Highlights

Understanding the mechanisms of spindle positioning and elongation in animal cell

Research

Cell division is an essential process for the maintenance of life, required for the development during embryogenesis and adult life forms. Eukaryotic cells segregate their genome and cellular constituents during mitosis. Cells undergo significant changes during mitosis, including genome condensation, nuclear envelope breakdown, and mitotic spindle formation for chromosome segregation. However, during mitotic exit, cells must reverse these processes by rebuilding the nuclear envelope, decondensing their genome, and restoring the genome's and nucleus's three-dimensional (3D) architecture to resume transcription. How cells precisely control and coordinate mitotic entry and exit programs is not well understood. The primary goal of our laboratory is to understand how many of these processes are spatiotemporally regulated using interdisciplinary cell biological, biochemical, and biophysical approaches. 

Mechanisms of spindle positioning

During mitosis, a diamond-shaped microtubule-based structure is responsible for the alignment of the mitotic spindle at a particular orientation. This precise alignment ultimately determines the future plane of cell division. The orchestration of the mitotic spindle's accurate orientation during metaphase hinges on an evolutionarily conserved membrane-anchored complex known as the ternary complex (NuMA/LGN/Gαi). This complex helps in localizing dynein/dynactin at the cell membrane. The dynein/dynactin motor's activity on the astral microtubules plays a pivotal role in generating cortical force, thereby facilitating the alignment of the mitotic spindle. Despite accumulating a wealth of knowledge over the years, the molecular intricacies governing the spatiotemporal dynamics of this complex during mitosis remain elusive. Our current research endeavors aim to delve into the intricacies of how the ternary complex and the cortical levels of dynein are precisely regulated in space and time during the process of mitosis.

Decoding the mechanisms of cytokinetic furrow formation

During cytokinesis initiation, protein complexes assemble at the equatorial membrane, forming a narrow RhoA zone that regulates cytokinetic furrow formation. Yet, the confinement of RhoA/actomyosin networks to this membrane remains enigmatic. Our recent study reveals that NuMA/dynein/dynactin complexes are polarly localized during anaphase, while RhoA regulators, Ect2 and centralspindlin, occupy the equatorial membrane, mutually excluding each other. Investigating the molecular basis of this membrane compartmentalization is paramount, as it orchestrates synchronized chromosome separation and furrow induction, offering insights into intricate cellular dynamics. Our ongoing research aims to dissect the molecular basis of this phenomenon, unraveling the intricacies orchestrating chromosome segregation with cytokinetic furrow initiation.

Mechanisms of genome decondensation and 3D chromatin organization

Post-mitosis, the crucial restoration of interphase nuclear and 3D genome organization is indispensable for transcription resumption. Yet, the intricacies linking chromatin decompaction to the re-establishment of interphase nuclear and genome organization remain elusive. Recent observations reveal a fascinating connection: the evolutionarily conserved protein NuMA, abundant in the mammalian nucleus, directly associates with DNA, aiding in genome decondensation at mitotic exit. However, the precise mechanisms employed by NuMA to promote genome decondensation remain unknown. Our research is geared towards unraveling this mystery as we endeavor to comprehend the orchestrated cellular dance that leads to genome decondensation, shedding light on NuMA's role in this intricate process.

Research highlights 

  • Exploring the mechanisms of nuclear envelope breakdown at mitotic entry
  • Dissecting the mechanisms of genome decondensation at mitotic exit
  • Investigating how cytokinetic furrow forms during mitosis
  • Studying the pathways of spindle assembly and spindle orientation

