scholarly journals Ecm29-Dependent Proteasome Localization Regulates Cytoskeleton Remodeling at the Immune Synapse

2021 ◽  
Vol 9 ◽  
Author(s):  
Jorge Ibañez-Vega ◽  
Felipe Del Valle ◽  
Juan José Sáez ◽  
Fanny Guzman ◽  
Jheimmy Diaz ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Spreading ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Asymmetric Distribution ◽  
Retrograde Flow ◽  
Cortical Actin ◽  
Immune Synapse ◽  
Cytoskeleton Remodeling ◽  
And Shifts

The formation of an immune synapse (IS) enables B cells to capture membrane-tethered antigens, where cortical actin cytoskeleton remodeling regulates cell spreading and depletion of F-actin at the centrosome promotes the recruitment of lysosomes to facilitate antigen extraction. How B cells regulate both pools of actin, remains poorly understood. We report here that decreased F-actin at the centrosome and IS relies on the distribution of the proteasome, regulated by Ecm29. Silencing Ecm29 decreases the proteasome pool associated to the centrosome of B cells and shifts its accumulation to the cell cortex and IS. Accordingly, Ecm29-silenced B cells display increased F-actin at the centrosome, impaired centrosome and lysosome repositioning to the IS and defective antigen extraction and presentation. Ecm29-silenced B cells, which accumulate higher levels of proteasome at the cell cortex, display decreased actin retrograde flow in lamellipodia and enhanced spreading responses. Our findings support a model where B the asymmetric distribution of the proteasome, mediated by Ecm29, coordinates actin dynamics at the centrosome and the IS, promoting lysosome recruitment and cell spreading.

scholarly journals Ecm29-dependent proteasome localization regulates cytoskeleton remodeling at the immune synapse

2020 ◽  
Author(s):  
Jorge Ibañez-Vega ◽  
Felipe Del Valle ◽  
Juan José Saez ◽  
Jheimmy Diaz ◽  
Andrea Soza ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Spreading ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Asymmetric Distribution ◽  
Retrograde Flow ◽  
Cortical Actin ◽  
Immune Synapse ◽  
Cytoskeleton Remodeling ◽  
And Shifts

AbstractThe formation of an immune synapse (IS) enables B cells to capture membrane-tethered antigens, where cortical actin cytoskeleton remodeling regulates cell spreading and depletion of F-actin at the centrosome promotes the recruitment of lysosomes to facilitate antigen extraction. How B cells regulate both pools of actin, remains poorly understood. We report here that decreased F-actin at the centrosome and IS relies on the distribution of the proteasome, regulated by Ecm29. Silencing Ecm29 decreases the proteasome pool associated to the centrosome of B cells and shifts its accumulation to the cell cortex and IS. Accordingly, Ecm29-silenced B cells display increased F-actin at the centrosome, impaired centrosome and lysosome repositioning to the IS and defective antigen extraction and presentation. Ecm29-silenced B cells, which accumulate higher levels of proteasome at the cell cortex, display decreased actin retrograde flow in lamellipodia and enhanced spreading responses. Our findings support a model where B the asymmetric distribution of the proteasome, mediated by Ecm29, coordinates actin dynamics at the centrosome and the IS, promoting lysosome recruitment and cell spreading.

Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics

2002 ◽  
Vol 115 (8) ◽  
pp. 1703-1715 ◽  
Author(s):  
Derek T. Warren ◽  
Paul D. Andrews ◽  
Campbell W. Gourlay ◽  
Kathryn R. Ayscough
Keyword(s):  
Immunofluorescence Microscopy ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Binding Studies ◽  
Endocytic Machinery ◽  
Single Complex ◽  
Actin Patch ◽  
First Time

Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p(YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.

scholarly journals Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

2010 ◽  
Vol 21 (20) ◽  
pp. 3529-3539 ◽  
Author(s):  
Tim Ting Chiu ◽  
Nish Patel ◽  
Alisa E. Shaw ◽  
James R. Bamburg ◽  
Amira Klip
Keyword(s):  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Muscle Cells ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Glut4 Translocation ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Rac Gtpase ◽  
Skeletal Myoblasts

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

scholarly journals The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse

2021 ◽  
Vol 9 ◽  
Author(s):  
Madison Bolger-Munro ◽  
Kate Choi ◽  
Faith Cheung ◽  
Yi Tian Liu ◽  
May Dang-Lawson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Cell Spreading ◽  
B Cell Antigen Receptor ◽  
Actin Remodeling ◽  
Immune Synapse ◽  
Cell Antigen ◽  
Bcr Signaling

