scholarly journals Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin

2013 ◽  
Vol 200 (3) ◽  
pp. 301-320 ◽  
Author(s):  
Vanesa M. Tomatis ◽  
Andreas Papadopulos ◽  
Nancy T. Malintan ◽  
Sally Martin ◽  
Tristan Wallis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Late Phase ◽  
Dependent Manner ◽  
Myosin Vi ◽  
Actin Network ◽  
Cortical Actin ◽  
Specific Effects ◽  
Active Pool ◽  
Kinase Phosphorylation

Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.

ENA/VASP proteins regulate exocytosis by mediating myosin VI-dependent recruitment of secretory granules to the cortical actin network

2017 ◽  
Vol 84 ◽  
pp. 100-111 ◽  
Author(s):  
Vanesa M. Tomatis ◽  
Peter Josh ◽  
Andreas Papadopulos ◽  
Rachel S. Gormal ◽  
Vanessa Lanoue ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Myosin Vi ◽  
Actin Network ◽  
Cortical Actin

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

scholarly journals Coupled myosin VI motors facilitate unidirectional movement on an F-actin network

2009 ◽  
Vol 187 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Sivaraj Sivaramakrishnan ◽  
James A. Spudich
Keyword(s):  
Single Molecule ◽  
Membrane Trafficking ◽  
Cell Periphery ◽  
Myosin Vi ◽  
Actin Network ◽  
Cortical Actin ◽  
Actin Cortex ◽  
Unidirectional Movement ◽  
Transport Vesicle ◽  
Pointed End

Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesicle-size cargo over 10 µm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.

scholarly journals Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

2017 ◽  
Vol 114 (7) ◽  
pp. 1595-1600 ◽  
Author(s):  
Thomas A. Masters ◽  
Folma Buss
Keyword(s):  
Actin Filaments ◽  
Leucine Zipper ◽  
Adaptor Protein ◽  
Cell Cortex ◽  
Myosin Vi ◽  
Actin Network ◽  
Ph Domain ◽  
Cortical Actin ◽  
Directed Movement ◽  
Interacting Protein

Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end–directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end–directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.

scholarly journals The TRE17 Oncogene Encodes a Component of a Novel Effector Pathway for Rho GTPases Cdc42 and Rac1 and Stimulates Actin Remodeling

2003 ◽  
Vol 23 (6) ◽  
pp. 2151-2161 ◽  
Author(s):  
Jeffrey M. Masuda-Robens ◽  
Sara N. Kutney ◽  
Hongwei Qi ◽  
Margaret M. Chou
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Actin Remodeling ◽  
Membrane Recruitment ◽  
Truncation Mutant ◽  
Effector Pathway

ABSTRACT The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.

scholarly journals Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

2001 ◽  
Vol 12 (5) ◽  
pp. 1353-1365 ◽  
Author(s):  
Rüdiger Rudolf ◽  
Thorsten Salm ◽  
Amin Rustom ◽  
Hans-Hermann Gerdes
Keyword(s):  
Secretory Granules ◽  
Late Phase ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Chromogranin B ◽  
Cooperative Action ◽  
Cytoskeletal Elements ◽  
Golgi Network

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

scholarly journals Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis

2011 ◽  
Vol 22 (10) ◽  
pp. 1716-1726 ◽  
Author(s):  
Kouichi Mizuno ◽  
José S. Ramalho ◽  
Tetsuro Izumi
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Small Gtpase ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Insulin Granules ◽  
Myosin Va ◽  
Exogenous Expression ◽  
Mutation Analyses

Exophilin8/MyRIP/Slac2-c is an effector protein of the small GTPase Rab27a and is specifically localized on retinal melanosomes and secretory granules. We investigated the role of exophilin8 in insulin granule trafficking. Exogenous expression of exophilin8 in pancreatic β cells or their cell line, MIN6, polarized (exophilin8-positive) insulin granules at the cell corners, where both cortical actin and the microtubule plus-end–binding protein, EB1, were present. Mutation analyses indicated that the ability of exophilin8 to act as a linker between Rab27a and myosin Va is essential for its granule-clustering activity. Moreover, exophilin8 and exophilin8-associated insulin granules were markedly stable and immobile. Total internal reflection fluorescence microscopy indicated that exophilin8 restricts the motion of insulin granules at a region deeper than that where another Rab27a effector, granuphilin, accumulates docked granules directly attached to the plasma membrane. However, the exophilin8-induced immobility of insulin granules was eliminated upon secretagogue stimulation and did not inhibit evoked exocytosis. Furthermore, exophilin8 depletion prevents insulin granules from being transported close to the plasma membrane and inhibits their fusion. These findings indicate that exophilin8 transiently traps insulin granules into the cortical actin network close to the microtubule plus-ends and supplies them for release during the stimulation.

scholarly journals Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization

2020 ◽  
pp. jbc.RA120.012703
Author(s):  
Ashim Rai ◽  
Duha Vang ◽  
Michael Ritt ◽  
Sivaraj Sivaramakrishnan
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
High Rate ◽  
Myosin Vi ◽  
Actin Network ◽  
Cortical Actin ◽  
Myosin V ◽  
Kinetic Measurements ◽  
Actin Cortex ◽  
Run Lengths

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single molecule motility shows thatsaturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable to constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and “stop-and-go” motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers result in actin remodeling and foci formation. In contrast, Dab2 MIRmyosinVI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

scholarly journals Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

2015 ◽  
Vol 210 (5) ◽  
pp. 785-800 ◽  
Author(s):  
Marion Gabel ◽  
Franck Delavoie ◽  
Valérie Demais ◽  
Cathy Royer ◽  
Yannick Bailly ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Electron Tomography ◽  
Secretory Granules ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Annexin A2 ◽  
Cortical Actin ◽  
Lipid Microdomains ◽  
Lipid Domain

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites.

scholarly journals Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase–activating protein Gem-interacting protein

2012 ◽  
Vol 23 (10) ◽  
pp. 1902-1916 ◽  
Author(s):  
Jennifer L. Johnson ◽  
Jlenia Monfregola ◽  
Gennaro Napolitano ◽  
William B. Kiosses ◽  
Sergio D. Catz
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Vesicular Transport ◽  
Gtpase Activating Protein ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Actin Remodeling ◽  
Actin Depolymerization ◽  
Rhoa Activity ◽  
Rhoa Gtpase

Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase–activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin.

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