The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized.

To better understand the human Katanins and more broadly the mechanisms controlling mammalian microtubule-severing, we first analyzed the human Katanin interactome (Katan-ome) through biochemical tandem affinity purifications and mass proteomic analyses. This revealed that all Katanin subunits could reciprocally co-purify with each other, with the exception of KATNAL2 that only purified with KATNA1. These interactions were verified by in vitro and in cell reciprocal co-immunoprecipitations, with the exception of the KATNAL2-KATNA1 interaction. Analysis of the cell cycle subcellular localization of all Katanin subunits showed that KATNA1, KATNAL1 and KATNB1 localized to the cytoplasm during interphase and to the spindle poles during mitosis. Surprisingly, KATNBL1 localized to the nucleus during interphase (dependent on an N-terminal nuclear localization signal) and to the spindle poles during mitosis. In cell microtubule-severing assays with each A subunit revealed that unlike KATNA1 and KATNAL1, which showed microtubule-severing activity, KATNAL2 lacked detectable microtubule-severing activity. Interestingly, in vitro microtubule-severing assays showed that full-length KATNBL1 regulated KATNAL1 microtubule-severing activity in a concentration dependent manner. Furthermore, KATNB1 was able to compete the KATNBL1-KATNA1 and KATNBL1-KATNAL1 interactions.

These results indicate that KATNBL1 is a regulator of microtubule-severing activity and that it cooperates/competes with KATNB1 for binding to KATNA1 and KATNAL1 and that KATNB1 and KATNBL1 together regulate Katanin A subunit microtubule-severing activity.

Mitotic spindle assembly is a highly complex and specifically orchestrated event that occurs once per cell division. It relies on a multitude of protein complexes, protein-protein interactions, and regulatory mechanisms. To date, many proteins that associate with microtubules and function in mitotic spindle assembly have been identified and characterized. A strategy in the treatment of cancer has been to inhibit cell division with antimitotics, a group of natural and synthetic small molecules that arrest cells in mitosis, induce apoptosis, and cause cell death. My research focuses on finding potential cancer therapeutics by identifying novel antimitotic proteins, and by developing anti-leukemia small molecule drugs.

Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to regulating cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.

Targeting of the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug-screening efforts to identify novel anti-leukemia agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on FACS analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that relies on the use of chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of leukemia CCRF-CEM cells to each of 181,420 drug-like compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified a leukemia specific inhibitors (Leusin-1). Leusin-1 arrests cells in G2-phase and trigger an apoptotic cell death. More importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemia, non blood-born cancers, or normal cells. Leusin-1 represents novel leukemia specific inhibitors and could be used to develop new anti-leukemia therapies.

Mitotic cell division is a process requiring a highly coordinated dance between many enzymes, substrates and metabolites to result in the segregation of identical sets of daughter chromosomes. A hallmark of cancer is the ability to perturb this process in ways that increase the proliferation of cancer cells. We have studied several aspects of cellular division in order to further elucidate how cancer cell progression can occur in human disease. Mammalian cell division is a biological process that has been studied for decades and important discoveries often coincide with the development of noveltools and techniques. I have developed a new cell-based high-throughput screening tool that combines CRISPR/Cas9 technology with the Fluorescence Ubiquitin Cell Cycle Indicator system (FUCCI) for assessing the cell cycle effects of knocking out genes of interest. This tool provides a genome-encoded cell cycle phase indicator system and dox-inducible Cas9 for use with guide RNA libraries for future screens. Many of the proteins our lab studies were initially identified through either proteomic analysis or genomic screening. While these approaches have yielded interesting hits, they have focused exclusively on protein-based regulation of cell cycle progression and division. To this end we performed a high-throughput screen of 1,200 different naturally occurring metabolites in order to find novel affectors of the cell cycle and have identified 180 putative. These results will provide the basis for future projects analyzing these metabolites and their roles in cell cycle regulation. Ribosome biogenesis has long been linked to cell proliferation and in my studies, I characterized Rexo4, an exonuclease responsible for processing nascent ribosomal RNA. Recent studies suggest that Rexo4 is a biomarker for cancer disease and is seen to be upregulated in cancer cells at both the mRNA and protein level. My work has determined that Rexo4 is a requirement for cell cycle progression in mammalian cells and that both its nucleolar localization and exonuclease activity are required for cell proliferation.