When B cells encounter membrane-bound antigens, the formation and coalescence of B cell antigen receptor (BCR) microclusters amplifies BCR signaling. The ability of B cells to probe the surface of antigen-presenting cells (APCs) and respond to APC-bound antigens requires remodeling of the actin cytoskeleton. Initial BCR signaling stimulates actin-related protein (Arp) 2/3 complex-dependent actin polymerization, which drives B cell spreading as well as the centripetal movement and coalescence of BCR microclusters at the B cell-APC synapse. Sustained actin polymerization depends on concomitant actin filament depolymerization, which enables the recycling of actin monomers and Arp2/3 complexes. Cofilin-mediated severing of actin filaments is a rate-limiting step in the morphological changes that occur during immune synapse formation. Hence, regulators of cofilin activity such as WD repeat-containing protein 1 (Wdr1), LIM domain kinase (LIMK), and coactosin-like 1 (Cotl1) may also be essential for actin-dependent processes in B cells. Wdr1 enhances cofilin-mediated actin disassembly. Conversely, Cotl1 competes with cofilin for binding to actin and LIMK phosphorylates cofilin and prevents it from binding to actin filaments. We now show that Wdr1 and LIMK have distinct roles in BCR-induced assembly of the peripheral actin structures that drive B cell spreading, and that cofilin, Wdr1, and LIMK all contribute to the actin-dependent amplification of BCR signaling at the immune synapse. Depleting Cotl1 had no effect on these processes. Thus, the Wdr1-LIMK-cofilin axis is critical for BCR-induced actin remodeling and for B cell responses to APC-bound antigens.

scholarly journals Calcium-mediated actin reset (CaAR) mediates acute cell adaptations

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Pauline Wales ◽  
Christian E Schuberth ◽  
Roland Aufschnaiter ◽  
Johannes Fels ◽  
Ireth García-Aguilar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dynamic Equilibrium ◽  
Calcium Influx ◽  
Cell Spreading ◽  
Cell Types ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Cellular Morphogenesis ◽  
Wide Range ◽  
A Cell

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.

scholarly journals Vamp-7–dependent secretion at the immune synapse regulates antigen extraction and presentation in B-lymphocytes

2017 ◽  
Vol 28 (7) ◽  
pp. 890-897 ◽  
Author(s):  
Dorian Obino ◽  
Jheimmy Diaz ◽  
Juan José Sáez ◽  
Jorge Ibañez-Vega ◽  
Pablo J. Sáez ◽  
...  
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Snare Protein ◽  
Contact Site ◽  
Microtubule Network ◽  
Immune Synapse ◽  
Mhc Ii ◽  
Cytoskeleton Remodeling ◽  
Cellular Machinery ◽  
Ag Contact

Recognition of surface-tethered antigens (Ags) by B-cells leads to the formation of an immune synapse that promotes Ag uptake for presentation onto MHC-II molecules. Extraction of immobilized Ags at the immune synapse of B-cells relies on the local secretion of lysosomes, which are recruited to the Ag contact site by polarization of their microtubule network. Although conserved polarity proteins have been implicated in coordinating cytoskeleton remodeling with lysosome trafficking, the cellular machinery associated with lysosomal vesicles that regulates their docking and secretion at the synaptic interface has not been defined. Here we show that the v-SNARE protein Vamp-7 is associated with Lamp-1+ lysosomal vesicles, which are recruited and docked at the center of the immune synapse of B-cells. A decrease in Vamp-7 expression does not alter lysosome transport to the synaptic interface but impairs their local secretion, a defect that compromises the ability of B-cells to extract, process, and present immobilized Ag. Thus our results reveal that B-cells rely on the SNARE protein Vamp-7 to promote the local exocytosis of lysosomes at the immune synapse, which is required for efficient Ag extraction and presentation.

scholarly journals Novel Proteins Linking the Actin Cytoskeleton to the Endocytic Machinery in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (10) ◽  
pp. 3646-3661 ◽  
Author(s):  
H. Dewar ◽  
D. T. Warren ◽  
F. C. Gardiner ◽  
C. G. Gourlay ◽  
N. Satish ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Elevated Temperatures ◽  
Adaptor Protein ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

scholarly journals The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

2021 ◽  
Vol 9 ◽  
Author(s):  
Nikola Deretic ◽  
Madison Bolger-Munro ◽  
Kate Choi ◽  
Libin Abraham ◽  
Michael R. Gold
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Cell Antigen Receptor ◽  
Retrograde Flow ◽  
Glia Maturation Factor ◽  
Actin Remodeling ◽  
Immune Synapse ◽  
Actin Networks ◽  
Maturation Factor ◽  
Bcr Signaling

Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that reducing the levels of GMFγ in human B cell lines via transfection with a specific siRNA impairs the ability of B cells to spread on antigen-coated surfaces, decreases the velocity of actin retrograde flow, diminishes the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and decreases APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.

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