Group Member

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Aqreeb Ul Nisa Khan

Email : aqreeb@iisc.ac.in

Designation : phd_student

Category : Molecular and Cellular Biology

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Dwaipayan Chakrabarty

Email : dwaipayan@iisc.ac.in

Designation : phd_student

Category : Molecular and Cellular Biology

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Jyotsna Mahla

Email : jyotsnamahla@iisc.ac.in

Designation : Phd Student

Category : Molecular and Cellular Biology

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Kuheli Adhikary

Email : kuheli@iisc.ac.in

Designation : phd_student

Category : Molecular and Cellular Biology

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Madhumitha B

Email : madhumitha@iisc.ac.in

Designation : phd_student

Category : Molecular and Cellular Biology

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Manjula G S

Email : manjulags@iisc.ac.in

Designation : support_staff

Category : Molecular and Cellular Biology

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Monika Verma

Email : monikaverma@iisc.ac.in

Designation : phd_student

Category : Molecular and Cellular Biology

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Tanya Maisuriya

Email : tanyam@iisc.ac.in

Designation : Research Assistant

Category : Molecular and Cellular Biology

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Vignesh O

Email : vignesh@iisc.ac.in

Designation : Phd Student

Category : Molecular and Cellular Biology

Publications


2023

Kapoor, S., Adhikary, K. and Kotak, S.
PP2A-B55SUR-6 promotes nuclear envelope breakdown in C. elegans embryos, Cell Reports, 42(12)

DOI

Nuclear envelope (NE) disassembly during mitosis is critical to ensure faithful segregation of the genetic material. NE disassembly is a phosphorylation-dependent process wherein mitotic kinases hyper-phosphorylate lamina and nucleoporins to initiate nuclear envelope breakdown (NEBD). In this study, we uncover an unexpected role of the PP2A phosphatase B55SUR-6 in NEBD during the first embryonic division of Caenorhabditis elegans embryo. B55SUR-6 depletion delays NE permeabilization and stabilizes lamina and nucleoporins. As a result, the merging of parental genomes and chromosome segregation is impaired. NEBD defect upon B55SUR-6 depletion is not due to delayed mitotic onset or mislocalization of mitotic kinases. Importantly, we demonstrate that microtubule-dependent mechanical forces synergize with B55SUR6 for efficient NEBD. Finally, our data suggest that the lamin LMN-1 is likely a bona fide target of PP2A-B55SUR-6. These findings establish a model highlighting biochemical crosstalk between kinases, PP2A-B55SUR-6 phosphatase, and microtubule-generated mechanical forces in timely NE dissolution.


2022

Sana, S., Rajeevan, A, and Kotak, S.
Membrane compartmentalization of Ect2/Cyk4/Mklp1 and NuMA/dynein regulates cleavage furrow formation, Journal of Cell Biology, 221(12)

DOI

In animal cells, spindle elongation during anaphase is temporally coupled with cleavage furrow formation. Spindle elongation during anaphase is regulated by NuMA/dynein/dynactin complexes that occupy the polar region of the cell membrane and are excluded from the equatorial membrane. How NuMA/dynein/dynactin are excluded from the equatorial membrane and the biological significance of this exclusion remains unknown. Here, we show that the centralspindlin (Cyk4/Mklp1) and its interacting partner RhoGEF Ect2 are required for NuMA/dynein/dynactin exclusion from the equatorial cell membrane. The Ect2-based (Ect2/Cyk4/Mklp1) and NuMA-based (NuMA/dynein/dynactin) complexes occupy mutually exclusive membrane surfaces during anaphase. The equatorial membrane enrichment of Ect2-based complexes is essential for NuMA/dynein/dynactin exclusion and proper spindle elongation. Conversely, NuMA-based complexes at the polar region of the cell membrane ensure spatially confined localization of Ect2-based complexes and thus RhoA. Overall, our work establishes that membrane compartmentalization of NuMA-based and Ect2-based complexes at the two distinct cell surfaces restricts dynein/dynactin and RhoA for coordinating spindle elongation with cleavage furrow formation.


