Moving Microtubules in Vivo Model

Travis, Jeffrey

Abstract

The role of microtubules in human physiology and pathology is incompletely understood. It is clear microtubules are the structural elements of nerve cells along which axoplasmic transport occurs. Distribution of normal axoplasmic transport is thought to be the pathological basis of some neurological disorders. In fact, fast axoplasmic transport represents the physiological adaptation of the general cellular process of microtubule-associated intracellular transport. Recent studies have shown that microtubules dissociated from the axoplasm of the squid giant axon are capable of autonomous movement. It is likely that microtubule movements plays an important role in axoplasmic transport. However, to date there is no definitive evidence that axoplasmic microtubules actually move in situ. Indeed, there is no hard evidence that any cytoplasmic (i.e. non- ciliary or non-mitotic) microtubules are motile. The present application proposes experiments designed to test the hypothesis that microtubules actively translocate in the living cell. The investigator intends to use the pseudopodial extensions of Allogromia, a marine protist, as a paradigm for axoplasmic transport. The protocols described herein will generate short microtubules in optically favorable cell processes. The microtubules of Allogromia lamellipodia will be disassembled by a rapid cold block. Upon rewarming, the motility of the nascent microtubules will be recorded by digitally enhanced differential interference microscopy and analyzed. Simultaneous monitoring of the microtubule translocations and the change in polymer length will allow the principal investigator to distinguish microtubule assembly/disassembly from active translocations. This allow the quantitative analysis of microtubule motility in vivo, and an unambiguious test of the moving microtubule hypothesis.