Motor proteins and cytoskeletal dynamics in T cells, B cells and mesenchymal cells

National Heart, Lung, And Blood Institute

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Abstract

B-cell activation and immune synapse (IS) formation with membrane-bound antigens are actin-dependent processes that scale positively with the strength of antigen-induced signals. Importantly, ligating the B-cell integrin, LFA-1, with ICAM-1 promotes IS formation when antigen is limiting. Whether the actin cytoskeleton plays a specific role in integrin-dependent IS formation is unknown. Here we show using super-resolution imaging of primary B cells that LFA-1: ICAM-1 interactions promote the formation of an actomyosin network that dominates the B-cell IS. This network is created by the formin mDia1, organized into concentric, contractile arcs by myosin 2A, and flows inward at the same rate as B-cell receptor (BCR): antigen clusters. Consistently, individual BCR microclusters are swept inward by individual actomyosin arcs. Under conditions where integrin is required for synapse formation, inhibiting myosin impairs synapse formation, as evidenced by reduced antigen centralization, diminished BCR signaling, and defective signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network plays a key role in the mechanism by which LFA-1 co-stimulation promotes B-cell activation and IS formation. Myosin 10 (Myo10) is an actin-based motor protein that can also interact with microtubules via a Myth4 domain. This interaction appears particularly important for mitosis, as Myo10 localizes to spindles and spindle poles in early frog embryos to promote spindle pole integrity, spindle length, spindle anchoring, and mitotic progression. Myo10 also cooperates with dynein to position the mitotic spindle in HeLa cells, and with the kinesin HSET to cluster supernumerary centrosomes in cancer cells. Here we characterized Myo10s contribution to mitosis using Myo10 knockout HeLa cells and MEFs isolated from two Myo10 knockout mouse lines. Most notably, all three exhibit a pronounced increase in the frequency of multipolar spindles. Staining of metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is PCM fragmentation, which creates y-tubulin-positive, centriole-negative microtubule asters that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing centrosome clustering. These results argue that Myo10 supports spindle bipolarity by maintaining PCM integrity in both normal and cancer cells, and by promoting supernumerary centrosome clustering in cancer cells. Filopodia are thin cell surface projections composed of bundled actin filaments whose barbed ends all point to the filopodia tip. Several properties, including their ability to form integrin based-adhesions, have implicated filopodia in ECM sensing, pathfinding, and cell migration (Arjonen et al 2011). Myosin 10 (Myo10) is a barbed end-directed myosin with a strong preference for walking on bundled actin filaments (Ricca and Rock 2010). Consistently, Myo10 moves robustly to filopodial tips, which represent its primary location in interphase cells (Kerber et al 2009). Given this, and given that it promotes filopodia formation, Myo10 has been appropriately coined the filopodial myosin (Kerber and Cheney 2011). Bringing things full circle, Myo10 contains an integrin-binding FERM domain that allows it to participate in integrin activation at filopodial tips (Jacquemet Biorxiv). Here we used Myo10 knockout (KO) and Halo-Myo10 knockin (KI) HeLa cell lines to investigate the possibility that Myo10 also contributes to adhesion during mitosis. Quantitative imaging of metaphase KI cells showed that endogenous Myo10 localizes together with active integrin almost exclusively at two sites of ECM contact: the bottom of the rounded cell body and the distal ends/tips of retraction fibers (RFs). Importantly, these later structures have been implicated for decades in the adhesion of mitotic cells (Mitchison 1992). More importantly, time lapse imaging showed that 90% of HeLa cell RFs arise from preexisting, Myo10-positive filopodia. Measurements of fluorescence intensity within the TIRF field showed that Myo10 increases and talin decreases between mitotic entry and metaphase, and that this reverses at telophase. This result is consistent with mounting evidence that focal adhesion components like talin mostly disappear during mitosis (Dix et al 2018), and suggests that Myo10 largely replaces talin during mitosis as the clutch component connecting integrins to actin. Measurements of IRM signal, RF failure rate, and active integrin content within RFs all showed that Myo10 does indeed support adhesion during mitosis. Together, these data provide insight into how HeLa cells remain adhered during mitosis, and they show that HeLa cell RFs are in fact filopodia. This latter conclusion, which we confirmed by staining for the filopodia markers VASP and fascin, and by imaging Myo10 moving out metaphase RFs at 0.5 um/s, reveals a self-organizing property of these adhesive structures: their ability to support adhesion during mitosis is hardwired by the fact that they preexist as Myo10-dependent adhesive filopodia, and their barbed end-out organization licenses Myo10-dependent adhesion reinforcement during mitosis.