2020

Keshri, R., Rajeevan, A., and Kotak, S.
PP2A-B55 counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA, Journal of Cell Science, 133(14)

DOI

Proper orientation of the mitotic spindle is critical for accurate development and morphogenesis. In human cells, spindle orientation is regulated by the evolutionarily conserved protein NuMA, which interacts with dynein and enriches it at the cell cortex. Pulling forces generated by cortical dynein orient the mitotic spindle. Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) negatively regulates its cortical localization. Thus, only NuMA not phosphorylated at T2055 localizes at the cell cortex. However, the identity and the mechanism of action of the phosphatase complex involved in T2055 dephosphorylation remains elusive. Here, we characterized the PPP2CA-B55γ (PPP2R2C)–PPP2R1B complex that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle orientation. In vitro reconstitution experiments revealed that this complex is sufficient for T2055 dephosphorylation. Importantly, we identified polybasic residues in NuMA that are critical for T2055 dephosphorylation, and for maintaining appropriate cortical NuMA levels for accurate spindle elongation. Furthermore, we found that Cdk1- mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation at T2055 are reversible events. Altogether, this study uncovers a novel mechanism by which Cdk1 and its counteracting PP2A-B55γ complex orchestrate spatiotemporal levels of cortical force generators for flawless mitosis.

Rajeevan, A., Keshri, R., and Kotak, S.
NuMA interaction with chromatin is vital for proper chromosomes decondensation at the mitotic exit, Molecular Biology of the Cell, 31(0)

DOI

NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding–deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindleindependent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA


2019

Kapoor, S and Kotak, S.
Centrosome Aurora A regulates RhoGEF ECT-2 localization and ensures a single PAR-2 polarity axis in C. elegans embryos, Development, 146(0)

DOI

Proper establishment of cell polarity is essential for development. In the one-cell C. elegans embryo, a centrosome-localised signal provides spatial information for polarity establishment. It is hypothesised that this signal causes local inhibition of the cortical actomyosin network, and breaks symmetry to direct partitioning of the PAR proteins. However, the molecular nature of the centrosomal signal that triggers cortical anisotropy in the actomyosin network to promote polarity establishment remains elusive. Here, we discover that depletion of Aurora A kinase (AIR-1 in C. elegans) causes pronounced cortical contractions on the embryo surface, and this creates more than one PAR-2 polarity axis. This function of AIR-1 appears to be independent of its role in microtubule nucleation. Importantly, upon AIR-1 depletion, centrosome positioning becomes dispensable in dictating the PAR-2 axis. Moreover, we uncovered that a Rho GEF, ECT-2, acts downstream of AIR-1 in regulating contractility and PAR-2 localisation, and, notably, AIR-1 depletion influences ECT-2 cortical localisation. Overall, this study provides a novel insight into how an evolutionarily conserved centrosome Aurora A kinase inhibits promiscuous PAR-2 domain formation to ensure singularity in the polarity establishment axis.


2018

Sana, S*., Keshri, R*., Rajeevan, A., Kapoor, S., and Kotak, S.
Plk1 regulates spindle orientation by phosphorylating NuMA in human cells, Life Science Alliance, 0(0)

DOI

Proper orientation of the mitotic spindle defines the correct division plane and is essential for accurate cell division and development. In metazoans, an evolutionarily conserved complex comprising of NuMA/LGN/Gαi regulates proper orientation of the mitotic spindle by orchestrating cortical dynein levels during metaphase. However, the molecular mechanisms that modulate the spatiotemporal dynamics of this complex during mitosis remain elusive. Here, we report that acute inactivation of Polo-like kinase 1 (Plk1) during metaphase enriches cortical levels of dynein/NuMA/LGN and thus influences spindle orientation. We establish that this impact of Plk1 on cortical levels of dynein/NuMA/LGN is through NuMA, but not via dynein/LGN. Moreover, we reveal that Plk1 inhibition alters the dynamic behavior of NuMA at the cell cortex. We further show that Plk1 directly interacts and phosphorylates NuMA. Notably, NuMA-phosphorylation by Plk1 impacts its cortical localization, and this is needed for precise spindle orientation during metaphase. Overall, our finding connects spindle-pole pool of Plk1 with cortical NuMA and answers a long-standing puzzle about how spindle-pole Plk1 gradient dictates proper spindle orientation for error-free mitosis.

Alumni
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Ashwathi Rajeevan

ashwathir@alum.iisc.ac.in

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Riya Keshri

riyakeshri@alum.iisc.ac.in

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Shrividya Sana

shrividhyas@alum.iisc.ac.in

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Sukriti Kapoor

sukritis@alum.iisc.ac.in

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