The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for many cellular processes. Recent work has shown that acto-myosin dynamics modify the local membrane organization, but the molecular details are not well understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, we detect, image and distinguish actin and myosin filaments. As a result, we can follow single, membrane attached actin filaments diffusing within the acto-myosin network, revealing differing types of motion depending on filament length. We go on to quantify myosin II filament dwell times and processivity as a function of ATP concentration, providing evidence for the predicted ensemble behavior of myosin head domains. Simultaneous observation of long-term network flow and organization enables us to link changes in myosin II filament dynamics with decreasing ATP concentrations to a switch in the acto-myosin network from a remodeling, fluid state to contractile behavior, and to observe the formation of vortices so far only predicted by theory.

Recent in-vivo studies have revealed that several membrane proteins are driven to form nanoclusters by active contractile flows arising from F-actin and myosin at the cortex. The mechanism of clustering was shown to be arising from the dynamic patterning of transient contractile platforms (asters) generated by actin and myosin. Myosin-II, which assemble as minifilaments consisting of tens of myosin heads, are rather bulky structures and hence a concern could be that steric considerations might obstruct the emergence of nanoclustering. Here, using coarse-grained, agent-based simulations that respect the size of constituents, we find that in the presence of steric hindrance, the patterns exhibited by actomyosin in two dimensions, do not resemble the steady state patterns observed in our in-vitro reconstitution of actomyosin on a supported bilayer. We then perform simulations in a thin rectangular slab, allowing the separation of a layer of actin filaments from those of myosin-II minifilaments. This recapitulates the observed features of in-vitro patterning. Using super resolution microscopy, we find direct evidence for stratification in our in-vitro system. Our study suggests the possibility that molecular stratification may be an important organising feature of the cortical cytoskeleton in-vivo. † Equal contribution

Caveolin-3 is the major structural protein of caveolae in muscle cells. Mutations in the CAV3 gene cause different type of muscle disorders mostly characterized by defects in membrane integrity and repair, deregulation in the expression of various muscle proteins and deregulation of several muscle associated signaling pathways.We show here that myotubes derived from patients bearing the CAV3 P28L and R26Q mutations present a lack of functional caveolae at the plasma membrane which results in an abnormal mechanoresponse. Mutant myotubes can no longer buffer the increase of membrane tension induced by mechanical stress and present an hyperactivation of the IL6/STAT3 signaling pathway at rest and under mechanical stress. The impaired mechanical regulation of the IL6/STAT3 signaling pathway by caveolae leads to chronic activation and a higher expression of muscle specific genes. These defects could be reversed by reassembling a pool of functional All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/281113 doi: bioRxiv preprint first posted online Mar. 20, 2018; caveolae through expression of wild type Cav3. Our findings bring more mechanistic insight into human Cav3 associated muscle disorders and show a general defect in the mechanoresponse of CAV3 P28L and R26Q myotubes.

Actin cytoskeletal dynamics are tightly regulated by a range of signaling pathways. Through different binding domains, Neural Wiscott-Aldrich syndrome protein (NWASP) plays an important role in connecting multiple signaling inputs to actin polymerization via the actin related protein (Arp2/3) complex. Multiple binding partners can activate NWASP by opening up its autoinhibited conformation in which the C-terminal VCA (verproline-centralacidic) domain is blocked; this open functional form can then bind to Arp2/ 3 complex and nucleate actin branch polymerization. In-vitro experiments showed a synergistic NWASP activation when a ligand combination is used (Cdc42 þ PIP2 or Nck þ PIP2); that is, combinations of 2 binding partners produce much stronger activation than the sum of the individual effects. Because PIP2 is a membrane lipid, membrane recruitment of NWASP could be a major regulator in Arp2/3 dependent actin nucleation in the cell. To understand these spatiotemporal controls, we developed mesoscale kinetic Langevin dynamics models using the SpringSaLaD software. We used coarse grain models of NWASP and its different binding partners to perform virtual binding experiments. Our simulations reproduced the synergistic dual activation by Cdc42 þ PIP2 and Nck þ PIP2. They also offered mechanistic insights on (1) how PIP2 might act to facilitate the NWASP activation by localizing the molecules to the membrane surface; (2) what structural domains in NWASP play pivotal roles in this process; (3) how the geometric arrangements of SH3 domains in Nck could control NWASP activation. Our simulation also recapitulated the experimentally observed requirement for allosteric NWASP binding to Arp2/3. Thus, our study is able to provide a biophysical basis for the complex regulation of NWASP and how it can shape downstream Arp2/3-mediated actin dynamics. (Supported by grant R01GM132859 from NIGMS.

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