The research in the Varma lab is focused on understanding how accurate chromosome segregation is achieved during mitosis and how defects in this process leads to cancer initiation and propagation. A dividing cell assembles kinetochores and a mitotic spindle at the onset of mitosis. The kinetochores serve as sites where spindle microtubules come in physical contact with chromosomes, and are hence extremely important for accurate chromosome segregation. Improper kinetochore microtubule (kMT) attachments lead to erroneous chromosome segregation, chromosome loss, and inevitably aneuploidy, which is the leading cause of cancer in tissue cells, birth defects, and miscarriages during human embryonic development.
Over a decade of research had identified the kinetochore-bound Ndc80 complex as the key requirement for direct physical contact with spindle microtubules. However, what is still not understood well is how kinetochores and the Ndc80 complex remain stably attached to highly dynamic microtubule plus-ends during mitotic metaphase and subsequent chromosome segregation in anaphase. Work in yeast model system has provided us with important insights into the possible mechanism governing this process, but we still do not have a clear mechanistic picture in vertebrate systems. Work in our lab will thus focus on understanding the molecular mechanisms involved in controlling and regulating kMT attachments in vertebrate cells.
To this end, we are interested in understanding how the function of the Ndc80 complex is regulated for accomplishing proper attachments between kinetochores and microtubules. Our previous work has discovered the DNA replication licensing protein, Cdt1, to be required for robust kMT attachments in human cells in addition to its established role in DNA replication origin licensing. Cdt1 contributes to this process by binding to the loop region of the Ndc80 complex, but the precise nature of this interaction is unknown. Current work is directed towards understanding how Cdt1 and the loop domain of the Ndc80 complex coordinate in the formation of these robust kMT attachments using cell biological and biochemical approaches. We are also interested in understanding what other molecular players function with the loop domain and/or Cdt1 to contribute to this process. These studies are likely to lead to the discovery of novel molecular pathways for kMT attachments, the dysfunction of which results in chromosomal missegregation and aneuploidy.
We are also very interested to delineate the intricate mechanism that link kMT attachment formation with the activation and silencing of the spindle assembly checkpoint (SAC) which is also absolutely critical for accurate chromosome segregation. SAC is an essential surveillance mechanism at kinetochores that monitors the successful attachment between each individual kinetochore and spindle microtubule in a dividing cell before the onset of anaphase chromosome segregation. SAC is defective in cancer cells, hence promoting them to divide uncontrollably. Previous work using super-resolution microscopy has illustrated that at metaphase kinetochores exhibiting active SAC signaling, the proteins of SAC module are located within 5-10 nm of the KMN Network module, the essential components required for stable kMT attachments. Their adjacent location relative to one another might enable these modules to signal directly to each other during checkpoint silencing. Future work will focus on elucidating the molecular details of the association between SAC proteins and the KMN network using high-resolution microscopy and siRNA-mediated epistatic analysis.
Our recent studies have identified several mutants of Ndc80 and Cdt1 that produces severe defects in various stages of chromosome alignment and segregation. We are in the process of analyzing the behavior of these mutants in both normal diploid human cells and in various cancer cell models including breast, blood and prostate to understand how they contribute to cancer initiation and propagation. The level of many proteins involved in kMT attachments are altered in cancer cells which is likely to produce weaker or hyperstable attachments and contributing to chromosome mis-segregation. We are currently assessing how the control of gene expression of these proteins at the transcriptional and epigenetic levels control the accuracy of chromosome segregation to prevent tumor initiation. Imbalance in kMT attachments from opposite spindle poles could also lead to structural anomalies in chromosomes and we are probing this as a cause of genomic instability leading to cancer. Additionally, some of the spindle assembly checkpoint (SAC) proteins are mutated in cancer and we are examining the molecular mechanism of how these mutations could lead to chromosomal instability induced by aneuploidy during chromosome segregation.