Major cellular functions such as protein synthesis and folding, lipid synthesis, intracellular calcium storage, and nuclear transport of macromolecules all depend on the endoplasmic reticulum (ER). Failures in ER structure or function have been linked to a host of chronic diseases such as type-2 diabetes and autism, and neurodegenerative diseases such as hereditary spastic paraplegia (HSP) and Alzheimer's disease. During mitosis, ER morphology changes dramatically, a phenomenon necessary for proper membrane partitioning and nuclear membrane formation. Relatively little is known about how mitotic ER changes occur and how they are regulated, or which molecular mechanisms are responsible for these changes. Mitotic cyclin:Cdk kinase complexes are master regulators of nuclear and cytoskeletal dynamics during cell division, yet their involvement in mitotic ER reorganization is unknown. Additionally, since members of the Ras superfamily of small GTPases control changes during mitosis, they are likely to drive mitotic ER dynamics. Indeed, the Rab5 GTPase was recently found to have a role in mitotic ER structure independent of its endocytic role. Therefore, exploring how these proteins affect ER morphology is a key issue to be addressed. The goal of this project is to define the regulation of ER reorganization during mitosis by mitotic cyclin:Cdk and to define a molecular pathway that coordinates mitotic ER reorganization with Rab5 GTPase activity. These processes will be studied in detail in the embryo of the model organism, Drosophila melanogaster. The early Drosophila embryo is ideal for studying mitosis due to its rapid, abbreviated cell cycle, with initial rounds of mitosis occurring every ~15 minutes. Specifically, cell cycle progression or Rab5 activity will be modulated through established methods, and their effects on ER morphology assessed via timelapse confocal imaging of GFP-tagged ER markers.
The aims of the proposal are as follows: (1) Determine the role of mitotic cyclins on mitotic ER reorganization in the early Drosophila embryo. (2) Investigate how modulation of Rab5 activity affects mitotic ER reorganization.
This proposal has broad and significant relevance to major chronic human diseases like type-2 diabetes, autism, and Alzheimer's disease, due to the fundamental roles of the ER in cellular function. Completion of the proposed experiments would yield a basic molecular understanding of how ER morphology is regulated during the cell cycle. This particular knowledge would contribute to the development of therapies for diseases like hereditary spastic paraplegia.
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