scholarly journals Lsb5p interacts with actin regulators Sla1p and Las17p, ubiquitin and Arf3p to couple actin dynamics to membrane trafficking processes

2005 ◽  
Vol 387 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Rosaria COSTA ◽  
Derek T. WARREN ◽  
Kathryn R. AYSCOUGH
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Active Form ◽  
Actin Dynamics ◽  
Binding Motif ◽  
Pheromone Receptor ◽  
Cargo Proteins ◽  
Endocytic Machinery ◽  
Vacuolar Protein ◽  
First Time

The importance of coupling the process of endocytosis to factors that regulate actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified. Sla1p is a well-characterized yeast protein that binds both to activators of actin dynamics, Las17p and Pan1p, and to cargo proteins, such as the pheromone receptor Ste2p. Previously, we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localizes to the plasma membrane. Lsb5p has a similar structure to the GGA [Golgi-localized, γ-ear-containing, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS [Vps27p (vacuolar protein sorting protein 27), Hrs, Stam] domain and a GAT (GGA and Tom1) domain. It does not, however, contain either a γ-adaptin ear or a clathrin-binding motif. In the present study, we have further defined its interaction site with both Sla1p and with Las17p, two regulators of actin dynamics. The site of interaction with Sla1p involves the Sla1 HD1 (homology domain 1), which also was shown previously to interact with the pheromone receptor Ste2p. We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin. Finally, we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the correct cortical site in cells. Taken together, our data provide further evidence for the role of Lsb5p in membrane-trafficking events at the plasma membrane and also demonstrate for the first time an interaction of Arf3 with the endocytic machinery in yeast.

Interactions between Sla1p, Lsb5p and Arf3p in yeast endocytosis

2005 ◽  
Vol 33 (6) ◽  
pp. 1273-1275 ◽  
Author(s):  
R. Costa ◽  
K.R. Ayscough
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Cell Signalling ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Pheromone Receptor ◽  
Cargo Proteins ◽  
Endocytic Machinery ◽  
Adp Ribosylation Factor ◽  
Uptake Of Nutrients

Endocytosis is critical for controlling the protein–lipid composition of the plasma membrane, uptake of nutrients as well as pathogens, and also plays an important role in regulation of cell signalling. While a number of pathways for endocytosis have been characterized in different organisms, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognized for many years in budding yeast, and is increasingly supported by studies in mammalian cells. Our studies have focused on proteins that we have shown to act at the interface between the actin cytoskeleton and the endocytic machinery. In particular, we have studied interactions of Sla1p, which binds to both activators of actin dynamics, i.e. Abp1p, Las17p and Pan1p, and to cargo proteins such as the pheromone receptor Ste2p. More recently we have mapped the interaction of Sla1p with Lsb5p, a protein that has a similar structure to the GGA [Golgi-localizing, γ-adaptin ear homology domain, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS (Vps27p/Hrs/STAM)-domain and a GAT (GGAs and TOM1) domain. We show that Lsb5p can interact with yeast Arf3p (orthologous with mammalian Arf6) and we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the cell cortex.

scholarly journals A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

2021 ◽  
Vol 7 (13) ◽  
pp. eabc6345
Author(s):  
Shrawan Kumar Mageswaran ◽  
Wei Yuan Yang ◽  
Yogaditya Chakrabarty ◽  
Catherine M. Oikonomou ◽  
Grant J. Jensen
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Membrane Damage ◽  
Light And Electron Microscopy ◽  
Actin Dynamics ◽  
Endosomal Sorting ◽  
Plasma Membrane Damage ◽  
Cryo Electron Tomography ◽  
Vacuolar Protein

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

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 Regulated Exocytosis in Neuroendocrine Cells: A Role for Subplasmalemmal Cdc42/N-WASP-induced Actin Filaments

2004 ◽  
Vol 15 (2) ◽  
pp. 520-531 ◽  
Author(s):  
Stéphane Gasman ◽  
Sylvette Chasserot-Golaz ◽  
Magali Malacombe ◽  
Michael Way ◽  
Marie-France Bader
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Secretory Granules ◽  
Small Gtpases ◽  
Neuroendocrine Cells ◽  
Actin Dynamics ◽  
Cell Periphery ◽  
Regulated Exocytosis

In neuroendocrine cells, actin reorganization is a prerequisite for regulated exocytosis. Small GTPases, Rho proteins, represent potential candidates coupling actin dynamics to membrane trafficking events. We previously reported that Cdc42 plays an active role in regulated exocytosis in chromaffin cells. The aim of the present work was to dissect the molecular effector pathway integrating Cdc42 to the actin architecture required for the secretory reaction in neuroendocrine cells. Using PC12 cells as a secretory model, we show that Cdc42 is activated at the plasma membrane during exocytosis. Expression of the constitutively active Cdc42L61 mutant increases the secretory response, recruits neural Wiskott-Aldrich syndrome protein (N-WASP), and enhances actin polymerization in the subplasmalemmal region. Moreover, expression of N-WASP stimulates secretion by a mechanism dependent on its ability to induce actin polymerization at the cell periphery. Finally, we observed that actin-related protein-2/3 (Arp2/3) is associated with secretory granules and that it accompanies granules to the docking sites at the plasma membrane upon cell activation. Our results demonstrate for the first time that secretagogue-evoked stimulation induces the sequential ordering of Cdc42, N-WASP, and Arp2/3 at the interface between granules and the plasma membrane, thereby providing an actin structure that makes the exocytotic machinery more efficient.

