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 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 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 Exophilin-8 assembles secretory granules for exocytosis in the actin cortex via interaction with RIM-BP2 and myosin-VIIa

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Fushun Fan ◽  
Kohichi Matsunaga ◽  
Hao Wang ◽  
Ray Ishizaki ◽  
Eri Kobayashi ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Direct Interaction ◽  
Null Mouse ◽  
Cell Periphery ◽  
Actin Network ◽  
Actin Cortex ◽  
Granule Membrane ◽  
Mouse Pancreatic Islets ◽  
Myosin Va ◽  
Myosin Viia

Exophilin-8 has been reported to play a role in anchoring secretory granules within the actin cortex, due to its direct binding activities to Rab27 on the granule membrane and to F-actin and its motor protein, myosin-Va. Here, we show that exophilin-8 accumulates granules in the cortical F-actin network not by direct interaction with myosin-Va, but by indirect interaction with a specific form of myosin-VIIa through its previously unknown binding partner, RIM-BP2. RIM-BP2 also associates with exocytic machinery, Cav1.3, RIM, and Munc13-1. Disruption of the exophilin-8–RIM-BP2–myosin-VIIa complex by ablation or knockdown of each component markedly decreases both the peripheral accumulation and exocytosis of granules. Furthermore, exophilin-8-null mouse pancreatic islets lose polarized granule localization at the β-cell periphery and exhibit impaired insulin secretion. This newly identified complex acts as a physical and functional scaffold and provides a mechanism supporting a releasable pool of granules within the F-actin network beneath the plasma membrane.

scholarly journals Actin kinetics shapes cortical network structure and mechanics

2016 ◽  
Vol 2 (4) ◽  
pp. e1501337 ◽  
Author(s):  
Marco Fritzsche ◽  
Christoph Erlenkämper ◽  
Emad Moeendarbary ◽  
Guillaume Charras ◽  
Karsten Kruse
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Length Distribution ◽  
Living Cells ◽  
Stochastic Simulations ◽  
Cortical Actin ◽  
Animal Cells ◽  
Cellular Mechanics ◽  
Cellular Processes ◽  
Actin Cortex

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

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

scholarly journals Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

2019 ◽  
Author(s):  
Soma Godó ◽  
Klaudia Barabás ◽  
Ferenc Lengyel ◽  
Dávid Ernszt ◽  
Tamás Kovács ◽  
...  
Keyword(s):  
Single Molecule ◽  
Glutamatergic Neurotransmission ◽  
Gonadal Steroid ◽  
Actin Network ◽  
Cortical Actin ◽  
Surface Movement ◽  
Surface Mobility ◽  
Rapid Effect ◽  
17Β Estradiol ◽  
Single Molecule Analysis

AbstractThe gonadal steroid 17β-estradiol (E2) rapidly alters glutamatergic neurotransmission, but its direct effect on the AMPA receptor (AMPAR) remains unknown. Live-cell single-molecule imaging experiments revealed that E2 rapidly and dose-dependently alters the surface movement of AMPAR via membrane estrogen receptors with distinct effects on somas and neurites. The effect of E2 on the surface mobility of AMPAR depends on the integrity of the cortical actin network.

scholarly journals Coupled Myosin VI Motors Facilitate Unidirectional Movement on an F-actin Network

2010 ◽  
Vol 98 (3) ◽  
pp. 723a
Author(s):  
Sivaraj Sivaramakrishnan ◽  
James Spudich
Keyword(s):  
Myosin Vi ◽  
Actin Network ◽  
Unidirectional Movement

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.

scholarly journals Cracking up: symmetry breaking in cellular systems

2006 ◽  
Vol 175 (5) ◽  
pp. 687-692 ◽  
Author(s):  
Ewa Paluch ◽  
Jasper van der Gucht ◽  
Cécile Sykes
Keyword(s):  
Cell Membrane ◽  
Cellular Systems ◽  
Common Mechanism ◽  
Cell Cortex ◽  
Actin Network ◽  
Cortical Actin ◽  
Animal Cells ◽  
Actin Cortex ◽  
Myosin Motors ◽  
Local Relaxation

The shape of animal cells is, to a large extent, determined by the cortical actin network that underlies the cell membrane. Because of the presence of myosin motors, the actin cortex is under tension, and local relaxation of this tension can result in cortical flows that lead to deformation and polarization of the cell. Cortex relaxation is often regulated by polarizing signals, but the cortex can also rupture and relax spontaneously. A similar tension-induced polarization is observed in actin gels growing around beads, and we propose that a common mechanism governs actin gel rupture in both systems.

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