Membrane hemifusion is a stable intermediate of exocytosis

Membrane fusion during exocytosis requires that two initially distinct bilayers pass through a hemifused intermediate in which the proximal monolayers are shared. Passage through this intermediate is an essential step in the process of secretion, but is difficult to observe directly in vivo. Here we study membrane fusion in the sea urchin egg, in which thousands of homogeneous cortical granules are associated with the plasma membrane prior to fertilization. Using fluorescence redistribution after photobleaching, we find that these granules are stably hemifused to the plasma membrane, sharing a cytoplasmic-facing monolayer. Furthermore, we find that the proteins implicated in the fusion process-the vesicle-associated proteins VAMP/synaptobrevin, synaptotagmin, and Rab3-are each immobile within the granule membrane. Thus, these secretory granules are tethered to their target plasma membrane by a static, catalytic fusion complex that maintains a hemifused membrane intermediate.     

The Mechanical Properties of Early Drosophila Embryos Measured by High-Speed Video Microrheology

In early development, Drosophila melanogaster embryos form a syncytium, i.e., multiplying nuclei are not yet separated by cell membranes, but are interconnected by cytoskeletal polymer networks consisting of actin and microtubules. Between division cycles 9 and 13, nuclei and cytoskeleton form a two-dimensional cortical layer. To probe the mechanical properties and dynamics of this self-organizing pre-tissue, we measured shear moduli in the embryo by high-speed video microrheology. We recorded position fluctuations of injected micron-sized fluorescent beads with kHz sampling frequencies and characterized the viscoelasticity of the embryo in different locations. Thermal fluctuations dominated over nonequilibrium activity for frequencies between 0.3 and 1000 Hz. Between the nuclear layer and the yolk, the cytoplasm was homogeneous and viscously dominated, with a viscosity three orders of magnitude higher than that of water. Within the nuclear layer we found an increase of the elastic and viscous moduli consistent with an increased microtubule density. Drug-interference experiments showed that microtubules contribute to the measured viscoelasticity inside the embryo whereas actin only plays a minor role in the regions outside of the actin caps that are closely associated with the nuclei. Measurements at different stages of the nuclear division cycle showed little variation.