Determination of the functions of Rab32, Rab38, and their effector Myosin Vc in the biogenesis of melanosomes
Date
2013
Authors
Bultema, Jarred, author
Di Pietro, Santiago, advisor
Ross, Eric, committee member
DeLuca, Jennifer, committee member
Chen, Chaoping, committee member
Reddy, Anireddy, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
In mammals, pigment produced within specialized cells is responsible for skin, hair, and eye coloration. Melanocytes are specialized cells that produce pigment within an organelle known as the melanosome. Melanosomes are a member of a specialized class of organelles, known as Lysosome-related organelles (LRO), which are responsible for a number of critical functions in mammals such as pigmentation, blood clotting, lung function, and immune function. LROs are related to the ubiquitous lysosome, and are formed using the same molecular mechanisms as lysosomes that rely upon the Adaptor Protein complexes -1 (AP-1) and -3 (AP-3), and the Biogenesis of Lysosome-related Organelles Complex (BLOC)-2 (BLOC-2). These protein complexes are critical for the trafficking of specialized cargoes to melanosomes required for proper melanin synthesis. But, these complexes are also used for the formation of lysosomes, and no mechanism is known to distinguish between trafficking to lysosomes and melanosomes. The melanosome serves as a model system to study the formation of LROs, and insights from the study of melanosomes help explain the biogenesis of other LROs. In this dissertation, I present the finding that Rab32 and Rab38 function as melanosome-specific trafficking factors that allow for the use of AP-3, AP-1, and BLOC-2 in melanosome biogenesis. Using biochemical approaches, I show that Rab32 and Rab38 bind directly to AP-3, AP-1, and BLOC-2 on membranes. In microscopy experiments, I demonstrate that Rab32 and Rab38 localize to early endosomal subdomains where AP-3, AP-1, and BLOC-2 function. Using a combination of biochemical and microscopic approaches, I show that Rab32 and Rab38 serve partially redundant functions in trafficking of specialized cargoes to melanosomes. I report the discovery that Myosin Vc, a class V myosin motor, interacts with Rab32 and Rab38 and serves novel functions in melanosomes trafficking. I show, using biochemical approaches, that Myosin Vc directly binds to several melanosomal Rab proteins and serves as an effector of these proteins in melanosome biogenesis. Using a combination of approaches, I demonstrate that depletion of Myosin Vc from melanocyte cells causes defects in the trafficking of cargoes to melanosomes, but also causes severe defects in the secretion of mature melanosomes. With biochemical and microscopic approaches, I compare the function and localization of Myosin Vc in melanocytes to related proteins Myosin Va and Myosin Vb, and provide evidence to suggest that all three of these proteins function in distinct steps of melanosome trafficking. My results answer outstanding questions about the use of ubiquitous trafficking machinery (AP-3, AP-1, and BLOC-2) in trafficking to a specialized organelle. I provide evidence to answer outstanding questions about the mechanism of action of Rab32 and Rab38 in melanosome trafficking through my studies with Myosin Vc. I also establish new areas of research in the comparison of Myosin Va, Myosin Vb, and Myosin Vc in melanosome trafficking. My results address numerous unknown areas in melanosome biogenesis, expand the knowledge of melanosome biogenesis, and provide numerous new avenues of research to explore to understand specialized trafficking to LROs.
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Subject
Myosin Vc
Rab38
LRO
lysosome related organelle
melanosome
Rab32