Proper spindle positioning and orientation are essential for asymmetric cell division and require microtubule-actin filament (F-actin) interactions in many systems. Such interactions are particularly important in meiosis, where they mediate nuclear anchoring, as well as meiotic spindle assembly and rotation, two processes required for asymmetric cell division. Myosin-10 proteins are phosphoinositide-binding, actin-based motors that contain carboxy-terminal MyTH4 and FERM domains of unknown function. Here we show that Xenopus laevis myosin-10 (Myo10) associates with microtubules in vitro and in vivo, and is concentrated at the point where the meiotic spindle contacts the F-actin-rich cortex. Microtubule association is mediated by the MyTH4-FERM domains, which bind directly to purified microtubules. Disruption of Myo10 function disrupts nuclear anchoring, spindle assembly and spindle-F-actin association. Thus, this myosin has a novel and critically important role during meiosis in integrating the F-actin and microtubule cytoskeletons.

The actin filament (F-actin) cytoskeleton associates dynamically with the plasma membrane and is thus ideally positioned to participate in endocytosis. Indeed, a wealth of genetic and biochemical evidence has confirmed that actin interacts with components of the endocytic machinery, although its precise function in endocytosis remains unclear. Here, we use 4D microscopy to visualize the contribution of actin during compensatory endocytosis in Xenopus laevis eggs. We show that the actin cytoskeleton maintains exocytosing cortical granules as discrete invaginated compartments, such that when actin is disrupted, they collapse into the plasma membrane. Invaginated, exocytosing cortical granule compartments are directly retrieved from the plasma membrane by F-actin coats that assemble on their surface. These dynamic F-actin coats seem to drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments. Active Cdc42 and N-WASP are recruited to exocytosing cortical granule membranes before F-actin coat assembly and coats assemble by Cdc42-dependent, de novo actin polymerization. Thus, F-actin may power fusion pore resealing and function in two novel endocytic capacities: the maintenance of invaginated compartments and the processing of endosomes.

We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription-factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.