THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS

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  • March 19, 2019
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  • Leano, Jonathan
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle formation, with mutants displaying chromosome aggregation, aberrant monopolar spindle morphologies, and aneuploidy. How CLASP binds the microtubule lattice to regulate MT dynamics and facilitate proper spindle assembly remains unknown; however, it has been postulated that cryptic TOG domains underlie CLASP's ability to regulate MT dynamics. In this work, we report the crystal structure of the first cryptic TOG domain (TOG2) from human CLASP1, confirming the presence of a TOG array in CLASP. CLASP1 TOG2 displays a bent architecture at the tubulin-binding surface that contrasts with the flat tubulin-binding surface from XMAP215 family TOG domains. Mutating key tubulin-binding determinants along the tubulin-binding surface of TOG2 abrogated the ability of CLASP to 1) rescue mitotic bipolar spindle formation in Drosophila S2 cells 2) associate CLASP with the MT lattice, and 3) promote in vitro MT polymerization. These findings highlight the mechanistic use of a cryptic TOG domain in CLASP to facilitate bipolar spindle formation and MT polymerization. Determining the crystal structure of TOG1 and the second cryptic TOG-like domain (TOG3) is ongoing. Structural characterization of CLASP's array of TOG domains we shows that differential TOG domain architecture confers distinct functions for each TOG domain including MT lattice association, MT polymerization, and MT stabilization. In addition, CLASP's C-terminal domain (CTD) associates with the coiled-coil regions of various associating factors to recruit CLASP at specific cellular locations and is also a necessary component for CLASP dimerization. To determine the role of CLASP CTD in promoting dimerization and interacting with known CLASP-associating factors, we are structurally and biochemically characterizing the interaction between CLASP CTD and the coiled-coil (CC) domain of CLIP-170, a known CLASP-associating factor. CLASP CTD and CLIP-170 CC form a complex in SEC-MALS and ITC experiments. In addition, CLASP CTD alone exists as a monomer, suggesting that CLASP CTD is necessary, but not sufficient, for dimerization. Further analysis to structurally characterize the interaction between CLASP CTD and CLIP-170 CC is an ongoing goal for this thesis work.
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  • In Copyright
Advisor
  • Slep, Kevin
  • Ke, Hengming
  • Crews, Stephen
  • Lee, Andrew
  • Cheney, Richard
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2014
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  • Chapel Hill, NC
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  • There are no restrictions to this item.
Date uploaded
  • April 23, 2015

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