uncover groups of genes that are co-regulated in particular processes. Here we undertake such an analysis on CAP, a protein that participates in the regulation of the actin cytoskeleton and in cAMP signaling in Dictyostelium. microarray analysis revealed that loss of CAP altered the expression of many cytoskeletal components. One of these, the Rho GDP-dissociation inhibitor RhoGDI1, was analyzed further. RhoGDI1 null cells expressed lower amounts of CAP, which failed to accumulate predominantly at the cell cortex. To further position CAP in the corresponding signal transduction pathways we studied CAP localization and cellular functioning in mutants that have defects in several signaling components. CAP showed correct localization and dynamics in all analyzed strains except in mutants with deficient cAMP dependent protein kinase A activity, where CAP preferentially accumulated in crown shaped structures. Ectopic expression of CAP improved the efficiency of phagocytosis in Gbeta-deficient cells and restored the pinocytosis, morphology and actin distribution defects in a PI3 kinase double mutant (pi3k1/2 null). Our results show that CAP acts at multiple crossroads and links signaling pathways to the actin cytoskeleton either by physical interaction with cytoskeletal components or through regulation of their gene expression.

  20. End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae.

    PubMed

    Wesp, A; Hicke, L; Palecek, J; Lombardi, R; Aust, T; Munn, A L; Riezman, H

    1997-11-01

    end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

  1. End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

    PubMed Central

    Wesp, A.; Hicke, L.; Palecek, J.; Lombardi, R.; Aust, T.; Munn, A.L.; Riezman, H.

    1997-01-01

    end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis. PMID:9362070

  2. Investigation of hippocampal synaptic transmission and plasticity in mice deficient in the actin-binding protein Drebrin

    PubMed Central

    Willmes, Claudia G.; Mack, Till G. A.; Ledderose, Julia; Schmitz, Dietmar; Wozny, Christian; Eickholt, Britta J.

    2017-01-01

    The dynamic regulation of the actin cytoskeleton plays a key role in controlling the structure and function of synapses. It is vital for activity-dependent modulation of synaptic transmission and long-term changes in synaptic morphology associated with memory consolidation. Several regulators of actin dynamics at the synapse have been identified, of which a salient one is the postsynaptic actin stabilising protein Drebrin (DBN). It has been suggested that DBN modulates neurotransmission and changes in dendritic spine morphology associated with synaptic plasticity. Given that a decrease in DBN levels is correlated with cognitive deficits associated with ageing and dementia, it was hypothesised that DBN protein abundance instructs the integrity and function of synapses. We created a novel DBN deficient mouse line. Analysis of gross brain and neuronal morphology revealed no phenotype in the absence of DBN. Electrophysiological recordings in acute hippocampal slices and primary hippocampal neuronal cultures showed that basal synaptic transmission, and both long-term and homeostatic synaptic plasticity were unchanged, suggesting that loss of DBN is not sufficient in inducing synapse dysfunction. We propose that the overall lack of changes in synaptic function and plasticity in DBN deficient mice may indicate robust compensatory mechanisms that safeguard cytoskeleton dynamics at the synapse. PMID:28198431

  3. Dynamics of the actin-binding protein drebrin in motile cells and definition of a juxtanuclear drebrin-enriched zone.