The assay details are as followed. It is an assay using fixation and immunostaining. Is it possible to use EGFP-tubulin to do it live?
MT regrowth assay
MTs were depolymerised on ice for 40 min or with nocodazole (10 uM) for 2 h. Regrowth was induced by incubation in prewarmed medium (37°C). For nocodazole washout assays, cells were rinsed five times with ice-cold medium and then moved to a dish with warm (37°C) medium as described (Efimov et al, 2007). All the MT regrowth experiments were carried out at room temperature.
In Efimov et al 2007, a few details are added:
For nocodazole washout experiments, free tubulin was removed prior to fixation by 40” incubation at 370C in extraction buffer (PIPES 60mM, HEPES 25mM, EGTA 10mM, MgCl2 2mM, Saponin 0.1% pH6.9, supplemented with nocodazole (0.25 nM) and taxol (0.25nM), modified from (Zhai and Borisy, 1994). Sheep polyclonal TGN46 antibody was from Serotec. Alexa568, Alexa488 and Alexa350-conjugated highly cross-absorbed goat-anti-mouse IgG antibodies and Alexa568 Alexa488-conjugated goat-anti-rabbit IgG antibodies and Alexa568-conjugated goat-anti-rat IgG antibodies (Molecular Probes) were used as secondary antibodies.
EMBO J. 2009 Apr 22;28(8):1016-28.
Microtubule nucleation at the cis-side of the Golgi apparatus requires AKAP450 and GM130.
Rivero S, Cardenas J, Bornens M, Rios RM.
Departamento de Señalización Celular, Centro Andaluz de Biología Molecular y Medicina Regenerativa, CABIMER-CSIC, Sevilla, Spain.
We report that microtubule (MT) nucleation at the Golgi apparatus requires AKAP450, a centrosomal gamma-TuRC-interacting protein that also forms a distinct network associated with the Golgi. Depletion of AKAP450 abolished MT nucleation at the Golgi, whereas depletion of the cis-Golgi protein GM130 led to the disorganisation of AKAP450 network and impairment of MT nucleation. Brefeldin-A treatment induced relocalisation of AKAP450 to ER exit sites and concomitant redistribution of MT nucleation capacity to the ER. AKAP450 specifically binds the cis-side of the Golgi in an MT-independent, GM130-dependent manner. Short AKAP450-dependent growing MTs are covered by CLASP2. Like for centrosome, dynein/dynactin complexes are necessary to anchor MTs growing from the Golgi. We further show that Golgi-associated AKAP450 has a role in cell migration rather than in cell polarisation of the centrosome-Golgi apparatus. We propose that the recruitment of AKAP450 on the Golgi membranes through GM130 allows centrosome-associated nucleating activity to extend to the Golgi, to control the assembly of subsets of MTs ensuring specific functions within the Golgi or for transporting specific cargos to the cell periphery.
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