Research Projects

Cell division is fundamental to the development and maintenance of all life forms. In eukaryotic cells, mitosis ensures accurate segregation of sister chromatids through the formation of a microtubule-based structure called the mitotic spindle. In human cells, spindle assembly is regulated by the conserved spindle pole protein NuMA (Nuclear Mitotic Apparatus). We recently demonstrated that the pole-localized Aurora A kinase dynamically regulates NuMA localization and its material properties. Inactivation of Aurora A leads to abnormal NuMA accumulation at the spindle poles and causes a shift in its state from dynamic to solid-like. Notably, this transition occurs in metaphase and anaphase phases, where global protein synthesis is largely suppressed, suggesting that a phosphatase antagonizes Aurora A phosphorylation of NuMA. We further showed that phosphorylation of NuMA at serine 1969 (S1969) by Aurora A is critical for maintaining its dynamic behavior. A phospho-deficient mutant (S1969A) mimics the effects of Aurora A inhibition, leading to non-dynamic NuMA accumulation at the poles. Therefore, objective 1 of this proposal is to identify and characterize the phosphatase complex responsible for counteracting Aurora A-mediated phosphorylation of NuMA at S1969. To accomplish this, we will 1) generate and validate a phospho-specific antibody against phosphorylated S1969 2) perform a candidate-based RNAi screen targeting mitotic phosphoprotein phosphatases (PPPs) to identify phosphatase complex involved in S1969 dephosphorylation and 3) biochemically characterize the identified PPP complex to understand how this phosphatase complex dephosphorylate NuMA at T2055, in other words, what are the molecular mechanisms. Following chromosome segregation, the nuclear envelope must reassemble to restore interphase nuclear architecture and support gene expression. NuMA returns to the nucleus post-mitosis and facilitates chromatin decondensation—a process that depends on its ability to bind DNA. We previously showed that DNA-binding deficient NuMA mutants form higher-order structures (puncta and fibrils) that deform nuclear shape. However, the molecular basis for the formation of these assemblies remains unknown. Thus, in the realm of objective 2, we will investigate the emergent properties of NuMA and understand how DNA binding suppresses its tendency to self-assemble. This will be addressed by 1) performing ultrastructural analysis of DNA-binding–deficient NuMA mutants using transmission electron microscopy (TEM) and 2) mapping domains in NuMA responsible for oligomerization and fibril formation by expressing domain-deletion and point-mutant constructs. This project combines advanced cell biological, biochemical, and biophysical approaches to investigate how NuMA coordinates mitotic spindle assembly and nuclear reorganization. Since Aurora A is frequently upregulated in many cancers, our findings will have broad implications for understanding mitotic dysregulation in tumorigenesis. Furthermore, the phospho-specific antibody against NuMA-S1969 will be a valuable diagnostic tool for assessing Aurora A activity in patients' tumor samples.