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Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va-) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the(More)
Cyclin G-associated kinase (GAK), the ubiquitous form of the neuronal-specific protein auxilin 1, is an essential cofactor for Hsc70-dependent uncoating of clathrin-coated vesicles. Total internal reflectance microscopy was used to determine the timing of GAK binding relative to dynamin and clathrin binding during invagination of clathrin-coated pits.(More)
Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for(More)
During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing(More)
In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end-tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a(More)
Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity(More)
Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for(More)
Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here we demonstrate that WPBs are often found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of(More)
T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking(More)
Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of(More)