Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells

Baird, Michelle A.; Billington, Neil; Wang, Aibing; Adelstein, Robert S.; Sellers, James R.; Fischer, R.; Waterman, Clare M.

doi:10.1091/mbc.e16-05-0335

Cited by 53 publications

(60 citation statements)

References 65 publications

(56 reference statements)

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“…Interestingly, studies on synapse organization on lipid bilayers show that actin organizes in concentric structures crossed by radial fibres [22] through continuous centripetal flow [12], [14]. In B lymphocytes interacting with antigen-coated gels, we did not observe such actin flows but collective pulsatile actomyosin contractions, similar in features and timescales to the ones described in several other systems [23]- [26]. Are these pulsatile contractions sufficient to form actin patches and thus localize antigen extraction at the centre of the synapse?…”

supporting

confidence: 69%

Actomyosin-driven force patterning controls endocytosis at the immune synapse

Kumari

Saéz

Lankar

et al. 2018

Preprint

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An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules [1]. Although mechanics has been shown to play an important role in this process [2], how forces organize and impact on synapse function is unknown. We found that mechanical forces are spatio-temporally patterned at the immune synapse: global contractile forces are observed at the synapse periphery and local point-like forces are detected at its centre. The global contractile forces result from a pulsatile centripetal actomyosin flow that leads to formation of F-actin protrusions from which the central pointlike forces emerge. Noticeably, these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis. Accordingly, deletion of the myosin IIA gene leads to impaired B cell responses. The interplay between global and local forces governed by the actomyosin cytoskeleton therefore controls the endocytic function of the immune synapse and might constitute a more general mechanism in the physical regulation of cell-cell interactions.Cells are endowed with the ability to internalize substrate-bound molecules, which they recognize through specific surface receptors. While the role of substrate mechanics has been extensively investigated in the context of adhesion, its impact on receptor endocytosis remains unclear. A typical case of coupling between substrate mechanics and juxtacrine signalling (i.e. by direct contact) occurs at the immunological synapse, i.e. the tight contact zone that forms between a lymphocyte and an antigen-presenting cell [1], [3]. In the case of B lymphocytes, synapse formation results from the engagement of the B-cell antigen receptor (BCR) by antigens exposed at the surface of neighbouring cells in vivo. The immune synapse provides a platform that facilitates signalling and leads to antigen internalization within B cells [4]-[6] which is needed for B cells to ultimately produce high-affinity antibodies and generate immune memory ([7], [8]). As, in general, endocytosis often involves surfacetethered rather than soluble molecules when occurring in tissues, antigen internalization at the B cell synapse provides a valuable model to study the impact of mechanics in this process. Different experimental systems have been developed as surrogate antigen-presenting cells to study antigen extraction at B cell synapse ex vivo: planar lipid bilayers [9], plasma membrane sheets [10] and polystyrene beads [11]. On bilayers, the immune synapse consists of a set of concentric patterns in which molecules and cytoskeletal components are partitioned: a distal supramolecular antigen cluster (dSMAC) with an actin ring, a peripheral supramolecular antigen cluster (pSMAC) enriched for adhesion molecules and a central supramolecular antigen cluster (cSMAC) in which antigens concentrate [9]. The first antigen ext...

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supporting

confidence: 69%

Actomyosin-driven force patterning controls endocytosis at the immune synapse

Kumari

Saéz

Lankar

et al. 2018

Preprint

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“…Likewise, here, we show that actomyosin fibers that are not attached via integrins pulsate. This supports results from cell culture experiments showing localized and stochastic pulses of non-muscle myosin 2 assembly and disassembly in adherent cells, which are independent of integrin-ECM engagement 32 . Our results also reinforce our recently proposed model, in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies 16 .…”

Section: Discussionsupporting

confidence: 87%

“…We have previously shown that mosaic egg chambers containing integrin mutant FCs developed stratified epithelia at both poles 21 . We also showed that mutant cells in ectopic layers showed polarity and cell shape defects 20, 32 . These results have led us to propose that integrins might play a role in regulating cell shape and polarity.…”

Section: Resultsmentioning

confidence: 63%

Integrins control tissue morphogenesis and homeostasis by sustaining the different types of intracellular actin networks

