scholarly journals Actin bundle architecture and mechanics regulate myosin II force generation

2020 ◽  
Author(s):  
Kimberly L. Weirich ◽  
Samantha Stam ◽  
Ed Munro ◽  
Margaret L. Gardel
Keyword(s):  
Actin Filaments ◽  
Myosin Ii ◽  
Force Generation ◽  
Biological Processes ◽  
Actin Bundle ◽  
Force Magnitude ◽  
Cargo Transport ◽  
Mixed Polarity ◽  
Bundle Compliance ◽  
Filament Spacing

AbstractThe actin cytoskeleton is a soft, structural material that underlies biological processes such as cell division, motility, and cargo transport. The cross-linked actin filaments self-organize into a myriad of architectures, from disordered meshworks to ordered bundles, which are hypothesized to control the actomyosin force generation that regulates cell migration, shape, and adhesion. Here, we use fluorescence microscopy and simulations to investigate how actin bundle architectures with varying polarity, spacing, and rigidity impact myosin II dynamics and force generation. Microscopy reveals that mixed polarity bundles formed by rigid cross-linkers support slow, bidirectional myosin II filament motion, punctuated by periods of stalled motion. Simulations reveal that these locations of stalled myosin motion correspond to sustained, high forces in regions of balanced actin filament polarity. By contrast, mixed polarity bundles formed by compliant, large cross-linkers support fast, bidirectional motion with no traps. Simulations indicate that trap duration is directly related to force magnitude, and that the observed increased velocity corresponds to lower forces resulting from both the increased bundle compliance and filament spacing. Our results indicate that the properties of actin structures regulate the dynamics and magnitude of myosin II forces, highlighting the importance of architecture and mechanics in regulating forces in biological materials.

scholarly journals Active cargo positioning in antiparallel transport networks

2019 ◽  
Author(s):  
Mathieu Richard ◽  
Carles Blanch-Mercader ◽  
Hajer Ennomani ◽  
Wenxiang Cao ◽  
Enrique M. De La Cruz ◽  
...  
Keyword(s):  
Transport Properties ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Actin Network ◽  
Cargo Transport ◽  
Stochastic Transport ◽  
Mixed Polarity ◽  
Micro Patterns

ABSTRACTCytoskeletal filaments assemble into dense parallel, antiparallel or disordered networks, providing a complex environment for active cargo transport and positioning by molecular motors. The interplay between the network architecture and intrinsic motor properties clearly affects transport properties but remains poorly understood. Here, by using surface micro-patterns of actin polymerization, we investigate stochastic transport properties of colloidal beads in antiparallel networks of overlapping actin filaments. We found that 200-nm beads coated with myosin-Va motors displayed directed movements towards positions where the net polarity of the actin network vanished, accumulating there. The bead distribution was dictated by the spatial profiles of local bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Remarkably, beads coated with heavy mero-myosin-II motors showed a similar behavior. However, although velocity gradients were steeper with myosin II, the much larger bead diffusion observed with this motor resulted in less precise positioning. Our observations are well described by a three-state model, in which active beads locally sense the net polarity of the network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk. The precision of bead positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. Our results unveiled physical rules for cargo transport and positioning in networks of mixed polarity.Significance statementCellular functions rely on small groups of molecular motors to transport their cargoes throughout the cell along polar filaments of the cytoskeleton. Cytoskeletal filaments self-assemble into dense networks comprising intersections and filaments of mixed polarity, challenging directed motor-based transport. Using micro-patterns of actin polymerization in-vitro, we investigated stochastic transport of colloidal beads in antiparallel networks of overlapping actin filaments. We found that beads coated with myosin motors sensed the net polarity of the actin network, resulting in active bead positioning to regions of neutral polarity with a precision depending on the motor type. A theoretical description of our experimental results provides the key physical rules for cargo transport and positioning in filament networks of mixed polarity.

