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Caveate: a novel tool to understand the role of the midbody during cytokinesis


The successful completion of cytokinesis depends on the regulated assembly of the midbody, a compact structure formedtransiently in the thread-like cytoplasmic bridge between two daughter cells. Several proteins have been described which localize to the midbody, many of which are also observed at the centrosome, but a complete survey of its composition is lacking largely impart to its transient nature. A molecular description of this structure may however help understand its dynamic formation and role in cytokinesis.The host lab has identified and cloned a novel caveolin termed "Caveate" that in contrast to other caveolins is not membrane bound and has a diffuse localization in interphase but is recruited to the midbody during cytokinesis. The first part of the proposal aims to further characterize caveate biochemically and determine its binding partners as well as perform simultaneous imaging of YFP-caveate and CFP-microtubules/actin to investigate the dynamic assembly of the midbody. Interestingly however is the observation that GFP-caveate blocks cytokinesis at the midbody stage suggesting it has a possible role during final stage of cell separation. This will be investigated in cultured cells using RNA interference as well as through micro-injection of antibodies against caveate and any resulting phenotypes will be assessed. As GFP-caveate dominantly blocks midbody disassembly it provides the perfect reagent to "trap" this transient structure. Concurrently with the characterization of caveate we will use GFP-caveate as a tool to fluorescently label and enrich for midbodies to allow their purification for the first time. Midbody preparations will be subjected to two-dimensional gel electrophoresis and resulting proteins will be identified by mass spectrometry. Interesting candidate proteins will be studied further, particularly those with suspected links to cytoskeletal and membrane dynamics. These studies will include antibody production for immunofluorescence, protein interaction mapping, and GFP-tagging of the proteins for live-cell mic...


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