Publications by authors named "Geethu Emily Thomas"

5 Publications

  • Page 1 of 1

Kinetochore-microtubule interactions in chromosome segregation: lessons from yeast and mammalian cells.

Biochem J 2017 10 18;474(21):3559-3577. Epub 2017 Oct 18.

School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, CET Campus, Thiruvananthapuram, Kerala 695016, India

Chromosome congression and segregation require robust yet dynamic attachment of the kinetochore with the spindle microtubules. Force generated at the kinetochore-microtubule interface plays a vital role to drive the attachment, as it is required to move chromosomes and to provide signal to sense correct attachments. To understand the mechanisms underlying these processes, it is critical to describe how the force is generated and how the molecules at the kinetochore-microtubule interface are organized and assembled to withstand the force and respond to it. Research in the past few years or so has revealed interesting insights into the structural organization and architecture of kinetochore proteins that couple kinetochore attachment to the spindle microtubules. Interestingly, despite diversities in the molecular players and their modes of action, there appears to be architectural similarity of the kinetochore-coupling machines in lower to higher eukaryotes. The present review focuses on the most recent advances in understanding of the molecular and structural aspects of kinetochore-microtubule interaction based on the studies in yeast and vertebrate cells.
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http://dx.doi.org/10.1042/BCJ20170518DOI Listing
October 2017

Human SAS-6 C-Terminus Nucleates and Promotes Microtubule Assembly in Vitro by Binding to Microtubules.

Biochemistry 2015 Oct 7;54(41):6413-22. Epub 2015 Oct 7.

School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram , CET Campus, Thiruvananthapuram 695016, Kerala, India.

Centrioles are essential components of the animal centrosome and play crucial roles in the formation of cilia and flagella. They are cylindrical structures composed of nine triplet microtubules organized around a central cartwheel. Recent studies have identified spindle assembly abnormal protein SAS-6 as a critical component necessary for formation of the cartwheel. However, the molecular details of how the cartwheel participates in centriolar microtubule assembly have not been clearly understood. In this report, we show that the C-terminal tail (residues 470-657) of human SAS-6, HsSAS-6 C, the region that has been shown to extend toward the centriolar wall where the microtubule triplets are organized, nucleated and induced microtubule polymerization in vitro. The N-terminus (residues 1-166) of HsSAS-6, the domain known to be involved in formation of the central hub of the cartwheel, did not, however, exert any effect on microtubule polymerization. HsSAS-6 C bound to the microtubules and localized along the lengths of the microtubules in vitro. Microtubule pull-down and coimmunoprecipitation (Co-IP) experiments with S-phase synchronized HeLa cell lysates showed that the endogenous HsSAS-6 coprecipitated with the microtubules, and it mediated interaction with tubulin. Isothermal calorimetry titration and size exclusion chromatography showed that HsSAS-6 C bound to the αβ-tubulin dimer in vitro. The results demonstrate that HsSAS-6 possesses an intrinsic microtubule assembly promoting activity and further implicate that its outer exposed C-terminal tail may play critical roles in microtubule assembly and stabilizing microtubule attachment with the centriolar cartwheel.
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http://dx.doi.org/10.1021/acs.biochem.5b00978DOI Listing
October 2015

+TIP EB1 downregulates paclitaxel‑induced proliferation inhibition and apoptosis in breast cancer cells through inhibition of paclitaxel binding on microtubules.

Int J Oncol 2015 Jan 7;46(1):133-46. Epub 2014 Oct 7.

School of Biology, Indian Institute of Science Education and Research, CET Campus, Thiruvananthapuram 695016, Kerala, India.

Microtubule plus‑end‑binding protein (+TIP) EB1 has been shown to be upregulated in breast cancer cells and promote breast tumor growth in vivo. However, its effect on the cellular actions of microtubule‑targeted drugs in breast cancer cells has remained poorly understood. By using cellular and biochemical assays, we demonstrate that EB1 plays a critical role in regulating the sensitivity of breast cancer cells to anti‑microtubule drug, paclitaxel (PTX). Cell viability assays revealed that EB1 expression in the breast cancer cell lines correlated with the reduction of their sensitivity to PTX. Knockdown of EB1 by enzymatically‑prepared siRNA pools (esiRNAs) increased PTX‑induced cytotoxicity and sensitized cells to PTX‑induced apoptosis in three breast cancer cell lines, MCF‑7, MDA MB‑231 and T47D. Apoptosis was associated with activation of caspase‑9 and an increase in the cleavage of poly(ADP‑ribose) polymerase (PARP). p53 and Bax were upregulated and Bcl2 was downregulated in the EB1‑depleted PTX‑treated MCF‑7 cells, indicating that the apoptosis occurs via a p53‑dependent pathway. Following its upregulation, the nuclear accumulation of p53 and its association with cellular microtubules were increased. EB1 depletion increased PTX‑induced microtubule bundling in the interphase cells and induced formation of multiple spindle foci with abnormally elongated spindles in the mitotic MCF‑7 cells, indicating that loss of EB1 promotes PTX‑induced stabilization of microtubules. EB1 inhibited PTX‑induced microtubule polymerization and diminished PTX binding to microtubules in vitro, suggesting that it modulates the binding sites of PTX at the growing microtubule ends. Results demonstrate that EB1 downregulates inhibition of PTX‑induced proliferation and apoptosis in breast cancer cells through a mechanism in which it impairs PTX‑mediated stabilization of microtubule polymerization and inhibits PTX binding on microtubules.
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http://dx.doi.org/10.3892/ijo.2014.2701DOI Listing
January 2015

