| dc.description.abstract | In animal cells, the duplicated genetic material is aligned on a microtubule-based structure known as the mitotic spindle during mitosis. At the mitotic exit, the mitotic spindle elongates, and the sister chromatids get separated. The separation of sister chromatids is followed by the cleavage furrow formation and its ingression, which eventually partition the cytoplasmic constituents and genetic material into newly formed daughter cells. How the chromosome separation is coordinated with cleavage furrow formation is incompletely understood. Also, when animal cells enter mitosis, the chromatin gets highly condensed, and the transcription is chiefly paused. However, when cells exit mitosis, the chromatin should get decondensed in a tightly regulated manner to ensure proper landscaping of chromosome territories, which makes it competent enough for DNA-based processes like replication and transcription. The accurate functioning of these processes is critical for the development and for stem cell divisions. In this study, we have linked the function of an evolutionarily conserved protein, nuclear mitotic apparatus (NuMA), in cleavage furrow formation and chromatin decondensation at the mitotic exit. In the first part of my thesis, we have tried to characterize the function of chromatin-localized NuMA in regulating chromatin decondensation. In the second part, we have attempted to provide insight into how spatial localization of NuMA at the plasma membrane coordinates chromosome separation with cleavage furrow formation.
1). NuMA regulates chromatin decondensation at the mitotic exit and nuclear shape in interphase cells
NuMA is a highly abundant (~10^6 copies) protein of interphase nuclei. Few studies hint that nuclear NuMA may have a role in chromatin organization, and it is hypothesized to be a part of the nuclear structural framework. In this regard, the loss of NuMA's function based on antibody-based microinjections was associated with nuclear shape defects. However, since the depletion of NuMA is linked with multiple mitotic abnormalities, it remained unclear whether the nuclear shape defects seen upon NuMA depletion is an indirect effect due to impairment of NuMA's mitotic function or a direct outcome of the absence of NuMA in the nucleus. Further, whether NuMA is bound to chromatin in the nucleus was also unknown. Even if NuMA is bound to chromatin, what mechanisms ensure its release upon mitotic entry was unknown. In this work, by utilizing fluorescence recovery after photobleaching (FRAP) and biochemical analysis, we report that NuMA is transiently bound to chromatin in the nucleus. We show that NuMA, which is bound to DNA, is released in late prophase upon nuclear envelope breakdown (NEBD) by the action of Cdk1-CyclinB kinase. Importantly, we identify evolutionarily conserved sequences rich in basic amino acids, arginine, and lysine, at the C-terminus of NuMA that aid in its direct interaction with DNA. In the absence of such interaction, NuMA becomes significantly mobile in the nucleus. Notably, the expression of the DNA-binding deficient mutant of NuMA delays chromatin decondensation at the mitotic exit. Furthermore, we discovered that DNA binding deficient NuMA polymerizes into high-order structures such as fibrillar networks, which perturbs nuclear shape. The DNA-binding property of NuMA prevents the formation of these higher-order structures and thus helps in maintaining the proper nuclear architecture. Overall, this study links the chromatin binding ability of NuMA with the proper chromatin decondensation at mitotic exit and maintenance of nuclear shape in interphase, independent of its mitotic role.
2). Polarized membrane distribution of NuMA/dynein and Ect2/Cyk4/Mklp1 regulate cleavage furrow formation
Animal cells partition their genetic material and cellular constituents through cytokinesis. The initiation of cytokinesis is regulated by the activation of small GTPase RhoA that helps in myosin II activation and actin polymerization at the equatorial membrane, resulting in cleavage furrow formation. RhoA is spatiotemporally regulated by a heterotetrameric complex known as centralspindlin consisting of a dimer of kinesin-6 member Mklp1 and a dimer of RhoGAP Cyk4. The centralspindlin complex localizes at the spindle midzone and promotes the localization of its downstream effectors RhoGEF Ect2 which directly activates RhoA and regulates cytokinesis. However, how a precise RhoA zone at the equatorial membrane is established and maintained remained unclear.
In anaphase, the mitotic protein NuMA is enriched at the polar membrane via its direct interaction with membrane phospholipids, PtIns(4)P and PtIns(4,5)P2 and is vital for proper spindle elongation by cortically anchoring the dynein/dynactin complex. However, despite the presence of PtIns(4)P and PtIns(4,5)P2 throughout the membrane, the NuMA/dynein complexes are restricted to the polar membrane and are excluded from the equatorial membrane, which is mutually exclusively occupied by RhoA. The mechanism of equatorial membrane exclusion of NuMA/dynein complex and its biological relevance remained unknown. In this work, we uncovered that Ect2, Cyk4, and Mklp1 are critical in restricting NuMA/dynein to the polar cortical region. In the absence of Ect2, Cyk4, or Mklp1, NuMA/dynein complex occupies the equatorial cortex, which impacts proper spindle elongation. Further, we show that Ect2 is in complex with Cyk4 and Mklp1 in anaphase cells. We establish that the membrane localization, but not the spindle midzone localization of the Ect2/Cyk4/Mklp1 complex, is critical for NuMA/dynein exclusion and, thus, for proper spindle elongation. Conversely, we show that polar membrane localization of the NuMA/dynein complex confines RhoA to a narrow zone at the equatorial membrane, which ensures cleavage furrow formation and cytokinesis. Overall our work provides insight into the mechanism that restricts NuMA/dynein and Ect2/Cyk4/Mklp1 to mutually exclusive membrane surfaces, which ensures proper chromatin segregation and cleavage furrow formation in animal cells. This coordination is critical for an error-free cell division program. | en_US |
