Speaker: Abesh Bera, graduate student

Major: Microbiology

Major professor: GDCB Professor Mohan Gupta

Department: Genetics, Development and Cell Biology

Title: "Peer-to-peer regulation of cytoplasmic microtubules and alpha-tubulin isotype mediated regulation of interpolar microtubules facilitate anaphase during mitosis"

Abstract: Microtubules (MTs) are essential, intrinsically dynamic cytoskeletal filaments ubiquitously present in eukaryotes. MTs are polymerized from tubulin protein, which is a heterodimer composed of structurally similar a- and b-subunits. Many prokaryotes also harbor dynamic filaments polymerized from a tubulin ancestral homolog named FtsZ. Large numbers of MTs can work collaboratively to accomplish complex functions like cell division, cell migration, force generation and intracellular cargo transport, with a high degree of fidelity to ensure cell viability and genome stability. The collective behavior of MT populations is thought to largely result as emergent properties of the plethora of in vivo MT organizers and regulatory proteins. These include specific tubulin isotypes, MT-associated proteins (MAPs), tubulin isotype stoichiometry, tubulin post-translational modifications, and even inter-cytoskeletal cross-talk. Failure in one or more of these regulatory layers can significantly impair cell viability or health. Indeed, dysregulation of MT function or regulation is known to cause pathological defects in processes like mitosis/meiosis, oogenesis/oocyte maturation, and embryo/neurodevelopment. However, the mechanisms that coordinate the actions of populations of dynamic MTs to engineer complex cellular machines or how subsets of MTs faithfully cooperate within the overall cellular network remain largely unknown. 

For my research, I utilized Saccharomyces cerevisiae, a simple eukaryotic budding yeast model with high genetic tractability, fewer tubulin isotypes, no known tubulin post-translational modifications and easily trackable astral MTs that perform conserved functions during mitosis. Leveraging this powerful and convenient genetic model, I uncovered a novel “peer-to-peer” regulatory mechanism controlling MT occupancy and number inside the daughter cell, or bud, during anaphase spindle positioning. We further elucidated the physiological significance behind this unique regulation. Through quantitative fluorescence imaging of mitotic cells, we discovered that biasing the prevalence of only one MT inside the bud allows that MT to be specifically enriched with the spindle positioning MAP, Kip2: a plus end-directed processive kinesin motor, and aids in dynein trafficking to the plus-end in budding yeast. We further uncovered a previously unknown, anaphase-specific role of another conserved spindle positioning MAP, Kar9. We discovered Kar9 dynamically redistributes on MT plus ends in a spatially differential manner based on the net MT occupancy status of the bud - thus mediating the peer-to-peer regulation of multiple MTs between the mother and bud compartments during anaphase spindle positioning. 

While we showed cytoplasmic MTs are regulated in a novel, peer-to-peer fashion during anaphase spindle positioning, we discovered that within the nucleus, and specifically on interpolar MTs, each a-tubulin isotype, TUB1 and TUB3, differentially affects the function of the two essential and highly conserved, budding yeast orthologs of the tumor overexpressed genes (TOG)-containing proteins, STU1 and STU2, respectively. Utilizing strains expressing only one of the a-tubulin isotypes at levels comparable to total tubulin in normal cells, we demonstrate synergistic and epistatic genetic relationships, as well as localization biases, between the TOG proteins and the tubulin isotypes. Furthermore, we show that the TOG-containing protein, Stu2, confers relatively higher elongation rate and/or stability to spindles in Tub1-only cells prior to mitotic exit. 

Overall, my dissertation elucidates how cytoplasmic and nuclear MTs safeguard cells against erroneous spindle positioning and premature mitotic exit. Understanding these fundamental processes in a simple eukaryotic system like budding yeast will provide novel and valuable paradigms for how different subsets of MTs faithfully cooperate yet perform distinct tasks within the overall MT network. These insights are crucial to fully understand how MTs perform diverse and essential processes throughout eukaryotic biology.