scholarly journals Type I myosins anchor actin assembly to the plasma membrane during clathrin-mediated endocytosis

2019 ◽  
Vol 218 (4) ◽  
pp. 1138-1147 ◽  
Author(s):  
Ross T.A. Pedersen ◽  
David G. Drubin
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Force Generation ◽  
Type I ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Actin Networks ◽  
Wide Range ◽  
Assembly Factors ◽  
Myosin Motors

The actin cytoskeleton generates forces on membranes for a wide range of cellular and subcellular morphogenic events, from cell migration to cytokinesis and membrane trafficking. For each of these processes, filamentous actin (F-actin) interacts with membranes and exerts force through its assembly, its associated myosin motors, or both. These two modes of force generation are well studied in isolation, but how they are coordinated in cells is mysterious. During clathrin-mediated endocytosis, F-actin assembly initiated by the Arp2/3 complex and several proteins that compose the WASP/myosin complex generates the force necessary to deform the plasma membrane into a pit. Here we present evidence that type I myosin is the key membrane anchor for endocytic actin assembly factors in budding yeast. By mooring actin assembly factors to the plasma membrane, this myosin organizes endocytic actin networks and couples actin-generated forces to the plasma membrane to drive invagination and scission. Through this unexpected mechanism, myosin facilitates force generation independent of its motor activity.

scholarly journals Actin Assembly Factors Regulate the Gelation Kinetics and Architecture of F-actin Networks

2013 ◽  
Vol 104 (8) ◽  
pp. 1709-1719 ◽  
Author(s):  
Tobias T. Falzone ◽  
Patrick W. Oakes ◽  
Jennifer Sees ◽  
David R. Kovar ◽  
Margaret L. Gardel
Keyword(s):  
Actin Assembly ◽  
Actin Networks ◽  
Gelation Kinetics ◽  
Assembly Factors

scholarly journals ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

2003 ◽  
Vol 162 (4) ◽  
pp. 647-659 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Membrane Trafficking ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Dense Core Vesicle ◽  
Type I ◽  
Endosomal Membranes ◽  
Phosphatidylinositol 4 ◽  
Adp Ribosylation Factor

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.

scholarly journals Initial ciliary assembly in Chlamydomonas requires Arp2/3-dependent recruitment from a ciliary protein reservoir in the plasma membrane

2020 ◽  
Author(s):  
Brae M Bigge ◽  
Nicholas E Rosenthal ◽  
David Sept ◽  
Courtney M Schroeder ◽  
Prachee Avasthi
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Long Range ◽  
Protein Targeting ◽  
Basal Bodies ◽  
Cell Periphery ◽  
Filamentous Actin ◽  
Actin Networks ◽  
Ciliary Protein ◽  
Broad Approach

ABSTRACTCilia are organelles important for signaling and motility. They are composed of microtubules ensheathed in plasma membrane. The mechanisms related to ciliogenesis also require another cytoskeletal element, actin, which has been shown to be important for organizing the basal bodies and transition zone at the base of cilia and for short- and long-range trafficking. However, most studies of actin’s role in ciliogenesis have taken a broad approach by knocking out all filamentous actin until now. Here, we more delicately dissect the interplay between actin and cilia by specifically focusing on actin networks nucleated by the Arp2/3 complex in Chlamydomonas. We find that knocking out Arp2/3-mediated actin networks dramatically impairs ciliary assembly and maintenance in these cells, and these defects are due to a problem with incorporation and gating of existing ciliary proteins, particularly in the early stages of assembly. We also show that cells lacking the Arp2/3 complex have more dramatic defects in ciliary maintenance using material from non-Golgi sources. Finally, we find relocalization of a ciliary membrane protein from the cell periphery to the cilia by internalization is dependent on actin and the Arp2/3 complex. Based on these results, we propose a new model of ciliary protein targeting during early ciliogenesis in which proteins previously targeted from the Golgi to the plasma membrane are reclaimed from this reservoir by Arp2/3-mediated networks.

scholarly journals Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins

2019 ◽  
Vol 116 (9) ◽  
pp. 3594-3603 ◽  
Author(s):  
Christof Litschko ◽  
Stefan Brühmann ◽  
Agnes Csiszár ◽  
Till Stephan ◽  
Vanessa Dimchev ◽  
...  
Keyword(s):  
Cell Polarization ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Multicellular Development ◽  
Actin Cortex ◽  
Image Velocimetry ◽  
Genes Encoding ◽  
And Migration ◽  
Myosin Motors

