Mechanisms and Regulation of Organelle Transport in Xenopus Melanophores
Rogers, Stephen Leonard
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Permalink
https://hdl.handle.net/2142/86756
Description
Title
Mechanisms and Regulation of Organelle Transport in Xenopus Melanophores
Author(s)
Rogers, Stephen Leonard
Issue Date
2000
Doctoral Committee Chair(s)
Vladimir I. Gelfand
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Cell
Language
eng
Abstract
Organelle transport in eukaryotic cells is a process essential to many cellular functions. Understanding the mechanisms that drive and regulate these movements is, therefore, an important goal for cell biology. We have addressed these issues by studying pigment granule motility in Xenopus melanophores. These dermal cells are able to cyclically transport pigment granules, termed melanosomes, either to aggregate to the cell center or disperse throughout the cell in response to hormonal stimulus to effect color changes in the animal. We developed protocols for the biochemical purification of melanosomes from an immortalized melanophore cell line. Isolated melanosomes possessed the motor proteins cytoplasmic dynein and kinesin-II and were able to move along microtubules in vitro. These motors retained their regulated state following purification. Melanosomes were also found to possess the actin-based motor myosin-V and to move on actin filaments in a cell-free system. Using Xenopus egg extracts arrested in interphase or metaphase, we also demonstrated that actin-based melanosome motility is regulated in a cell cycle-dependent manner. In metaphase extracts, motility was inhibited as compared to interphase. This inhibition correlated with the dissociation of myosin-V from melanosomes and was not due to inhibition of its motor activity. In addition, cytoplasmic dynein, but not kinesin-II, was also found to dissociate from melanosomes under these conditions. Myosin-V was also found to be hyperphosphorylated in metaphase extracts, suggesting a possible molecular mechanism contributing to its mitotic dissociation. In summary, this work demonstrates that microtubule- and actin-based organelle transport occurs in a coordinated manner within the cell and that this coordination may be due to parallel regulation.
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