Defining CLS-2 Function in Central Spindle Assembly in C. elegans Male Sperm Meiosis
By: Sebastian Gomez
Department: Biology
Faculty Advisor: Dr. Diana Chu
Male infertility is a serious concern for many Americans. To elucidate how infertility arises, we are using the Caenorhabditis elegans nematode to study unique molecular mechanisms across mitosis, oocyte meiosis, and male sperm meiosis that ensure proper chromosome segregation. In the case of mitosis and oocyte meiosis, a central spindle structure composed of microtubules assembles in the midzone and produces pushing/pulling forces between segregating chromosomes. The central spindle structure is initiated by the kinetochore protein, CLS-2. In the case of C. elegans male sperm meiosis, few microtubules are present in the midzone except those tied to the lagging unpaired X chromosome, a unique feature of male sperm meiosis. It is unknown whether the presence of the lagging unpaired X chromosome implies that there is a distinctive mechanism of chromosome segregation in male sperm meiosis. I hypothesize that CLS-2 does not play a significant role in central spindle assembly in male sperm meiosis. Instead, male sperm meiosis relies on the pulling forces of the kinetochore-connected microtubules rather than the push/pull forces generated by the central spindle. After conducting immunostaining experiments, I have discovered that in addition to CLS-2 localizing to kinetochores on DNA and centrosomes on the polar ends of a dividing sperm cell, CLS-2 also co-localizes with microtubules in the midzone region during anaphase I of male sperm meiosis which I believe is meant to stabilize the lagging X chromosome as it resolves to a polar end. We are investigating if this new localization pattern is consistent throughout the rest of male sperm meiosis. To observe the spatial/temporal relationship CLS-2 has with microtubules and DNA, I have created a new C. elegans strain that contains fluorescent markers on CLS-2, microtubules, and DNA. I have successfully depleted CLS-2 in live C. elegans males using the novel auxin-inducible degradation system and will be observing any differences in male sperm meiosis with respect to microtubules and DNA. Measuring rates of DNA movement will allow us to determine the role of CLS-2 in segregation dynamics. We also plan to observe whether removing the presence of the lagging X chromosome (like in the case of hermaphrodite sperm meiosis or tra-2 mutant worms) will have an influence on CLS-2 function. Understanding CLS-2 function can elucidate the molecular mechanisms required for forming healthy sperm.