Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells.
Curr Biol
; 26(13): 1728-1736, 2016 07 11.
Article
en En
| MEDLINE
| ID: mdl-27291054
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/ß-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 ß-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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tubulina (Proteína)
/
Proteínas Asociadas a Microtúbulos
Tipo de estudio:
Prognostic_studies
/
Risk_factors_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Curr Biol
Asunto de la revista:
BIOLOGIA
Año:
2016
Tipo del documento:
Article
País de afiliación:
Países Bajos