Cytoplasmic dynein is a retrograde microtubule-based motor enzyme whose function is believed to be critical in a range of processes including organelle transport and cell division. In order to function effectively within the cell, the activity of cytoplasmic dynein must be tightly and specifically regulated. The long-term objectives of this proposal are to identify and describe in molecular detail the mechanisms which regulate cytoplasmic dynein. Specifically, the current proposal focuses on the hypothesis that the intracellular targeting of cytoplasmic dynein is mediated by binding to an activator complex, and that this complex activates retrograde organelle transport by the enzyme. Two components of this activator complex, p150-Glued and centractin, have already been characterized. The specific alms of this research are: to complete the characterization of the cytoplasmic dynein activator complex by identifying the remaining major subunit of the complex, and isolating and sequencing the cDNA which encodes this subunit. The inter-relationships of the subunits will be probed by chemical cross-linking and binding analyses. The subcellular distribution of the complex will be determined, and the basis of its localization probed by examining the interaction of the complex with microtubules and cytoplasmic dynein. These studies will be complemented by a simultaneous cellular approach, in which the role of the complex in vivo will be probed by creating specific mutations in cultured cells, using the techniques of anti sense inhibition and cellular transfection. Preliminary data indicates that antisense inhibition of the complex results in significant disruption of the Golgi apparatus; these effects will be further characterized. We will also examine the cellular effects of expression of specific site mutation and deletion constructs in transfection assays. The effects of the induced mutations will be studied in detail using immunocytochemistry as well as functional assays. These studies will be accompanied by a detailed analysis of the cellular and developmental defects of mutations at the Glued locus in Drosophila, by immuno-cytochemistry and genetic analyses. It is anticipated that applying a directed range of approaches to this problem will more quickly advance our knowledge of the regulation and targeting of cytoplasmic dynein with the cell. Both of the subunits of the activator complex which have already been characterized are highly conserved through vertebrate evolution. One of the subunits has been identified as a novel form of actin, with distinct biochemical properties. Mutations in a homologue of another of the subunits, p150-Glued, have been determined to cause neurological defects which are lethal in Drosophila. This is consistent with the hypothesis that this complex plays a key role in retrograde axonal transport in neurons, which is required for normal neuronal function. And finally, the hypothesis that cytoplasmic dynein is involved in mitosis makes it essential that we better understand both the function and the regulation of this cellular motor.
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