scholarly journals The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation

2010 ◽  
Vol 84 (12) ◽  
pp. 5848-5859 ◽  
Author(s):  
Emily A. Bruce ◽  
Paul Digard ◽  
Amanda D. Stuart
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Influenza A Virus ◽  
Membrane Trafficking ◽  
Influenza A ◽  
Spherical Particles ◽  
Actin Dynamics ◽  
Apical Plasma Membrane ◽  
Thin Sections ◽  
Virus Particles

ABSTRACT Influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of pleomorphic spheres or vastly elongated filaments. For either type of virion, the factors responsible for separation of viral and cell membranes are not known. We find that cellular Rab11 (a small GTP-binding protein involved in endocytic recycling) and Rab11-family interacting protein 3 ([FIP3] which plays a role in membrane trafficking and regulation of actin dynamics) are both required to support the formation of filamentous virions, while Rab11 is additionally involved in the final budding step of spherical particles. Cells transfected with Rab11 GTP-cycling mutants or depleted of Rab11 or FIP3 content by small interfering RNA treatment lost the ability to form virus filaments. Depletion of Rab11 resulted in up to a 100-fold decrease in titer of spherical virus released from cells. Scanning electron microscopy of Rab11-depleted cells showed high densities of virus particles apparently stalled in the process of budding. Transmission electron microscopy of thin sections confirmed that Rab11 depletion resulted in significant numbers of abnormally formed virus particles that had failed to pinch off from the plasma membrane. Based on these findings, we see a clear role for a Rab11-mediated pathway in influenza virus morphogenesis and budding.

scholarly journals Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase

2008 ◽  
Vol 182 (6) ◽  
pp. 1153-1169 ◽  
Author(s):  
Ting Cai ◽  
Haojie Wang ◽  
Yiliang Chen ◽  
Lijun Liu ◽  
William T Gunning ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Long Range ◽  
Membrane Trafficking ◽  
Endocytic Trafficking ◽  
Binding Motif ◽  
Caveolin 1 ◽  
Pumping Function ◽  
Α1 Subunit

Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.

Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane

2007 ◽  
Vol 409 (2) ◽  
pp. 555-562 ◽  
Author(s):  
Satoshi Takida ◽  
Yusuke Maeda ◽  
Taroh Kinoshita
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Direct Evidence ◽  
Temperature Sensitive ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
P24 Proteins

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

scholarly journals Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Maryam Izadi ◽  
Eric Seemann ◽  
Dirk Schlobinski ◽  
Lukas Schwintzer ◽  
Britta Qualmann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filament ◽  
Network Formation ◽  
Actin Dynamics ◽  
Binding Motif ◽  
Dendritic Arbor ◽  
Filament Formation ◽  
Calmodulin Binding ◽  
Physical Linkage ◽  
Actin Nucleator

Local actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapins. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like’s newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like’s functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation in mammals.

scholarly journals Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells

2008 ◽  
Vol 182 (5) ◽  
pp. 845-853 ◽  
Author(s):  
Rita L. Nokes ◽  
Ian C. Fields ◽  
Ruth N. Collins ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
Bronchial Epithelial ◽  
Recycling Endosomes ◽  
Cargo Proteins ◽  
Vesicular Stomatitis ◽  
Golgi Network

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.

scholarly journals Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

2020 ◽  
Author(s):  
Marta Fratini ◽  
Praveen Krishnamoorthy ◽  
Irene Stenzel ◽  
Mara Riechmann ◽  
Kirsten Bacia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Pollen Tubes ◽  
Actin Dynamics ◽  
Tobacco Pollen ◽  
Dual Distribution ◽  
Cytoskeletal Dynamics

AbstractPollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is enriched in the subapical plasma membrane of pollen tubes and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P2-effects are specified is unclear. Spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P2-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labelling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P2. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P2 in pollen tubes. Despite localizing to one membrane region, AtPIP5K2 and NtPIP5K6 display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2 and NtPIP5K6 variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, PI4P 5-kinase variants targeted to nanodomains stabilized actin, suggesting a specific function of PtdIns(4,5)P2-nanodomains. A distinct role of nanodomain-associated AtPIP5K2 in actin regulation is further supported by proximity to and interaction with the Rho-GTPase NtRac5, and by functional interplay with elements of ROP-signalling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P2-functions to coordinate cytoskeletal dynamics and secretion in pollen tubes.

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