Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles

The cell’s dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament’s 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.

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Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments

Biochemistry and Biophysics Reports ◽

10.1016/j.bbrep.2021.101194 ◽

2022 ◽

Vol 29 ◽

pp. 101194

Author(s):

Arthur J. Michalek ◽

M. Yusuf Ali

Keyword(s):

Actin Filaments ◽

Critical Role ◽

Cargo Transport ◽

Myosin Va

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MYOSIN VA TRANSPORT OF LIPOSOMES IN THREE-DIMENSIONAL ACTIN NETWORKS IS MODULATED BY ACTIN FILAMENT DENSITY, POSITION, AND POLARITY

10.1101/526640 ◽

2019 ◽

Author(s):

Andrew T. Lombardo ◽

Shane R. Nelson ◽

Guy G. Kennedy ◽

Kathleen M. Trybus ◽

Sam Walcott ◽

...

Keyword(s):

Actin Filament ◽

Molecular Motors ◽

Actin Filaments ◽

Three Dimensional ◽

Fluorescent Reporter ◽

Actin Networks ◽

Cargo Transport ◽

Myosin Va ◽

Filament Network

The cell's dense three-dimensional (3D) actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position, using STORM microscopy, and its polarity by observing the movement of single fluorescent, reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350 nm fluid-like, liposomes transported by MyoVa teams (~10 motors) moving within each of the two networks. Compared to the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine-tune MyoVa-based intracellular cargo transport.

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F-actin disassembly factor MICAL1 binding to Myosin Va mediates cargo unloading during cytokinesis

Science Advances ◽

10.1126/sciadv.abb1307 ◽

2020 ◽

Vol 6 (45) ◽

pp. eabb1307 ◽

Cited By ~ 1

Author(s):

Fengfeng Niu ◽

Kang Sun ◽

Wenjie Wei ◽

Cong Yu ◽

Zhiyi Wei

Keyword(s):

Intracellular Trafficking ◽

Molecular Basis ◽

Complex Structure ◽

Actin Dynamics ◽

Actin Assembly ◽

Binding Partner ◽

Cargo Transport ◽

Binding Surface ◽

Myosin Va

Motor-mediated intracellular trafficking requires motors to position cargoes at proper locations. Myosin Va (MyoVa), an actin-based motor, is a classic model for studying cargo transport. However, the molecular basis underlying cargo unloading in MyoVa-mediated transport has remained enigmatic. We have identified MICAL1, an F-actin disassembly regulator, as a binding partner of MyoVa and shown that MICAL1-MyoVa interaction is critical for localization of MyoVa at the midbody. By binding to MICAL1, MyoVa-mediated transport is terminated, resulting in vesicle unloading at the midbody for efficient cytokinesis. The MyoVa/MICAL1 complex structure reveals that MICAL1 and F-actin assembly factors, Spires, share an overlapped binding surface on MyoVa, suggesting a regulatory role of F-actin dynamics in cargo unloading. Down-regulating F-actin disassembly by a MICAL1 mutant significantly reduces MyoVa and vesicles accumulating at the midbody. Collectively, our findings demonstrate that MyoVa binds to MICAL1 at the midbody destination and triggers F-actin disassembly to unload the vesicle cargo.

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Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1104298108 ◽

2011 ◽

Vol 108 (34) ◽

pp. E535-E541 ◽

Cited By ~ 49

Author(s):

M. Y. Ali ◽

G. G. Kennedy ◽

D. Safer ◽

K. M. Trybus ◽

H. L. Sweeney ◽

...

