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In JoVE (1)
Other Publications (3)
Articles by Anna Marie Sokac in JoVE
JoVE General
Imaging Cell Shape Change in Living Drosophila Embryos
1Program in Cell & Molecular Biology, Baylor College of Medicine (BCM), 2Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine (BCM)
Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.
Other articles by Anna Marie Sokac on PubMed
Cdc42-dependent Actin Polymerization During Compensatory Endocytosis in Xenopus Eggs
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.
Zygotically Controlled F-actin Establishes Cortical Compartments to Stabilize Furrows During Drosophila Cellularization
Cortical compartments partition proteins and membrane at the cell surface to define regions of specialized function. Here we ask how cortical compartments arise along the plasma membrane furrows that cellularize the early Drosophila embryo, and investigate the influence that this compartmentalization has on furrow ingression. We find that the zygotic gene product Nullo aids the establishment of discrete cortical compartments, called furrow canals, which form at the tip of incipient furrows. Upon nullo loss-of-function, proteins that are normally restricted to adjacent lateral regions of the furrow, such as Neurotactin and Discs large, spread into the furrow canals. At the same time, cortical components that should concentrate in furrow canals, such as Myosin 2 (Zipper) and Anillin (Scraps), are missing from some furrows. Depletion of these cortical components from the furrow canal compartments precipitates furrow regression. Contrary to previous models, we find that furrow compartmentalization does not require cell-cell junctions that border the furrow canals. Instead, compartmentalization is disrupted by treatments that reduce levels of cortical F-actin. Because the earliest uniform phenotype detected in nullo mutants is reduced levels of F-actin at furrow canals, we propose that Nullo compartmentalizes furrows via its regulation of F-actin, thus stabilizing furrows and insuring their ingression to complete cellularization.
Local Actin-dependent Endocytosis is Zygotically Controlled to Initiate Drosophila Cellularization
In early Drosophila embryos, several mitotic cycles proceed with aborted cytokinesis before a modified cytokinesis, called cellularization, finally divides the syncytium into individual cells. Here, we find that scission of endocytic vesicles from the plasma membrane (PM) provides a control point to regulate the furrowing events that accompany this development. At early mitotic cycles, local furrow-associated endocytosis is controlled by cell cycle progression, whereas at cellularization, which occurs in a prolonged interphase, it is controlled by expression of the zygotic gene nullo. nullo mutations impair cortical F-actin accumulation and scission of endocytic vesicles, such that membrane tubules remain tethered to the PM and deplete structural components from the furrows, precipitating furrow regression. Thus, Nullo regulates scission to restrain endocytosis of proteins essential for furrow stabilization at the onset of cellularization. We propose that developmentally regulated endocytosis can coordinate actin/PM remodeling to directly drive furrow dynamics during morphogenesis.
