The energy-dependent transport of membranes on microtubules plays a fundamental role in the pathways of membrane biogenesis and recycling common to all animal cells. In polarized epithelia membrane traffic is specialized to allow for selective delivery of lipids and proteins to the two distinct plasma membrane domains. Pharmacological studies suggest that microtubule-based transport is required for delivery of protein to the apical surface, movement of materials through the endocytic pathway and transepithelial transport (transcytosis). The primary goal of the proposed research is to test directly whether these membranes undergo microtubule- based transport in vivo and in vitro. Vesicle motility will be visualized using video-enhanced fluorescence and differential interference contrast (Nomarski) microscopy. The following questions will be addressed: 1. How are microtubules arranged in epithelia? MDCK microtubules are clustered into bundles rather than emanating from a common focus. Is this true in Caco-2 and WIF12-1? Do membranes, microtubules and/or microtubule-based motors colocalize? 2. Do Golgi-derived vesicles undergo microtubule-based transport? Cells will be treated with BODIPY-ceramide to label the Golgi apparatus and trans-Golgi network. Membrane vesicles will be isolated and their movement will be assayed in vitro. Anti-motor antibodies will be introduced into live cells to determine effects on membrane traffic. 3. Do endocytic and transcytotic vesicles move on microtubules? Cells will be incubated with fluorescent endocytic markers to label the endocytic pathway; highly specific probes will be used to label membrane proteins destined for transcytosis. Transcytosis will be visualized directly. It will be determined whether isolated fluorescently-labelled membranes are transported on microtubules in vitro.
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