Polarity sorting of actin filaments in cytochalasin-treated fibroblasts

1997 ◽  
Vol 110 (15) ◽  
pp. 1693-1704 ◽  
Author(s):  
A.B. Verkhovsky ◽  
T.M. Svitkina ◽  
G.G. Borisy
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Dynamic Network ◽  
Muscle Cells ◽  
Immunogold Labelling ◽  
Cultured Fibroblasts ◽  
Myosin S1 ◽  
Mixed Polarity ◽  
Myosin Interaction

The polarity of actin filaments is fundamental for the subcellular mechanics of actin-myosin interaction; however, little is known about how actin filaments are oriented with respect to myosin in non-muscle cells and how actin polarity organization is established and maintained. Here we approach these questions by investigating changes in the organization and polarity of actin relative to myosin II during actin filament translocation. Actin and myosin II reorganization was followed both kinetically, using microinjected fluorescent analogs of actin and myosin, and ultrastructurally, using myosin S1 decoration and immunogold labelling, in cultured fibroblasts that were induced to contract by treatment with cytochalasin D. We observed rapid (within 15 minutes) formation of ordered actin filament arrays: short tapered bundles and aster-like assemblies, in which filaments had uniform polarity with their barbed ends oriented toward the aggregate of myosin II at the base of a bundle or in the center of an aster. The resulting asters further interacted with each other and aggregated into bigger asters. The arrangement of actin in asters was in sharp contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the edge of the cell, actin filaments became oriented with their barbed ends toward the cell center; that is, the orientation was opposite to what was observed at the edge of nontreated cells. This rearrangement is indicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to their polarity during translocation. The results suggest that the myosin II-actin system of non-muscle cells is organized as a dynamic network where actin filament arrangement is defined in the course of its interaction with myosin II.

scholarly journals Actin bundle architecture and mechanics regulate myosin II force generation

2021 ◽  
Author(s):  
Kimberly L. Weirich ◽  
Samantha Stam ◽  
Edwin Munro ◽  
Margaret L. Gardel
Keyword(s):  
Myosin Ii ◽  
Force Generation ◽  
Actin Bundle

scholarly journals Active cargo positioning in antiparallel transport networks

2019 ◽  
Vol 116 (30) ◽  
pp. 14835-14842 ◽  
Author(s):  
Mathieu Richard ◽  
Carles Blanch-Mercader ◽  
Hajer Ennomani ◽  
Wenxiang Cao ◽  
Enrique M. De La Cruz ◽  
...  
Keyword(s):  
Transport Properties ◽  
Molecular Motors ◽  
Network Architecture ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Biased Random Walk ◽  
Cargo Transport ◽  
Stochastic Transport ◽  
Mixed Polarity ◽  
Myosin Va

Cytoskeletal filaments assemble into dense parallel, antiparallel, or disordered networks, providing a complex environment for active cargo transport and positioning by molecular motors. The interplay between the network architecture and intrinsic motor properties clearly affects transport properties but remains poorly understood. Here, by using surface micropatterns of actin polymerization, we investigate stochastic transport properties of colloidal beads in antiparallel networks of overlapping actin filaments. We found that 200-nm beads coated with myosin Va motors displayed directed movements toward positions where the net polarity of the actin network vanished, accumulating there. The bead distribution was dictated by the spatial profiles of local bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Remarkably, beads coated with heavy–mero-myosin II motors showed a similar behavior. However, although velocity gradients were steeper with myosin II, the much larger bead diffusion observed with this motor resulted in less precise positioning. Our observations are well described by a 3-state model, in which active beads locally sense the net polarity of the network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk. The precision of bead positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. Our results unveiled physical rules for cargo transport and positioning in networks of mixed polarity.

scholarly journals Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms

2003 ◽  
Vol 14 (3) ◽  
pp. 1002-1016 ◽  
Author(s):  
Nicole S. Bryce ◽  
Galina Schevzov ◽  
Vicki Ferguson ◽  
Justin M. Percival ◽  
Jim J.-C. Lin ◽  
...  
Keyword(s):  
Cell Size ◽  
Functional Properties ◽  
Actin Filament ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Molecular Composition ◽  
Cellular Migration ◽  
Stress Fibers ◽  
Human Homologue ◽  
Tropomyosin Isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5NM1), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5NM1was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.

scholarly journals Myosin Motors and Not Actin Comets Are Mediators of the Actin-based Golgi-to-Endoplasmic Reticulum Protein Transport

2003 ◽  
Vol 14 (2) ◽  
pp. 445-459 ◽  
Author(s):  
Juan M. Durán ◽  
Ferran Valderrama ◽  
Susana Castel ◽  
Juana Magdalena ◽  
Mónica Tomás ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Light Chain ◽  
Shiga Toxin ◽  
Actin Filaments ◽  
Protein Transport ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii ◽  
Myosin Motors