Mateos

Valencia-Expósito

Míguez

et al. 2019

Preprint

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29Forces generated by the actomyosin cytoskeleton are key contributors to the 30 generation of tissue shape. Within the cell, the actomyosin cytoskeleton organizes in 31 different types of networks, each of them performing distinct roles. In addition, 32 although they normally localize to precise regions of the cells, they are rarely 33 independent and often their dynamics influence each other. In fact, the reorganization of 34 a given structure can promote the formation of another, conversions that govern many 35 morphogenetic processes. In addition, maintenance of a specific actomyosin network 36 organization in a differentiated tissue might be equally important. Failure to do so could 37 lead to undesired cell state transitions, which in turn would have drastic consequences 38 on the homeostasis of the tissue. Still, little is known about the mechanisms that ensure 39 controlled transitions between actomyosin networks during morphogenesis or their 40 maintenance in a differentiated tissue. Here, we use the Drosophila follicular epithelium 41 to show that cell-ECM interactions mediated by integrins are necessary for the 42 establishment and maintenance of the different actomyosin networks present in these 43 epithelial cells. Elimination of integrins in a group of follicle cells results in changes in 44 the F-actin levels and physical properties of their intracellular actomyosin networks. 45Integrin mutant follicle cells have reduced number of basal stress fibers. They also show 46 increased cortical F-actin levels and tension, which interferes with proper basal surface 47 growth. Finally, clonal elimination of integrins also triggers non-autonomous 48 behavioural changes in neighbouring wild types cells, which now reorganize their actin 49 cytoskeleton and spread and overlay the mutant ones. Based on these results, we 50propose that cell-ECM interactions mediated by integrins regulate epithelia 51 morphogenesis and homesostasis by preserving the different types of intracellular actin 52 networks. 53 54 55 56 57 58 59 60 61 62 Forces generated by F-actin networks are important contributors to the generation of 64 cell and tissue shape. The architecture and mechanical properties of the F-actin network 65 are modulated by myosin II motors and actin binding proteins (reviewed in 1 . The 66 molecular composition of contractile actin networks and bundles is highly conserved 67 among eukaryotic species 2 . Nevertheless, their organization and dynamics change 68 across different cell types, their position within the cell and the differentiation state of 69 the cell. 70 There are two main ways in which actomyosin networks can be organized within 71 the cell, as cortical two-dimensional meshworks below the plasma membrane or as 72 stress-fibers. Studies over the last decade have assigned distinct roles for these two 73 types of networks. Thus, while pulsatile contraction of cortical actomyosin meshworks 74 has been mainly implicated in the cell shape changes underlying key morphogenetic 75 processes, such as gastrulatio...

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“…Further evidence of filament dynamicity was shown in an experiment in which NM2A with a photo-convertible tag was transfected into U2OS cells (35). The myosin in a small rectangle of the cell was photo-converted from green to red, and within 1 min red myosin began to coassemble with existing green myosin filaments as far away as 15 μm.…”

Section: Discussionmentioning

confidence: 91%

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Liu

Billington

Shu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLCphosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.C lass II myosins, the first of 37 known myosin classes (1, 2) to be discovered and originally thought to be specific for muscle, are now known to occur widely in nonmuscle cells in numerous species. Human nonmuscle cells express three different myosin 2 paralogs: nonmuscle myosins (NM) NM2A, NM2B, and NM2C. Like all class II myosins (3, 4), NM2 monomers are hexapeptides of two identical heavy chains (HC), two essential light chains (ELC), and two regulatory light chains (RLC). The HCs of NM2A, NM2B, and NM2C are encoded by different genes, have 60-80% sequence similarity, and isoforms of each are derived from alternative splicing (4).Each HC has an N-terminal globular motor domain with an ATPase site and an actin-binding site, followed by a lever arm that binds one ELC and one RLC, and a long tail that homodimerizes to form a coiled-coil α-helix. The HC ends with a short C-terminal nonhelical tailpiece (5). Like most class II myosins, NM2 monomers assemble into bipolar filaments by antiparallel association of their elongated coiled-coil helical tails. The filaments of NM2s are appreciably smaller than filaments of muscle myosins, with lengths of about 300 nm, consisting of ∼30 monomers for filaments of NM2A and NM2B and ∼14 monomers for filaments of NM2C (5).As described originally for smooth muscle myosin based on light-scattering data (6-8), filaments of unphosp...

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Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells

Cited by 53 publications

References 65 publications

Actomyosin-driven force patterning controls endocytosis at the immune synapse

Actomyosin-driven force patterning controls endocytosis at the immune synapse

Integrins control tissue morphogenesis and homeostasis by sustaining the different types of intracellular actin networks

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

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