Skeletal muscle development involves the formation of multi-nucleated myotubes. This is thought to proceed by the induction of differentiation (acquisition of fusion competence) of myoblast cells, their aggregation, and union of their plasma membranes. Various membrane proteins including N- and M-cadherins, N- and V-CAMs and integrins participate in myotube formation, but the molecular mechanisms of muscle cell fusion are poorly understood. Here we report the identification of three new, myoblast-expressed gene products, meltrin-alpha, beta and gamma, with homology to both viper haemorrhagic factors and fertilin (PH-30), a membrane protein involved in egg-sperm fusion. Meltrin-alpha, a member of the metalloproteinase/disintegrin protein family, appears to be required for myotube formation. Involvement of a fertilin-related protein in myogenesis suggests that there are common mechanisms in gamete and myoblast fusion.

Polo kinases play critical roles for proper M-phase progression. They are characterized by the presence of two regions of homology in the C-terminal non-catalytic domain, termed polo-box 1 (PB1) and polo-box 2 (PB2). Here we demonstrate that both PB1 and PB2 are required for targeting the catalytic activity of Plk1 to centrosomes, midbody, and kinetochores. Expression of either kinase-inactive PLK1⁄K82M or the C-terminal plk1⌬N induced a pre-anaphase arrest with elevated Cdc2 and Plk1 activity. Prophase-arrested cells exhibited randomly oriented spindle structures, whereas metaphase cells exhibited aberrant bipolar spindles with Mad2 localization at kinetochores of misaligned chromosomes. Microtubule nucleation activity of centrosomes was not compromised. In vivo time-lapse studies revealed that expression of plk1⌬N resulted in repeated cycles of bipolar spindle formation and disruption, suggestive of a defect in spindle stability. A prolonged arrest frequently led to the generation of micronucleated cells in the absence of sisterchromatid separation and centrosome duplication, indicating that micronucleation is not a result of accumulated cytokinesis failures. Interestingly, bypass of the mitotic arrest by dominantnegative spindle checkpoint components led to a failure in completion of cytokinesis. We propose that, in mammalian cells, the polo-box-dependent Plk1 activity is required for proper metaphase⁄anaphase transition and for cytokinesis.

In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation.

f Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SMspecific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP 2 -rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP 2 and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP 2 to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.