Mechanisms and Regulation of Organelle Transport in Xenopus Melanophores

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Rogers, Stephen Leonard

Contributors dc:contributor

• Vladimir I. Gelfand

• Biology, Cell

Language dc:language

eng

Identifier

(MiAaPQ)AAI9955664

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/86756