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

National Heart, Lung, And Blood Institute

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Abstract

HeLa cells and MDA-MB-231 cells utilize Myo10 and HSET differentially to maintain mitotic spindle bipolarity. To survive, cancer cells possessing more than one interphase centrosome must cluster the ensuing extra spindle poles to create a bipolar spindle. Such supernumerary centrosome clustering (SNCC) requires Myosin 10 (Myo10) and the pole-focusing kinesin HSET. We showed recently that Myo10 promotes SNCC in HeLa cells by promoting retraction fiber (RF)-based cell adhesion, and that it further supports spindle bipolarity by preventing the generation of extra spindle poles via pericentriolar material (PCM) fragmentation. Here we quantified the contribution that Myo10 and HSET make individually and together to SNCC and PCM/pole integrity in Hela cells and in MDA-MB-231 cells, which differ from HeLa in being more dependent on SNCC and less dependent on RF-based cell adhesion. As expected, knockdown (KD) of Myo10 and HSET individually increased the frequency of multipolar spindles in both cell types. Their effects were surprisingly not additive, however. For HeLa and MDA-MB-231 cells entering mitosis with more than one centrosome, the defect in SNCC was almost entirely responsible for their multipolar phenotype following KD of either Myo10 or HSET. For HeLa and MDA-MB-231 cells entering mitosis with one centrosome, PCM/pole fragmentation was the primary cause of multipolar spindles following HSET KD. Unlike HeLa, however, MDA-MB-231 cells exhibit very little PCM/pole fragmentation following Myo10 KD. This difference may be due to the smaller role that Myo10 plays in RF-based adhesion in MDA-MB-231. These and other results can inform efforts to target Myo10 and HSET to selectively kill cancer cells by increasing their frequency of multipolar divisions. Myosin 10-dependent protrusion coordination is required for efficient dendritic cell migration through complex environments. Initiating adaptive immune responses requires that dendritic cells (DCs) migrate through complex environments to deliver antigens to draining lymph nodes. To accomplish this, DCs extend multiple protrusions that, together with their nucleus, gauge tissue pore sizes. Commitment to the largest pore is coordinated with retraction of the other protrusions. Failure to coordinate protrusions, as occurs in DCs lacking Cdc42 or its GEF DOCK8, results in impaired migration and cell fragmentation. Here we show that DCs lacking myosin 10 (Myo10), which functions in Cdc42 pathways, also fail to coordinate their protrusions when migrating through complex environments in vitro. The resulting defects in migration and physical coherence are accompanied by miss-localization of the centrosome and myosin 2, both of which have been implicated in pore size selection and protrusion coordination. Competition for Myo10 at protrusion tips, combined with its ability to recruit Lamellipodin and VASP to the leading edge, may also contribute to protrusion coordination. Finally, and most importantly, the migration defects we observed in vitro are replicated in vivo, as Myo10 knockout DCs struggle to enter lymphatics and exhibit a profound defect in migration to draining lymph nodes. Together, our results argue that Myo10 plays a crucial role in initiating the adaptive immune response. A metaphase, septin-associated myosin filament cage ensures lumen integrity in intestinal organoids by promoting metaphase cell rounding and planar cell division. Here we investigated the relationships between myosin contractility, cell division plane and lumen integrity using mouse intestinal organoids, which normally maintain a single lumen as they grow. Attenuating myosin function inhibits interkinetic nuclear migration and metaphase cell rounding, causing the division plane to shift from planar to orthogonal and resulting in organoids possessing multiple lumens. Following the apical domain marker ZO-1 showed that new lumens originate at the interface between stacked daughter cells created by orthogonal divisions, and that subsequent lumen maturation involves passage through a transitional epithelial state. Super-resolution imaging of rounded metaphase cells revealed a never-before-seen myosin filament cage that we show resists surrounding tissue pressure and contributes to contractile ring formation. Finaly, septin filaments align with the myosin filament cage, and septin knockdown, like myosin inhibition, results in organoids possessing multiple lumens. We conclude that a septin-associated myosin filament cage maintains lumen integrity in intestinal organoids by supporting metaphase cell rounding, a prerequisite for planar cell division. Tropomyosin and integrin engagement cooperate to promote actomyosin arc-dependent force generation at the T cell immune synapse required for efficient target cell killing. Efficient target cell killing by CD8+ T cells requires the exertion of force on the target cell. This force may be generated by a contractile actomyosin arc network that comprises the medial, pSMAC portion of the T cell’s immune synapse. The pSMAC is also where the T cell’s integrin LFA-1 binds to ICAM on the target cell to drive T cell: target cell adhesion. Here we show that ligating LFA-1 with ICAM promotes the formation of the actomyosin arc network, and that a tropomyosin (Tpm3.1/3.2) decorates this network. Abrogating Tpm3.1/3.2 function using either a small molecule inhibitor or CRISPR-mediated gene deletion disrupts the concentric organization of the arc network, reduces its content of myosin 2A, reduces T cell traction force, reduces T cell: target cell adhesion, and reduces the ability of effector CD8+ T cells to kill antigen-pulsed target cells without attenuating signaling. These and other results argue that Tpm3.1 and LFA-1 ligation cooperate to promote the generation of actomyosin arc-dependent force at the T cell immune synapse required for efficient target cell killing, and they add to a growing appreciation for the role that positive feedback between adhesion and myosin force plays in promoting T and B cell effector functions. Myosin 10 supports adhesion during mitosis by linking actin to integrins at the tips of filopodia-derived retraction fibers. Filopodia support cell adhesion during interphase in part through the action of the integrin-binding MyTH4/FERM myosin, Myosin 10 (Myo10), which concentrates at filopodial tips. Whether Myo10-postive filopodia support adhesion during mitosis when conventional, Talin-based focal adhesions disassemble is unknown. Consistent with this possibility, Myo10 in HeLa cells localizes together with active integrin almost exclusively at the tips of metaphase retraction fibers, and time lapse imaging shows that the vast majority of these retraction fibers arise from preexisting, Myo10 tip-positive interphase filopodia. Fluorescence intensity measurements within the TIRF field as proxies for adhesion support show that between mitotic entry and metaphase the signals for Myo10 within filopodia/retraction fibers and Talin in focal adhesions increase and decrease, respectively, and that this reverses on mitotic exit. These observations suggest that Myo10 plays a significant role in promoting adhesion during mitosis. Consistently, measurements of retraction fiber failure frequency and active integrin content show that adhesion is attenuated in mitotic cells lacking Myo10. These defects are rescued by wild type Myo10 but not by a version incapable of binding β-1 integrin. Together, these data provide insight into how cells dividing in 2D remain adhered following focal adhesion disassembly, and they show that HeLa cell retraction fibers are in fact filopodia. This latter conclusion, which we confirmed in several ways, 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-positive adhesive filopodia, and their barbed end-out actin filament organization licenses Myo10-dependent adhesion reinforcement during mitosis.