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA−/E−/H− and racE− mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.

scholarly journals Cofilin recruitment and function during actin-mediated endocytosis dictated by actin nucleotide state

2007 ◽  
Vol 178 (7) ◽  
pp. 1251-1264 ◽  
Author(s):  
Voytek Okreglak ◽  
David G. Drubin
Keyword(s):  
Actin Filament ◽  
Membrane Trafficking ◽  
Adenosine Diphosphate ◽  
Living Cells ◽  
Actin Assembly ◽  
Actin Networks ◽  
Actin Turnover ◽  
And Function

Cofilin is the major mediator of actin filament turnover in vivo. However, the molecular mechanism of cofilin recruitment to actin networks during dynamic actin-mediated processes in living cells and cofilin's precise in vivo functions have not been determined. In this study, we analyzed the dynamics of fluorescently tagged cofilin and the role of cofilin-mediated actin turnover during endocytosis in Saccharomyces cerevisiae. In living cells, cofilin is not necessary for actin assembly on endocytic membranes but is recruited to molecularly aged adenosine diphosphate actin filaments and is necessary for their rapid disassembly. Defects in cofilin function alter the morphology of actin networks in vivo and reduce the rate of actin flux through actin networks. The consequences of decreasing actin flux are manifested by decreased but not blocked endocytic internalization at the plasma membrane and defects in late steps of membrane trafficking to the vacuole. These results suggest that cofilin-mediated actin filament flux is required for the multiple steps of endocytic trafficking.

scholarly journals Chlamydomonas reinhardtii formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly

2016 ◽  
Author(s):  
Jenna R. Christensen ◽  
Evan W. Craig ◽  
Michael J. Glista ◽  
David M. Mueller ◽  
Yujie Li ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Apical Cell ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Tubule Formation ◽  
Actin Networks ◽  
Cellular Processes ◽  
The Right

ABSTRACTThe regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. PRF1 further favors FOR1-mediated actin assembly by potently inhibiting Arp2/3 complex-mediated actin assembly. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.SUMMARY STATEMENTThe Chlamydomonas reinhardtii formin FOR1 initiates rapid assembly of fertilization tubule actin filaments from monomers associated with the actin-assembly inhibitor profilin PRF1.

scholarly journals Dictyostelium discoideumflotillin homologues are essential for phagocytosis and participate in plasma membrane recycling and lysosome biogenesis

2019 ◽  
Author(s):  
Cristina Bosmani ◽  
Frauke Bach ◽  
Florence Leuba ◽  
Nabil Hanna ◽  
Frédéric Burdet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Membrane Trafficking ◽  
Membrane Recycling ◽  
Lysosome Biogenesis ◽  
Wide Range ◽  
Particle Recognition ◽  
Associated Proteins ◽  
Soil Amoeba ◽  
Early Recruitment

ABSTRACTThe metazoan flotillins are lipid rafts residents involved in membrane trafficking and recycling of plasma membrane proteins.Dictyostelium discoideum, a social soil amoeba, uses phagocytosis to digest, kill and feed on bacteria.D. discoideumpossesses three flotillin-like proteins, termed VacA, VacB and the recently identified VacC. All three vacuolins gradually accumulate on postlysosomes and, like flotillins, are strongly associated with membranes and partly with lipid rafts. Vacuolins are absolutely required for uptake of various particles. Their absence impairs particle recognition possibly because of defective recycling of plasma membrane or cortex-associated proteins. In addition, vacuolins are involved in phagolysosome biogenesis, although this does not impact digestion and killing of a wide range of bacteria. Furthermore, vacuolin knockout affects early recruitment of the WASH complex on phagosomes, suggesting that vacuolins may be involved in the WASH-dependent plasma membrane recycling. Altogether, these results indicate that vacuolins act as the functional homologues of flotillins inD. discoideum.

scholarly journals Rho-GTPase–dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth

2008 ◽  
Vol 181 (7) ◽  
pp. 1155-1168 ◽  
Author(s):  
Yong Jik Lee ◽  
Amy Szumlanski ◽  
Erik Nielsen ◽  
Zhenbiao Yang
Keyword(s):  
Plasma Membrane ◽  
Tip Growth ◽  
Rho Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Latrunculin B ◽  
Dynamic Activity ◽  
Guanosine Triphosphatase ◽  
Spatiotemporal Coordination

