Determining the effects of chromatin-derived signals on dynein motor function during mitosis

Reynolds, Alice
In order to segregate chromosomes accurately during division, a cell must first assemble a bipolar mitotic spindle. This process is regulated in part by chromatin-mediated signaling cues that affect microtubule dynamics and microtubule motor function. The best characterized and arguably most important chromosome-mediated signaling pathway involves the small GTPase Ran and the nuclear receptor protein importin β (the Ran pathway). In these experiments we tested the hypothesis that the Ran pathway regulates the function of cytoplasmic dynein, a microtubule motor protein known to be critical for bipolar spindle assembly. We first investigated the possible interaction between importin β and dynein by adding recombinant, His-tagged importin β to mitotic cell-free extracts derived from Xenopus eggs. Ni-NTA agarose magnetic beads were then used to separate importin β and associated proteins from the rest of the extract. Western analysis confirmed the presence of dynein in the eluted fraction, suggesting that importin β interacts either directly with dynein or indirectly via dynein-associated proteins. Although more studies are needed to determine whether activation of the Ran pathway affects this interaction and/or dynein motor function, our preliminary data are the first to indicate a mitotic link between the Ran pathway and dynein.
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