SUMMARY
Successful embryonic development is dependent on the female gamete progressing correctly through meiosis.
Assembly and positioning of the meiotic spindle is a crucial part of this process, with gene knockouts that
impair these processes causing female infertility. Oocyte spindle organization and positioning is orchestrated
by actin, involving actin-associated proteins in a cytoplasmic meshwork and in the oocyte cortex. Our
research on actin-associated proteins in oocytes has identified nexilin as involved in these events, with data
presented here showing that RNAi-mediated knockdown of nexilin results in meiotic arrest and aberrant
organization of oocyte actin. We also have evidence that loss of nexilin affects the actin regulatory pathway
involving the LIM-domain containing kinase (LIMK) and its substrate, the actin-depolymerization factor
cofilin. The LIMK-cofilin pathway affects the depolymerization of F-actin filaments to monomeric G-actin, and
thus this is a promising mechanism by which nexilin could impact actin-dependent processes. Nexilin is of
broader relevance as well, due to its role in dilated and hypertropic cardiomyopathies (DCM and HCM,
respectively). Thus, the impact of the research proposed here is wide-ranging, with relevance to reproduction,
oocyte biology, muscle function, and cardiomyopathies. With onset of DCM typically being in one's 40s-60s,
we hypothesize that a function-disrupting mutation in the NEXN gene could be a cause of female infertility
during reproductive years, and then of heart disease later in life. Given that little is known about nexilin, our
overall goal is to elucidate the functions of nexilin, its connection to the LIMK-cofilin pathway, and how nexilin
dysfunction contributes to abnormalities in mammalian oocytes. We will achieve these goals with following
Specific Aims. In Aim 1, we will build on our data from RNAi-mediated knockdown nexilin in oocytes, and
develop an oocyte-specific nexilin conditional knockout (cKO) model, to analyze the effects of loss of nexilin
activity in oocytes, in vivo and in vitro. Aim 2 will use state-of-the-art studies in cellular mechanics, live-cell
imaging, and quantitative analyses to elucidate the mechanisms underlying the defects in spindle organization
and translocation associated with nexilin deficiency. This aim will test the hypotheses that aberrant spindle
positioning associated with deficiencies in nexilin or the LIMK-cofilin pathway are attributed to (a) aberrant
tension for cortical anchoring for spindle pulling to the oocyte periphery, or (b) defects in actin-based
movement of the spindle in the oocyte cytoplasm. Aim 3 will investigate how mutated forms of nexilin affect
oocytes, eggs, and early embryos. This work will be an invaluable assessment of the severity of different
disease-associated forms, and also provide answers to the question of if a woman has one of these NEXN
mutations, what would the effects be on her oocytes? Overall, this project will shed light on a poorly
understood but significant health-relevant protein by elucidating nexilin functions in oocytes and in general. In
turn, this work will translate to understanding nexilin functions in cardiomyocytes and other cell types.