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Sachin Kotak
Assistant Professor
Ph. D. (University of Frankfurt, Germany)
Room No:SE-12,
Phone: +91 80 22932292
E-mail: sachin.kotak@mcbl.iisc.ernet.in

How cells divide (or undergo mitosis) has fascinated researchers for over a century. Proper positioning and elongation of the mitotic spindle during metaphase and in anaphase are critical processes for determining the correct placement of the cytokinetic furrow. These processes further ensure that the cell fate determinants are appropriately segregated in daughter cells during asymmetric cell division, including in thestem cell lineages. A plethora of evidence from different experimental model systems indicates that spindle positioning is often dictated by an evolutionarily conserved ternary complex (NuMA/LGN/Gαi1-3in Homo sapiens and LIN-5/GPR-1/2/Gα in Caenorhabditis elegans). This complex promotes the binding of the minus-end directed microtubule-dependent motor protein dynein at the cell cortex. Such cortically anchored dynein is thought to regulate spindle positioning by exerting a pulling force on theastral microtubules.We have demonstrated that the levels of cortical dynein must be tightly regulated for proper spindle positioning both in human cells and C. elegans embryos. However, despite our basic understanding of the key players involved in spindle positioning, the mechanisms that spatially and temporally regulate the cortical levels of the ternary complex and/or dynein, as cells progress through mitosis, are not well delineated. Our main research objective is to understand the mechanisms of spindle positioning and elongation using two evolutionarily divergent cellular model systems of human cells and C. elegans embryos. These objectives will be achieved by utilizing multifaceted cell biology, biochemistry, and genetics approaches.

Uncovering the role of the plasma membrane lipids in mitotic spindle behavior
Our recent work has revealed that cortical levels of NuMA/dynein augment as human cells transit from metaphase to anaphase, and that this increase is essential for spindle elongation during anaphase [Fig. 1]. Further, we have demonstrated that cortical dynein enrichment during anaphase does not require Gαi1-3 and LGN, but is rather solely dependent on NuMA. Importantly, a novel piece of data which we obtained using several biochemical and cell-biological tools, indicates that NuMA directly interacts with PtIns(4)P [PIP] and PtIns (4,5)P2 [PIP2] phosphoinositides and that the cortical localization of NuMA during anaphase is dependent upon these membrane lipids [Fig. 1]. Intriguingly, NuMA is also excluded from the equatorial region of the cell cortex during anaphase in a manner that depends on the centralspindlin complex CYK4/MKLP1 [Fig. 1]. A major goal of our research is to unravel the mechanisms by which phosphoinositide-based lipids affect spindle positioning in human cells and in C. elegansone-cell stage embryos. In this realm, we are interested in addressing,(a) How the plasma membrane localization of these phosphoinositides varies through mitotic progression;(b) How such lipids interact with, and thus influence the localization of the ternary complex component NuMAand possibly its homologue LIN-5 in a temporal and spatial manner and, (c) How and why the centralspindlin complex excludesNuMA from the cortical equatorial zone during anaphase.

Identifying novel modulators of spindle positioning
Because of its large size (50 μm) and amenability to experimental manipulations, the C. elegansone-cell stage embryo has proven to be extremely useful for investigating metazoan spindle positioning. In the C. elegans embryo, under the influence of its intrinsic polarity cues, the spindle is initially set up in the embryo center, but is displaced towards the posterior during metaphase and anaphase, resulting in an asymmetric first cell division. Similar to human cells, spindle positioning in the one-cell stage of C. elegans embryo is dependent on the ternary complex (LIN5/GPR-1/2/Gα) and dynein. However, despite our basic understanding of the spindle positioning mechanisms in the C. elegans embryo, the nature of the factors influencing the cortical localization, as well as the activity of the ternary complex and dynein have not been addressed in a systematic manner. In order to identify novel factors involved in spindle positioning, we wouldutilizecutting edge genetic and proteomic approachesin C. elegans embryos to advance our understanding in this fascinating process.

Furthermore, since aberrant spindle positioning has been linked with neurological diseases as well as tumorigenesis, our findings may lead to the development of novel diagnostic and therapeutic tools for regenerative medicine, neurology and oncology.

Selected Publications:
Kotak, S., Busso, C., and Gönczy, P. (2014).NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane. EMBO J.  33, 1815-1830.

Kotak S. and Gönczy, P. (2014). NuMA phosphorylation dictates dynein- dependent spindle positioning. Cell Cycle 13, 177-178.

Kotak, S., Busso, C., and Gönczy, P. (2013).NuMA phosphorylation by CDK1 couples mitotic progression with cortical dynein function. EMBO J. 32, 2517-2529.

Kotak, S. and Gönczy, P. (2013). Mechanisms of spindle positioning: cortical force generators in the limelight. CurrOpin Cell Biol. 25(6), 741-748.

Kotak, S., Busso, C., and Gönczy, P. (2012). Cortical dynein is critical for proper spindle positioning in human cells.  J Cell Biol 199, 97-110.