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

scholarly journals An automated in vitro motility assay for high-throughput studies of molecular motors

Lab on a Chip ◽  
2018 ◽  
Vol 18 (20) ◽  
pp. 3196-3206 ◽  
Author(s):  
Till Korten ◽  
Elena Tavkin ◽  
Lara Scharrel ◽  
Vandana Singh Kushwaha ◽  
Stefan Diez
Keyword(s):  
High Throughput ◽  
Molecular Motors ◽  
Lab On A Chip ◽  
Force Generation ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Cargo Transport

Molecular motors, essential to force-generation and cargo transport within cells, are invaluable tools for powering nanobiotechnological lab-on-a-chip devices.

Development of macrociliary cells in Beroe. I. Actin bundles and centriole migration

1988 ◽  
Vol 89 (1) ◽  
pp. 67-80
Author(s):  
S. Tamm ◽  
S.L. Tamm
Keyword(s):  
Cell Membrane ◽  
Basal Body ◽  
Actin Filaments ◽  
Close Association ◽  
Directed Assembly ◽  
Double Layers ◽  
Basal Bodies ◽  
Apical Surface ◽  
Actin Bundle ◽  
Microfilament Bundle

Differentiation of macrociliary cells on regenerating lips of the ctenophore Beroe was studied by transmission electron microscopy. In this study of early development, we found that basal bodies for macrocilia arise by an acentriolar pathway near the nucleus and Golgi apparatus, in close association with plaques of dense fibrogranular bodies. Procentrioles are often aligned side-by-side in double layers with the cartwheel ends facing outward toward the surrounding plaques of dense granules. Newly formed basal bodies then disband from groups and develop a long striated rootlet at one end. At the same time, an array of microfilaments arises in the basal cytoplasm. The microfilaments are arranged in parallel strands oriented toward the cell surface. The basal body-rootlet units are transported to the apical surface in close association with the assembling actin filament bundle. Microfilaments run parallel to and alongside the striated rootlets, to which they often appear attached. Basal body-rootlet units migrate at the heads of trails of microfilaments, as if they are pushed upwards by elongation of their attached actin filaments. Near the apical surface the actin bundle curves and runs below the cell membrane. Newly arrived basal body-rootlets tilt upwards out of the microfilament bundle to contact the cell membrane and initiate ciliogenesis. The basal bodies tilt parallel to the flat sides of the rootlets, and away from the direction in which the basal feet point. The actin bundle continues to enlarge during ciliogenesis. These results suggest that basal body migration may be driven by the directed assembly of attached actin filaments.

scholarly journals Anchoring of actin to the plasma membrane enables tension production in the fission yeast cytokinetic ring

2019 ◽  
Vol 30 (16) ◽  
pp. 2053-2064 ◽  
Author(s):  
Shuyuan Wang ◽  
Ben O’Shaughnessy
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Tensile Force ◽  
Myosin Ii ◽  
Quantitative Evidence ◽  
Explicit Simulation ◽  
Adjacent Segments ◽  
Develop Tension

The cytokinetic ring generates tensile force that drives cell division, but how tension emerges from the relatively disordered ring organization remains unclear. Long ago, a musclelike sliding filament mechanism was proposed, but evidence for sarcomeric order is lacking. Here we present quantitative evidence that in fission yeast, ring tension originates from barbed-end anchoring of actin filaments to the plasma membrane, providing resistance to myosin forces that enables filaments to develop tension. The role of anchoring was highlighted by experiments on isolated fission yeast rings, where sections of ring became unanchored from the membrane and shortened ∼30-fold faster than normal. The dramatically elevated constriction rates are unexplained. Here we present a molecularly explicit simulation of constricting partially anchored rings as studied in these experiments. Simulations accurately reproduced the experimental constriction rates and showed that following anchor release, a segment becomes tensionless and shortens via a novel noncontractile reeling-in mechanism at about the velocity of load-free myosin II. The ends are reeled in by barbed end–anchored actin filaments in adjacent segments. Other actin anchoring schemes failed to constrict rings. Our results quantitatively support a specific organization and anchoring scheme that generate tension in the cytokinetic ring.

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