The cytoskeleton is essential to many aspects of life, cell division and motility being prime examples. The overarching goal of this proposal is to understand the roles of two polarity factors, Spire (Spir) and Cappuccino (Capu), in regulating the cytoskeleton and establishing the body axes in early Drosophila development. Spir and Capu are distinct types of actin nucleators, factors that build new actin filaments de novo. Mutations in either gene cause female sterility due to polarity and cytoskeletal defects, which indicates that they are involved in the same biological pathway. The most notable cytoskeletal defect is the loss of an actin mesh that traverses the Drosophila oocyte up until a dynamic process called cytoplasmic streaming begins. Interestingly, the Capu homolog, Fmn-2, was recently found to build an essential actin structure in mammalian oocytes suggesting functional conservation of Capu and perhaps Spir. In the proposed work, we will test three models of how Spir and Capu build actin structures and establish polarity in Drosophila oocytes: 1) the co-nucleation model, in which Spir and Capu nucleate collaboratively to build the actin mesh;2) the synergy model, in which Spir is not a nucleator but enhances nucleation by Capu indirectly;3) the crosslinking model, in which Spir and Capu regulate streaming by crosslinking the actin and microtubule cytoskeletons, as opposed to nucleating a mesh. The first and second models will be distinguished by using fluorescence microscopy, electron microscopy and other biochemical approaches to study the effects of Spir on actin filament dynamics. These experiments will allow direct observation of actin filaments to determine whether Spir primarily nucleates new filaments, severs existing filaments or alters filament dynamics. The first and third models will be distinguished using knowledge gained from in vitro experiments to design rational mutations in Spir and Capu. The mutations will be introduced into Drosophila to determine which activities of these two proteins are essential for their activities in vivo. Additional experiments will be performed to determine how the activities of these proteins are controlled. Distinguishing between these models will lead to a mechanistic understanding of two proteins, conserved throughout metazoan species. It will advance our knowledge of the cytoskeleton and how it is controlled. Given the co-existence of this pair of proteins in polar cells, including neurons and epithelial cells in mammals, it has been proposed that their role as polarity factors in Drosophila is conserved. Thus we anticipate that what is learned about Spir and Capu in Drosophila oogenesis will be applicable to our understanding of the cytoskeleton, cell polarity, fertility, development and health in many animals, ranging from Drosophila to humans. ) )

Public Health Relevance

The cytoskeleton is essential to many aspects of life, cell division and motility being prime examples. We are studying the role of the cytoskeleton in establishing the major body axes, the failure of which leads to birth defects and infertility. By studying normal function and regulation of the cytoskeleton during early development we will gain understanding and tools that enable diagnosis and treatment of a broad spectrum of diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM096133-03
Application #
8537948
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Nie, Zhongzhen
Project Start
2011-09-01
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2013
Total Cost
$273,289
Indirect Cost
$89,939
Name
University of California Los Angeles
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L et al. (2015) Filament assembly by Spire: key residues and concerted actin binding. J Mol Biol 427:824-39
Roth-Johnson, Elizabeth A; Vizcarra, Christina L; Bois, Justin S et al. (2014) Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly. J Biol Chem 289:4395-404
Vizcarra, Christina L; Bor, Batbileg; Quinlan, Margot E (2014) The role of formin tails in actin nucleation, processive elongation, and filament bundling. J Biol Chem 289:30602-13
Quinlan, Margot E (2013) Direct interaction between two actin nucleators is required in Drosophila oogenesis. Development 140:4417-25
Chen, Christine K; Sawaya, Michael R; Phillips, Martin L et al. (2012) Multiple forms of Spire-actin complexes and their functional consequences. J Biol Chem 287:10684-92