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

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. ISSN 0960-9822.

Full text not available from this repository.

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.

Item Type: Article
Subjects: Q Science > Q Science (General) > Q0002 General
Q Science > QD Chemistry > QD0001 General
Q Science > QP Physiology > QP0501 Animal biochemistry
Divisions: Faculty of Social and Applied Sciences > School of Human and Life Sciences
Depositing User: Dr Kristofer Leslie
Date Deposited: 14 Jul 2016 09:16
Last Modified: 28 Oct 2016 08:52
URI: https://create.canterbury.ac.uk/id/eprint/14707

Actions (login required)

Update Item (CReaTE staff only) Update Item (CReaTE staff only)

Downloads

Downloads per month over past year

View more statistics

Share

Connect with us

Last edited: 29/06/2016 12:23:00