Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells.
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.
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands.
Nuo
N
Yu
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands.
Luca
L
Signorile
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands.
Sreya
S
Basu
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands.
Sophie
S
Ottema
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands; Department of Radiation Oncology, Erasmus Medical Center, Rotterdam, the Netherlands.
Joyce H G
JHG
Lebbink
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands.
Kris
K
Leslie
Biomedical Imaging Group Rotterdam, Erasmus Medical Center, 3015 GE, Rotterdam, the Netherlands.
Ihor
I
Smal
Proteomics Centre, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands.
Dick
D
Dekkers
Proteomics Centre, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands.
Jeroen
J
Demmers
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: n.galjart@erasmusmc.nl.
Niels
N
Galjart
D000818:Animals; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000378:metabolism; D004847:Epithelial Cells; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D014404:Tubulin