Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.

SummaryFascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

Summary Nuclear movement is critical for developmental events, cell polarity and migration and is usually mediated by LINC complexes connecting the nucleus to cytoskeletal elements. Compared to active nuclear movement, relatively little is known about homeostatic positioning of nuclei including whether it is an active process. To explore homeostatic nuclear positioning, we developed a method to displace nuclei in adherent cells using centrifugal force. Nuclei displaced by centrifugation rapidly recentered by mechanisms that depended on cell context. In cell monolayers with wounds oriented orthogonal to the force, nuclei were displaced toward the front and back of the cells on the two sides of the wound. Nuclei recentered from both positions, but at different rates and cytoskeletal linkage mechanisms. Rearward recentering was actomyosin-, nesprin-2G- and SUN2-dependent, whereas forward recentering was microtubule-, dynein-, nesprin-2G- and SUN1-dependent. Nesprin-2G engaged actin through its N-terminus and microtubules through a novel dynein interacting site near its C-terminus. Both activities were necessary to maintain nuclear position in uncentrifuged cells. Thus, even when not moving, nuclei are actively maintained in position by engaging the cytoskeleton through the LINC complex.

Active positioning of the nucleus is integral to division, migration, and differentiation of mammalian cells1. Fibroblasts polarizing for migration orient their centrosomes by actin-dependent nuclear movement2. This nuclear movement depends on nesprin-2 giant (N2G), a large, actin-binding outer nuclear membrane component of transmembrane actin-associated (TAN) lines that couple nuclei to moving actin cables3. Here, we identify the diaphanous formin FHOD1 as an interaction partner of N2G. Silencing FHOD1 expression or expression of fragments containing binding sites of N2G or FHOD1 disrupted nuclear movement and centrosome orientation in polarizing fibroblasts. Unexpectedly, silencing of FHOD1 expression did not affect the formation or rearward flow of dorsal actin cables required for nuclear positioning. Rather, N2G-FHOD1 interaction provided a second connection to actin cables essential for TAN line formation and thus nuclear movement. These results reveal a unique function for a formin in coupling an organelle to actin filaments for translocation and suggest that TAN lines require multi-point attachments to actin cables to resist the large forces necessary to move the nucleus.

Summary Interactions between tumor cells and tumor-associated macrophages play critical roles in the initiation of tumor cell motility. To capture the cellular interactions of the tumor microenvironment with high-resolution imaging, we directly visualized tumor cells and their interactions with macrophages in zebrafish. Live-imaging in zebrafish revealed that macrophages are dynamic, yet maintain sustained contact with tumor cells. Additionally, the recruitment of macrophages to tumor cells promotes tumor cell dissemination. Using a Cre/LoxP strategy, we found that macrophages transfer cytoplasm to tumor cells in zebrafish and mouse models. Remarkably, macrophage cytoplasmic transfer correlated with melanoma cell dissemination. We further found that macrophages transfer cytoplasm to tumor cells upon cell contact in vitro. Thus, we present a model in which macrophage/tumor cell contact allows for the transfer of cytoplasmic molecules from macrophages to tumor cells corresponding to increased tumor cell motility and dissemination.