Isolation of functional tubulin dimers and of tubulin-associated proteins from mammalian cells

Journal article


Yuill, N., Signorile, L., Basu, S., Ottema, S., Lebbink, J., Leslie, K., Smal, I., Dekkers, D., Demmers, J. and Galjart, N. 2016. Isolation of functional tubulin dimers and of tubulin-associated proteins from mammalian cells. Current Biology. 26 (13), pp. 1728-1736. https://doi.org/10.1016/j.cub.2016.04.069
AuthorsYuill, N., Signorile, L., Basu, S., Ottema, S., Lebbink, J., Leslie, K., Smal, I., Dekkers, D., Demmers, J. and Galjart, N.
Abstract

The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated by tubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cells and describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains [4], bind tubulin efficiently, as does the Golgi-associated protein GCC185, which interacts with the CLASPs [5]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms.

Year2016
JournalCurrent Biology
Journal citation26 (13), pp. 1728-1736
PublisherElsevier
ISSN0960-9822
Digital Object Identifier (DOI)https://doi.org/10.1016/j.cub.2016.04.069
Official URLhttps://doi.org/10.1016/j.cub.2016.04.069
Publication dates
Online09 Jun 2016
Print11 Jul 2016
Publication process dates
Deposited14 Jul 2016
Accepted12 Apr 2016
Output statusPublished
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https://repository.canterbury.ac.uk/item/87w77/isolation-of-functional-tubulin-dimers-and-of-tubulin-associated-proteins-from-mammalian-cells

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