Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network.

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

Live-cell fluorescence imaging of echinoderm embryos.

The rapid development, simplicity and optical clarity of the sea urchin embryo make it an excellent model system for studying the dynamic events of early development. An ever-growing palette of fluorescent proteins and biosensors can now be applied to studying sea urchin development, and there are now a wide variety of imaging modes that can be employed to image sea urchin embryogenesis. However, when performing live-cell imaging, one must take into consideration the sensitivity of embryos (and fluorescent probes) to the intense light associated with confocal microscopes. Here, we discuss general considerations for keeping embryos viable on the microscope stage, as well as probes for imaging cellular membranes and the cytoskeleton. We compare the relative merits of different confocal microscopes for live imaging of embryos and describe the potential for live-cell super-resolution microscopy.