Molecular Organization and Function of Oocyte Spindles

An estimated 10-25% of human embryos contain incorrect chromosome numbers (aneuploidy), the leading cause of pregnancy loss and birth defects. Most critically, 25% of human oocytes are aneuploid, with this rate dramatically increasing after age 35, contributing to age-related fertility decline. The disproportionately high error rates in female versus male meiosis highlight fundamental gaps in understanding oocyte chromosome segregation. A critical knowledge gap exists in how oocytes assemble functional spindles without centrosomes—termed acentrosomal meiosis. While centrosomes organize microtubules in sperm meiosis, oocytes employ poorly understood alternative mechanisms. Additionally, although microtubules comprise diverse tubulin isotypes, their mechanistic significance in oocyte meiosis remains unexplored. Recent discoveries linking oocyte-specific tubulin TUBB8 mutations to human infertility disorders underscore the importance of understanding isotype-specific contributions to chromosome segregation. My goal is to elucidate the genetic and molecular mechanisms critical for acentrosomal meiosis and how tubulin isotype composition impacts chromosome segregation fidelity. My preliminary data using C. elegans β-tubulin isotypes TBB-1 and TBB-2 reveal these isotypes differentially affect spindle length, structural integrity of metaphase arrested spindles, microtubule motor sensitivity, and anaphase segregation velocity. I have remodeled existing tools, including a strain co- expressing endogenously tagged subunit of microtubule severing enzyme katanin (GFP::MEI-1) and mCherry::H2B, and single-β-tubulin substitution strains that also express GFP-tagged α-tubulin (GFP::TBA-2), providing unprecedented opportunities to dissect acentrosomal spindle assembly. I hypothesize that acentrosomal spindle assembly requires coordinated interactions between microtubule-interacting proteins and that such interactions are regulated by tubulin isotype composition. Using quantitative proteomics, forward genetic screens, in vitro biochemical analysis and high- resolution microscopy: Aim 1 investigates the molecular basis of differential β-tubulin isotype contributions through isotype-specific proteomics and biochemical characterization; Aim 2 identifies katanin-mediated mechanisms essential for acentrosomal spindle assembly using proximity labeling and genetic screening. This research aligns with my career objectives to become an independent investigator studying microtubule-dependent chromosome inheritance. The K99 phase will provide essential training in proteomics, biochemical reconstitution, and genetic screening—techniques underutilized in oocyte research. My mentoring team, led by Dr. Anne Villeneuve at Stanford, combines expertise in such essential techniques positioning me to master approaches that differentiate my research program. This project will establish the first comprehensive analysis of tubulin isotype-specific roles in any system and develop mechanisms enabling accurate chromosome segregation without centrosomes. The innovative methodological approaches will provide robust foundations for R01 applications, ultimately advancing diagnosis for reproductive health challenges affecting millions of families worldwide. Project Number: 1K99GM164664-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Emmanuel Nsamba | Institution: STANFORD UNIVERSITY, STANFORD, CA | Award Amount: $125,000 | Activity Code: K99 | Study Section: Special Emphasis Panel[ZRG1 EMS-S (90)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11352002