TY - JOUR
T1 - Structural analysis of the role of TPX2 in branching microtubule nucleation
AU - Alfaro-Aco, Raymundo
AU - Thawani, Akanksha
AU - Petry, Sabine
N1 - Funding Information:
We would like to thank David Octeau, Michael Rale, Ping Xu, and Matthew King for help with cloning, expression, and purification of various TPX2 constructs and with antibody preparation, Dr. Joshua Shaevitz for his advice on image analysis, and Dr. Simone Reber and Dr. Jay Gatlin for critical reading of the manuscript and helpful comments. This work was supported by the National Institutes of Health/National Institute of General Medical Sciences (4R00GM100013), the Pew Scholars Program in the Biomedical Sciences, the Sidney Kimmel Foundation, and the David and Lucile Packard Foundation (all to S. Petry), a Gilliam Fellowship for Advanced Study by the Howard Hughes Medical Institute (to R. Alfaro-Aco), and a Graduate Research Fellowship by the National Science Foundation (to R. Alfaro-Aco).
Publisher Copyright:
© 2017 Alfaro-Aco et al.
PY - 2017/4/1
Y1 - 2017/4/1
N2 - The mitotic spindle consists of microtubules (MTs), which are nucleated by the γ-tubulin ring complex (γ-TuRC). How the γ-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of Xenopus laevis TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2's ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified γ-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to γ-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate γ-TuRC to promote branching MT nucleation.
AB - The mitotic spindle consists of microtubules (MTs), which are nucleated by the γ-tubulin ring complex (γ-TuRC). How the γ-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of Xenopus laevis TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2's ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified γ-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to γ-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate γ-TuRC to promote branching MT nucleation.
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U2 - 10.1083/jcb.201607060
DO - 10.1083/jcb.201607060
M3 - Article
C2 - 28264915
AN - SCOPUS:85021849144
SN - 0021-9525
VL - 216
SP - 983
EP - 997
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 4
ER -