Showing papers by "Sandra L. Schmid published in 2004"
TL;DR: A key role for actin during clathrin-mediated endocytosis in mammalian cells is demonstrated, and stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathin-coated vesicle formation, including coated pit formation, constriction, and internalization.
Abstract: Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells
448 citations
TL;DR: Analysis of transferrin endocytosis rates in cells overexpressing mutant dynamins revealed a stronger correlation with both the basal and assembly-stimulated rates of GTP hydrolysis than with the calculated ratio of dynamin-GTP/free dynamin, suggesting that GTP binding is not sufficient, and GTP Hydrolysis is required for clathrin-mediated endocyTosis in vivo.
95 citations
Howard Hughes Medical Institute1, National Academy of Sciences2, National Institutes of Health3, Princeton University4, University of Texas Southwestern Medical Center5, Rockefeller University6, Harvard University7, Scripps Research Institute8, University of California, San Francisco9, Stanford University10
TL;DR: The concern that undeclared stocks of variola virus might exist and that they might be used as a bioterrorist weapon was heightened in late 2001 by the deliberate release of Bacillus anthracis, the agent of anthrax, in the weeks after the September 11, 2001, attacks as discussed by the authors.
Abstract: Smallpox, a devastating infectious disease dreaded throughout much of recorded history, is caused by the variola virus, a member of the poxviridae family. In the 20th century alone, smallpox deaths worldwide numbered in the millions. In 1980, after an intensive program of immunization with vaccinia virus, a related but relatively nonpathogenic virus, the World Health Organization (WHO) declared the disease eradicated. By 1983, all known stocks of variola virus were in two WHO collaborating centers: the U.S. Centers for Disease Control and Prevention (CDC) in Atlanta and (after a transfer in 1994) the Russian State Research Center of Virology and Biotechnology (the Vektor Institute) in Novosibirsk. The WHO Committee on Orthopoxvirus Infections voted on several occasions to recommend destruction of the stocks, but each time the decision was deferred to permit more research on live variola virus. A 1999 National Academies report summarized and assessed scientific needs for live variola virus (1).
The concern that undeclared stocks of variola virus might exist and that they might be used as a bioterrorist weapon (2) was heightened in late 2001 by the deliberate release of Bacillus anthracis , the agent of anthrax, in the weeks after the September 11, 2001, attacks. That concern prompted a voluntary national-preparedness effort to vaccinate healthcare workers, first responders, and members of the military against smallpox. However, given the substantial side effects, the risks associated with the smallpox vaccine, and the absence of information about an imminent bioterrorist attack, vaccination was not accepted by all members of those groups, nor was it recommended for the general public by the government (3).
Whatever the likelihood of covertly held variola virus stocks, an intentional release of the virus would pose a serious health threat and would probably provoke a …
85 citations
TL;DR: Overexpression of this assembly-incompetent mutant of dynamin 1 inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A, however, worm-like endocytic intermediates accumulated in cells expressing Dyn 1-I690K that were structurally distinct from long tubules that accumulated in Cells expressing Dyn2-K 44A.
Abstract: Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.
77 citations