Our procedures for """"""""deep-etch"""""""" EM will be modified in the coming period to include """"""""high-pressure slam-freezing"""""""", so that we can obtain new structural data on the molecular events that occur within the cytoplasm and membranes of virally-infected and virally-transformed whole cells. This will involve, first, completing the development of a new """"""""high pressure slammer"""""""" that we have invented (and making it available to the research community at large). Second, we will apply this new technology to the particularly vexing and challenging problem of how poxviruses are manufactured and disseminated by animal cells. Here we will use attenuated strains of Vaccinia virus (VV) that can be handled completely safely, to be provided by Dr. Bernard Moss of the NIH, who has generated a whole host of mutant VV's that will allow us to determine what phenotypic variations these mutations cause at the EM level, and thus to better understand the molecular mechanisms behind poxvirus infection. Third, we will exploit our new freezer's capacity to capture rapid membrane changes by imaging the dynamic """"""""invadopodia"""""""" of one type of cancer cell, the RSV-transformed BHK cell, which forms unique """"""""podosome rosettes"""""""" on its ventral surface that will serve as ideal 'test patterns' for us to identify transient physical changes in the organization of proteins and lipids (especially """"""""signaling rafts"""""""") the cell membrane, and to determine what membrane shape-changes are associated with these phase-dynamics. This and our next project will benefit from a further technical advance we have made, which permits us to freeze cells right through their coverslips and thus optimally image their ventral surfaces in the EM. In close work with Dr. Phyllis Hanson, who has developed a number of mutant ESCRT's and mutant AAA-ATPases that perturb MVB formation, we have used this technique to show that certain of her mutants redirect MVB budding to the cell surface, where we can easily 'capture' it in the EM. This promises to shed much light on the molecular mechanisms involved in various sorts of membrane budding, and to reveal the remarkable parallels between processes as seemingly different as multivesicular body formation and the budding of retro viruses from the cell surface.
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