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

National Heart, Lung, And Blood Institute

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Abstract

Myosin 10 at the tips of filopodia-derived retraction fibers supports adhesion during mitosis when conventional focal adhesions disassemble. Historically, adhesion during mitosis for cells grown in 2D has been attributed to retraction fibers (RFs), which are thought to arise from a combination of the cell rounding that occurs upon mitotic entry and the persistence of interphase focal adhesions (FAs). A recent study showed, however, that Talin, the main clutch component connecting actin to integrin, largely disappears from sites of adhesion in mitotic cells (Dix et al Dev Cell 2018). What then connects actin to integrin during mitosis? Here we show that endogenously tagged Myo10, an integrin-binding MyTH4/FERM domain myosin commonly referred to as the filopodial myosin, localizes together with active integrin and IRM signals at the tips of metaphase RFs. Consistent with the results of Dix et al, and with the idea that RFs are in fact filopodia, time lapse imaging shows that Talin-rich FAs at the cell perimeter vanish upon mitotic entry while pre-existing, Myo10-positive, interphase filopodia persist, such that 95% of them become RFs. In support of this, metaphase RFs stain for the filopodial crosslinker fascin and the filopodial tip marker VASP, and endogenous Myo10 moves out RFs at 0.7 um/s, consistent with the bundled, barbed end-out organization of actin found within filopodia. These results, together with the fact that fluorescence intensity measurements within the TIRF field as proxies for adhesion support show that Myo10 increases and Talin decreases between mitotic entry and metaphase, suggest that Myo10 at the tips of filopodia-derived RFs is replacing Talin as the main clutch component connecting actin to integrins during mitosis. Consistent with this idea, measurements of RF failure frequency and the content of active integrin within RFs indicate that adhesion is attenuated in dividing cells lacking Myo10. Moreover, these defects are rescued by WT Myo10 but not by a version that cannot bind integrin. Together, these data reveal a self-organizing property of mitotic RFs: their ability to support adhesion during mitosis is hardwired by the fact that they pre-exist as Myo10-dependent adhesive filopodia, and their barbed end-out organization licenses Myo10-dependent adhesion reinforcement during mitosis. Myosin 10 supports mitotic spindle bipolarity by promoting PCM integrity and supernumerary centrosome clustering. Myosin 10 (Myo10) is a member of the MyTH4/FERM domain family of unconventional, actin-based motor proteins. Previous studies have shown that Myo10 supports cell adhesion during interphase via its integrin-binding FERM domain and spindle pole integrity during mitosis via its microtubule-binding MyTH4 domain. Here we characterized Myo10s contribution to mitosis using Myo10 knockout HeLa cells and MEFs isolated from a Myo10 knockout mouse. Most notably, both of these knockout cells exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized 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 indicate, therefore, that Myo10 supports spindle bipolarity by maintaining PCM integrity in both normal and cancer cells, and by promoting supernumerary centrosome clustering in cancer cells. Finally, complementation experiments show that Myo10s FERM domain-dependent interaction with integrin is required for robust supernumerary centrosome clustering, and that its MyTH4 domain-dependent interaction with microtubules is essential for maintaining the integrity of spindle poles that mature normally. A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation. 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 mouse 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. Integrin ligation and tropomyosin promote the formation of the pSMAC actomyosin arc network in mouse CD8+ T cells. The ability of CD8+ T cells to kill virally-infected and transformed cells is critically dependent on their ability to form stable interactions with antigen-presenting cells (APCs). These interactions are mediated by a highly-organized structure at the T cell: APC interface known as the immunological synapse (IS). Mature synapses are composed of distal, peripheral and central supramolecular activation complexes (dSMAC, pSMAC, cSMAC). TCR microclusters engage antigen-bearing MHC at the IS periphery and are then transported across the dSMAC and pSMAC to the cSMAC. We showed previously that this centripetal microcluster transport is driven by the retrograde flow of an Arp2/3-generated, branched actin network in the dSMAC, and the contraction of formin-generated, myosin 2-rich, concentric actin arcs in the pSMAC. Perturbation of either of these actin structures inhibits microcluster centralization and dampens TCR signaling. We also showed that the adhesive ring formed by the T cells integrin LFA-1 that drives T cell: APC adhesion by binding to ICAM-1 on the APC surface colocalizes with the pSMAC actomyosin arcs, and that inhibiting the formation/organization of this actomyosin structure compromises T cell: APC adhesion. Here we investigated the contributions that LFA-1 ligation and the F-actin binding protein tropomyosin make to the synaptic recruitment of myosin 2A (M2A) and to the organization the pSMAC arcs. With regard to LFA-1 ligation, we found using effector mouse CD8+ T cells that the amount of M2A recruited to synapse, as well as the number and organization of the pSMAC actin arcs that M2A decorates, are both dramatically increased by the addition of ICAM-1 to the activating surface. With regard to tropomyosin, mouse CD8+ T cells express tropomyosin (Tpm) isoforms Tpm3.1 and Tpm4.2, both of which have been implicated in the recruitment and activation of myosin 2. We show that both isoforms are upregulated in effector CD8+ T cells and colocalize with pSMAC actomyosin arcs. Importantly, treatment with the Tpm3.1 inhibitor ATM3507 significantly reduces the amount of M2A recruited to the synapse and disrupts the organization of the pSMAC actin arcs. These results argue that LFA-1 ligation and tropomyosin work in concert to recruit and activate M2A at the synapse to allow the formation of an organized contractile pSMAC structure.