skip to main content

Explore Research Products in the NSF-PAR

This content will become publicly available on August 14, 2024

Title: Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

  more » « less
NSF-PAR ID:
10490831
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
DOI PREFIX: 10.1083
Date Published:
Journal Name:
Journal of Cell Biology
Volume:
223
Issue:
4
ISSN:
0021-9525
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ( , PLOS ONE)
    Prigent, Claude (Ed.)
    The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell. 
    more » « less
  2. ; ; ; ; ; ; ( , eLife)
    In most eukaryotic cells, actin filaments assemble into a shell-like actin cortex under the plasma membrane, controlling cellular morphology, mechanics, and signaling. The actin cortex is highly polymorphic, adopting diverse forms such as the ring-like structures found in podosomes, axonal rings, and immune synapses. The biophysical principles that underlie the formation of actin rings and cortices remain unknown. Using a molecular simulation platform called MEDYAN, we discovered that varying the filament treadmilling rate and myosin concentration induces a finite size phase transition in actomyosin network structures. We found that actomyosin networks condense into clusters at low treadmilling rates or high myosin concentrations but form ring-like or cortex-like structures at high treadmilling rates and low myosin concentrations. This mechanism is supported by our corroborating experiments on live T cells, which exhibit ring-like actin networks upon activation by stimulatory antibody. Upon disruption of filament treadmilling or enhancement of myosin activity, the pre-existing actin rings are disrupted into actin clusters or collapse towards the network center respectively. Our analyses suggest that the ring-like actin structure is a preferred state of low mechanical energy, which is, importantly, only reachable at sufficiently high treadmilling rates. 
    more » « less
  3. ; ; ; ; ; ; ( , Journal of Cell Biology)

    Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.

     
    more » « less
  4. ; ; ; ; ; ( , Proceedings of the National Academy of Sciences)

    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.

     
    more » « less
  5. ; ; ; ; ; ( , Cytoskeleton)
    Abstract

    Actomyosin networks are organized in space, direction, size, and connectivity to produce coordinated contractions across cells. We use theC. elegansspermatheca, a tube composed of contractile myoepithelial cells, to study how actomyosin structures are organized. FLN‐1/filamin is required for the formation and stabilization of a regular array of parallel, contractile, actomyosin fibers in this tissue. Loss offln‐1results in the detachment of actin fibers from the basal surface, which then accumulate along the cell junctions and are stabilized by spectrin. In addition, actin and myosin are captured at the nucleus by the linker of nucleoskeleton and cytoskeleton complex (LINC) complex, where they form large foci. Nuclear positioning and morphology, distribution of the endoplasmic reticulum and the mitochondrial network are also disrupted. These results demonstrate that filamin is required to prevent large actin bundle formation and detachment, to prevent excess nuclear localization of actin and myosin, and to ensure correct positioning of organelles.

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email