Sharyn Anne Endow
Email Address
gmssae@nus.edu.sg
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Publication A kinesin motor in a force-producing conformation(2010) Heuston, E; Bronner, C.E; Kull, F.J; Endow, S.A; DUKE-NUS MEDICAL SCHOOLBackground. Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. Results. Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central -sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. Conclusions. Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor. © 2010 Heuston et al; licensee BioMed Central Ltd.Publication Altered Nucleotide-Microtubule Coupling and Increased Mechanical Output by a Kinesin Mutant(2012) Liu H.-L.; Hallen M.A.; Endow S.A.; DEAN'S OFFICE (DUKE-NUS MEDICAL SCHOOL); DUKE-NUS MEDICAL SCHOOLKinesin motors hydrolyze ATP to produce force and do work in the cell - how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central ?-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central ?-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type - they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element - it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central ?-sheet, mediating free energy changes that lead to force production. © 2012 Liu et al.Publication Structural basis of small molecule ATPase inhibition of a human mitotic kinesin motor protein(Nature Publishing Group, 2017) Park, H.-W; Ma, Z; Zhu, H; Jiang, S; Robinson, R.C; Endow, S.A; DUKE-NUS MEDICAL SCHOOL; BIOCHEMISTRYKinesin microtubule motor proteins play essential roles in division, including attaching chromosomes to spindles and crosslinking microtubules for spindle assembly. Human kinesin-14 KIFC1 is unique in that cancer cells with amplified centrosomes are dependent on the motor for viable division because of its ability to cluster centrosomes and form bipolar spindles, but it is not required for division in almost all normal cells. Screens for small molecule inhibitors of KIFC1 have yielded several candidates for further development, but obtaining structural data to determine their sites of binding has been difficult. Here we compare a previously unreported KIFC1 crystal structure with new structures of two closely related kinesin-14 proteins, Ncd and KIFC3, to determine the potential binding site of a known KIFC1 ATPase inhibitor, AZ82. We analyze the previously identified kinesin inhibitor binding sites and identify features of AZ82 that favor binding to one of the sites, the ?4/?6 site. This selectivity can be explained by unique structural features of the KIFC1 ?4/?6 binding site. These features may help improve the drug-like properties of AZ82 and other specific KIFC1 inhibitors. © 2017 The Author(s).Publication Kinesins at a glance (Journal of Cell Science 123, (3420-3423))(2010) Endow, S.A.; Kull, F.J.; Liu, H.; DUKE-NUS MEDICAL SCHOOL[No abstract available]Publication Kinesins at a glance(2010) Endow, S.A; Kull, F.J; Liu, H; DUKE-NUS MEDICAL SCHOOL[No abstract available]Publication Arl2-and Msps-dependent microtubule growth governs asymmetric division(ROCKEFELLER UNIV PRESS, 2016-03-14) Chen, Keng; Koe, Chwee Tat; Xing, Zhanyuan Benny; Tian, Xiaolin; Rossi, Fabrizio; Wang, Cheng; Tang, Quan; Zong, Wenhui; Hong, Wan Jin; Taneja, Reshma; Yu, Fengwei; Gonzalez, Cayetano; Wu, Chunlai; Endow, Sharyn; Wang, Hongyan; Assoc Prof Reshma Taneja; PHYSIOLOGY; DUKE-NUS MEDICAL SCHOOL; BIOCHEMISTRY© 2016 Chen et al. Asymmetric division of neural stem cells is a fundamental strategy to balance their self-renewal and differentiation. It is long thought that microtubules are not essential for cell polarity in asymmetrically dividing Drosophila melanogaster neuroblasts (NBs; neural stem cells). Here, we show that Drosophila ADP ribosylation factor like-2 (Arl2) and Msps, a known microtubule-binding protein, control cell polarity and spindle orientation of NBs. Upon arl2 RNA intereference, Arl2-GDP expression, or arl2 deletions, microtubule abnormalities and asymmetric division defects were observed. Conversely, overactivation of Arl2 leads to microtubule overgrowth and depletion of NBs. Arl2 regulates microtubule growth and asymmetric division through localizing Msps to the centrosomes in NBs. Moreover, Arl2 regulates dynein function and in turn centrosomal localization of D-TACC and Msps. Arl2 physically associates with tubulin cofactors C, D, and E. Arl2 functions together with tubulin-binding cofactor D to control microtubule growth, Msps localization, and NB self-renewal. Therefore, Arl2-and Msps-dependent microtubule growth is a new paradigm regulating asymmetric division of neural stem cells.Publication Anastral spindle assembly and ?-tubulin in Drosophila oocytes(2011) Endow, S.A; Hallen, M.A; DUKE-NUS MEDICAL SCHOOLBackground: Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether ?-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for ?-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.Results: We show, for the first time, using a bright GFP fusion protein and live imaging, that the Drosophila maternally-expressed ?Tub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional ?Tub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of ?Tub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by ?Tub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding.Conclusions: The FRAP results, together with its mutant effects late in meiosis I, indicate that ?Tub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position ?-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division. © 2011 Endow and Hallen; licensee BioMed Central Ltd.Publication The kinesin-13 KLP10A motor regulates oocyte spindle length and affects EB1 binding without altering microtubule growth rates(2014) Do, K.K; Hoàng, K.L; Endow, S.A; DUKE-NUS MEDICAL SCHOOLKinesin-13 motors are unusual in that they do not walk along microtubules, but instead diffuse to the ends, where they remove tubulin dimers, regulating microtubule dynamics. Here we show that Drosophila kinesin-13 klp10A regulates oocyte meiosis I spindle length and is haplo-insufficient - KLP10A, reduced by RNAi or a loss-of-function P element insertion mutant, results in elongated and mispositioned oocyte spindles, and abnormal cortical microtubule asters and aggregates. KLP10A knockdown by RNAi does not significantly affect microtubule growth rates in oocyte spindles, but, unexpectedly, EB1 binding and unbinding are slowed, suggesting a previously unobserved role for kinesin-13 in mediating EB1 binding interactions with microtubules. Kinesin-13 may regulate spindle length both by disassembling subunits from microtubule ends and facilitating EB1 binding to plus ends. We also observe an increased number of paused microtubules in klp10A RNAi knockdown spindles, consistent with a reduced frequency of microtubule catastrophes. Overall, our findings indicate that reduced kinesin-13 decreases microtubule disassembly rates and affects EB1 interactions with microtubules, rather than altering microtubule growth rates, causing spindles to elongate and abnormal cortical microtubule asters and aggregates to form. © 2014, Company of Biologists Ltd. All rights reserved.