We are working to understand the molecular mechanisms of chromosome movement during mitosis. Several lines of cell biological evidence show that spindle microtubules (MTs) move as the cell repositions its chromosomes, and because MTs are essential for mitosis, it is widely believed that MT movements cause chromosome motion. We have recently found that antibodies to cytoplasmic dynein bind to mitotic kinetochores, centrosomes and the MTs that connect them. The work proposed here will test the hypothesis that spindle dynein is a molecular motor for MT and chromosome motion. We will use fluorescence analogue cytochemistry and immuno-electron microscopy to extend our understanding of dynein's localization in the spindle. We will pursue four strategies to try to perturb dynein function in vivo and learn whether this enzyme is essential for some aspect of mitosis: (1) Antibodies to dynein heavy and intermediate chains will be microinjected into living cells and the subsequent mitosis studied by light and electron microscopy. (2) Dynein will be modified to alter its motor action, then injected into cells to look for a competition with endogenous enzyme that inhibit dynein's function. (3) The genes for dynein's heavy chain(s) will be cloned from Dictyostelium and HeLa cells. The resulting sequences will be used to transform Dictyostelium and disrupt its dynein genes by homologous recombination or to perturb dynein in mammalian cells either by transfection with antisense DNA or by the introduction of a poisonous gene product. (4) Similar experiments will be carried out, using dynein's conserved intermediate chain. Both genes and proteins will be used to study the binding of cytoplasmic dynein with objects that it moves (""""""""A-end binding""""""""), to help understand the control of dynein's action in vivo. Finally, we have recently developed a system for the study of chromosome motion on disassembling MTs in vitro. Our data demonstrate that chromosomes and other particles attached to the plus ends of MTs that are fixed to a slide will move in the absence of nucleotide triphosphate when the concentration of tubulin is reduced. The mechanism for this movement will be studied, and we will investigate the interplay between tension- compression and assembly-disassembly. Our model system will be used to investigate the roles of dynein in MT-chromosome attachment, and to see whether the enzyme is required for a particle to hold on to a MT as it disassembles.
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