The dynamic activity of tip-localized filamentous actin (F-actin) in pollen tubes is controlled by counteracting RIC4 and RIC3 pathways downstream of the ROP1 guanosine triphosphatase promoting actin assembly and disassembly, respectively. We show here that ROP1 activation is required for both the polar accumulation and the exocytosis of vesicles at the plasma membrane apex. The apical accumulation of exocytic vesicles oscillated in phase with, but slightly behind, apical actin assembly and was enhanced by overexpression of RIC4. However, RIC4 overexpression inhibited exocytosis, and this inhibition could be suppressed by latrunculin B treatment or RIC3 overexpression. We conclude that RIC4-dependent actin assembly is required for polar vesicle accumulation, whereas RIC3-mediated actin disassembly is required for exocytosis. Thus ROP1-dependent F-actin dynamics control tip growth through spatiotemporal coordination of vesicle targeting and exocytosis.

scholarly journals Adaptive actin organization counteracts elevated membrane tension to ensure robust endocytosis

2020 ◽  
Author(s):  
Charlotte Kaplan ◽  
Sam J. Kenny ◽  
Shirley Chen ◽  
Johannes Schöneberg ◽  
Ewa Sitarska ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Force Generation ◽  
List Type ◽  
Membrane Tension ◽  
Actin Assembly ◽  
Actin Network ◽  
Coated Pits ◽  
Actin Organization ◽  
Tightly Coupled

AbstractClathrin-mediated endocytosis (CME) remains robust despite variations in plasma membrane tension. Actin assembly-mediated force generation becomes essential for CME under high membrane tension, but the underlying mechanisms are not understood. We investigated actin network ultrastructure at each stage of CME by super-resolution imaging. Actin and N-WASP spatial organization indicate that polymerization initiates at the base of clathrin-coated pits and that the actin network then grows away from the plasma membrane. Actin network organization is not tightly coupled to endocytic clathrin coat growth and deformation. Membrane tension-dependent changes in actin organization explain this uncoupling. Under elevated membrane tension, CME dynamics slow down and the actin network grows higher, resulting in greater coverage of the clathrin coat. This adaptive mechanism is especially crucial during the initial membrane curvature-generating stages of CME. Our findings reveal that adaptive force generation by the actin network ensures robust CME progression despite changes in plasma membrane tension.Highlights-Clathrin coat surface area and actin ultra-structure adapt to elevated membrane tension.-The actin network is nucleated at the base of the clathrin-coated pit and grows upward.-Actin ultra-structural organization is not tightly coupled to CME progression.-Actin force generation is required earlier in CME progression under elevated membrane tension.SummaryKaplan et al. revealed that actin assembly compensates for changes in plasma membrane tension by an adaptive force generating mechanism to ensure robust endocytosis. Under elevated membrane tension the network grows deeper, even in early endocytic stages, from the base upward.

scholarly journals Interferon Receptor Trafficking and Signaling: Journey to the Cross Roads

2021 ◽  
Vol 11 ◽  
Author(s):  
Natacha Zanin ◽  
Christine Viaris de Lesegno ◽  
Christophe Lamaze ◽  
Cedric M. Blouin
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Intracellular Signaling ◽  
Membrane Dynamics ◽  
Type I ◽  
Post Translational Modifications ◽  
Type I Ifns ◽  
Induced Signal ◽  
New Evidence

Like most plasma membrane proteins, type I interferon (IFN) receptor (IFNAR) traffics from the outer surface to the inner compartments of the cell. Long considered as a passive means to simply control subunits availability at the plasma membrane, an array of new evidence establishes IFNAR endocytosis as an active contributor to the regulation of signal transduction triggered by IFN binding to IFNAR. During its complex journey initiated at the plasma membrane, the internalized IFNAR complex, i.e. IFNAR1 and IFNAR2 subunits, will experience post-translational modifications and recruit specific effectors. These finely tuned interactions will determine not only IFNAR subunits destiny (lysosomal degradation vs. plasma membrane recycling) but also the control of IFN-induced signal transduction. Finally, the IFNAR system perfectly illustrates the paradigm of the crosstalk between membrane trafficking and intracellular signaling. Investigating the complexity of IFN receptor intracellular routes is therefore necessary to reveal new insight into the role of IFNAR membrane dynamics in type I IFNs signaling selectivity and biological activity.

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