Keyword(s):

Myosin Vi ◽

Cargo Transport ◽

Myosin Va

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Adapter Proteins Activate Myosin-Va during Cargo Transport

Biophysical Journal ◽

10.1016/j.bpj.2014.11.1644 ◽

2015 ◽

Vol 108 (2) ◽

pp. 302a

Author(s):

M. Yusuf Ali ◽

Elena Krementsova ◽

Maria Sckolnick ◽

David M. Warshaw ◽

Kathleen M. Trybus

Keyword(s):

Adapter Proteins ◽

Cargo Transport ◽

Myosin Va

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Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

10.1101/2021.08.10.455842 ◽

2021 ◽

Author(s):

Sam Walcott ◽

David M Warshaw

Keyword(s):

Phase Transition ◽

Actin Filament ◽

Actin Filaments ◽

Three Dimensional ◽

Fold Increase ◽

Coarse Grained ◽

Actin Network ◽

Cargo Delivery ◽

Myosin Va ◽

Filament Networks

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350nm vs. 1,750nm) transport by myoVa motors. 5,000 simulated liposomes transported within each network adopted one of three states: transport, tug of war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug of wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, Nc. Nc, is a geometric property of the actin network that depends only on the position and polarity of the actin filaments and the liposome diameter, as evidenced by a five-fold increase in liposome diameter resulting in a five-fold decrease in Nc. Thus, in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands.

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Small teams of myosin Vc coordinate their stepping for efficient cargo transport on actin bundles

10.1101/106658 ◽

2017 ◽

Author(s):

Elena B. Krementsova ◽

Ken’ya Furuta ◽

Kazuhiro Oiwa ◽

Kathleen M. Trybus ◽

M. Yusuf Ali

Keyword(s):

Actin Filaments ◽

Myosin Isoforms ◽

Back Side ◽

Dna Nanostructures ◽

Class V ◽

Actin Bundles ◽

Directional Motion ◽

Myosin Va ◽

Run Lengths

AbstractMyosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to transport cargo. Single molecules of myoVc cannot take multiple steps on single actin filaments, in stark contrast to the well-studied myosin Va (myoVa) isoform. Consistent with in vivo studies (1), only teams of myoVc motors can move continuously on actin bundles at physiologic ionic strength (2), raising the question of how motor motor interactions cause this preference. Here, using DNA nanostructures as synthetic cargos for linking defined numbers of myoVa or myoVc molecules, we compared the stepping behavior of myoVa versus myoVc teams, and myoVc stepping patterns on single actin filaments versus actin bundles. Run lengths of both myoVa and myoVc teams increased with motor number, but the run lengths of myoVc teams were longer on actin bundles than on filaments. By resolving the stepping behavior of individual myoVc motors with a Qdot bound to the motor domain, we found that coupling of two myoVc molecules significantly decreases futile back/side steps, which were frequently observed for single myoVc motors. Data showing how changes in the inter-motor distance of two coupled myoVc motors affected stepping dynamics suggested that mechanical tension coordinates the stepping behavior of two molecules for efficient directional motion. Our study thus provides a molecular basis to explain how teams of myoVc motors are suited to transport cargos such as zymogen granules on actin bundles.AbbreviationsMyoVcMyosin VcmyoVaMyosin VaQdotsQuantum dotsSDStandard DeviationSEStandard ErrorDNAdeoxyribonucleic acid

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Myosin Va Cargo Transport on Actin Bundles

Biophysical Journal ◽

10.1016/j.bpj.2009.12.3051 ◽

2010 ◽

Vol 98 (3) ◽

pp. 563a

Author(s):

Samantha Beck Previs ◽

Carol S. Bookwalter ◽

Kathleen M. Trybus ◽

David M. Warshaw

Keyword(s):

Actin Bundles ◽

Cargo Transport ◽

Myosin Va

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Subcellular localization of GFP-myosin-V in live mouse melanocytes

Journal of Cell Science ◽

10.1242/jcs.112.17.2853 ◽

1999 ◽

Vol 112 (17) ◽

pp. 2853-2865 ◽

Cited By ~ 2

Author(s):

V. Tsakraklides ◽

K. Krogh ◽

L. Wang ◽

J.C. Bizario ◽

R.E. Larson ◽

...

Keyword(s):

Subcellular Localization ◽

Actin Filaments ◽

Expression Patterns ◽

Intracellular Localization ◽

Motor Domain ◽

Chick Brain ◽

Myosin V ◽

Myosin Va ◽

Myosin Vb ◽

In Cells

Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH(2)-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin-Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to (gamma)-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules.

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