TACC3 protein regulates microtubule nucleation by affecting γ-tubulin ring complexes.

J Biol Chem 2014 Nov 22;289(46):31719-31735. Epub 2014 Sep 22.

School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, CET Campus, Thiruvananthapuram 695016, Kerala, India. Electronic address:

Centrosome-mediated microtubule nucleation is essential for spindle assembly during mitosis. Although γ-tubulin complexes have primarily been implicated in the nucleation process, details of the underlying mechanisms remain poorly understood. Here, we demonstrated that a member of the human transforming acidic coiled-coil (TACC) protein family, TACC3, plays a critical role in microtubule nucleation at the centrosome. In mitotic cells, TACC3 knockdown substantially affected the assembly of microtubules in the astral region and impaired microtubule nucleation at the centrosomes. The TACC3 depletion-induced mitotic phenotype was rescued by expression of the TACC3 C terminus predominantly consisting of the TACC domain, suggesting that the TACC domain plays an important role in microtubule assembly. Consistently, experiments with the recombinant TACC domain of TACC3 demonstrated that this domain possesses intrinsic microtubule nucleating activity. Co-immunoprecipitation and sedimentation experiments revealed that TACC3 mediates interactions with proteins of both the γ-tubulin ring complex (γ-TuRC) and the γ-tubulin small complex (γ-TuSC). Interestingly, TACC3 depletion resulted in reduced levels of γ-TuRC and increased levels of γ-TuSC, indicating that the assembly of γ-TuRC from γ-TuSC requires TACC3. Detailed analyses suggested that TACC3 facilitates the association of γ-TuSC-specific proteins with the proteins known to be involved in the assembly of γ-TuRC. Consistent with such a role for TACC3, the suppression of TACC3 disrupted localization of γ-TuRC proteins to the centrosome. Our findings reveal that TACC3 is involved in the regulation of microtubule nucleation at the centrosome and functions in the stabilization of the γ-tubulin ring complex assembly.
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http://dx.doi.org/10.1074/jbc.M114.575100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231652PMC
November 2014

Microtubule +TIP protein EB1 binds to GTP and undergoes dissociation from dimer to monomer on binding GTP.

Biochemistry 2014 Sep 21;53(34):5551-7. Epub 2014 Aug 21.

School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram , CET Campus, Thiruvananthapuram, Kerala 695016, India.

The +TIP protein EB1 autonomously tracks the growing plus end of microtubules and regulates plus-end dynamics. Previous studies have indicated that EB1 can recognize GTP-bound tubulin structures at the plus end, and it localizes on the microtubule surface at a site close to the exchangeable GTP-binding site of tubulin. Although the GTP-dependent structural change in tubulin has been demonstrated to be a critical determinant for recognition of plus ends by EB1, the effect of GTP on the structure of EB1 has remained unclear. Here, we have used spectroscopic, calorimetric, and biochemical methods to analyze the effect of GTP on EB1 in vitro. Isothermal titration calorimetry and tryptophan fluorescence quenching experiments demonstrated that EB1 binds to GTP with a dissociation constant ~30 μM. Circular dichroism measurements showed that EB1 undergoes changes in its secondary structure on binding GTP. Size-exclusion chromatography and urea-induced unfolding analyses revealed that GTP binding induces dissociation of the EB1 dimer to monomers. Size-exclusion chromatography followed by biochemical analysis further determined that EB1-GTP binding involves association of approximately one molecule of GTP per EB1 monomer. The results reveal a hitherto unknown GTP-dependent mechanism of dimer-to-monomer transition in EB1 and further implicate its possible role in regulating the stability of the EB1 dimer vs monomer as well as plus-end regulation in cells.
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http://dx.doi.org/10.1021/bi5007942DOI Listing
September 2014