Publications by authors named "K K Gireesh"

9 Publications

GTP-binding facilitates EB1 recruitment onto microtubules by relieving its auto-inhibition.

Sci Rep 2018 06 28;8(1):9792. Epub 2018 Jun 28.

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

Microtubule plus end-binding protein, EB1 is a key regulator of microtubule dynamics. Auto-inhibitory interaction in EB1 has previously been shown to inhibit its ability to bind to microtubules and regulate microtubule dynamics. However, the factors that promote its microtubule regulatory activity by over-coming the auto-inhibition are less known. Here, we show that GTP plays a critical role in promoting the microtubule-targeting activity of EB1 by suppressing its auto-inhibition. Our biophysical data demonstrate that GTP binds to EB1 at a distinct site in its conserved N-terminal domain. Detailed analyses reveal that GTP-binding suppresses the intra-molecular inhibitory interaction between the globular N-terminus and the C-terminal coiled-coil domain. We further show that mutation of the GTP-binding site residues in N-terminus weakens the affinity for GTP, but also for the C-terminus, indicating overlapping binding sites. Confocal imaging and biochemical analysis reveal that EB1 localization on the microtubules is significantly increased upon mutations of the GTP-binding site residues. The results demonstrate a unique role of GTP in facilitating EB1 interaction with the microtubules by relieving its intra-molecular inhibition. They also implicate that GTP-binding may regulate the functions of EB1 on the cellular microtubules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-28056-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023887PMC
June 2018

EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.

Nat Commun 2016 05 26;7:11665. Epub 2016 May 26.

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

Kinetochore couples chromosome movement to dynamic microtubules, a process that is fundamental to mitosis in all eukaryotes but poorly understood. In vertebrates, spindle-kinetochore-associated (Ska1-3) protein complex plays an important role in this process. However, the proteins that stabilize Ska-mediated kinetochore-microtubule attachment remain unknown. Here we show that microtubule plus-end tracking protein EB1 facilitates Ska localization on microtubules in vertebrate cells. EB1 depletion results in a significant reduction of Ska1 recruitment onto microtubules and defects in mitotic chromosome alignment, which is also reflected in computational modelling. Biochemical experiments reveal that EB1 interacts with Ska1, facilitates Ska1-microtubule attachment and together stabilizes microtubules. Structural studies reveal that EB1 either with Ska1 or Ska complex forms extended structures on microtubule lattice. Results indicate that EB1 promotes Ska association with K-fibres and facilitates kinetochore-microtubule attachment. They also implicate that in vertebrates, chromosome coupling to dynamic microtubules could be mediated through EB1-Ska extended structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms11665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894954PMC
May 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.575100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231652PMC
November 2014