Ice Recovery Assay for Detection of Golgi-Derived Microtubules

Abstract

Proper organization of the microtubule cytoskeleton is essential for many cellular processes including maintenance of Golgi organization and cell polarity. Traditionally, the centrosome is considered to be the major microtubule organizing center (MTOC) of the cell; however, microtubule nucleation can also occur through centrosome-independent mechanisms. Recently, the Golgi has been described as an additional, centrosome-independent, MTOC with distinct cellular functions. Golgi-derived microtubules contribute to the formation of an asymmetric microtubule network, control Golgi organization, and support polarized trafficking and directed migration in motile cells. In this chapter, we present an assay using recovery from ice treatment to evaluate the potential of the Golgi, or other MTOCs, to nucleate microtubules. This technique allows for clear separation of distinct MTOCs and observation of newly nucleated microtubules at these locations, which are normally obscured by the dense microtubule network present at steady-state conditions. This type of analysis is important for discovery and characterization of noncentrosomal MTOCs and, ultimately, understanding of their unique cellular functions.

Cep192 Controls the Balance of Centrosome and Non-Centrosomal Microtubules during Interphase

“…Unfortunately, this proved to be impossible owing to the severe fragmentation of the Golgi apparatus occurring when MTs are depolymerized in this cell line. However, this line of analysis was possible in RPE1 cells which maintain a relatively organized Golgi apparatus even in the total absence of MTs [18] . After 25 seconds of MT regrowth, Cep192 siRNA treated RPE1 Centrin-GFP cells displayed a 2.6-fold relative increase in the number of non-centrosomal MTs (neither end attached to the centrosome) with the majority of these having one end clearly associated with the Golgi apparatus ( Fig.…”

“…To quantify extra-centrosomal MT regrowth, RPE1 cells were plated on coverslips, depleted of siRNA for 72 hours and processed as described elsewhere, allowing 25 seconds of regrowth in 21°C media and 2 minutes in extraction buffer before fixation in methanol and immunostaining [18] , [19] . Extra-centrosomal MTs were counted by hand using ImageJ software.…”

Divergent microtubule assembly rates after short- versus long-term loss of end-modulating kinesins

“…The response of MTs to changes in the activity of regulators is influenced by the concentration of free assembly–competent tubulin, the concentration of MT ends, the nucleation capacity of the centrosome ( Zhou et al ., 2002 ), and possibly other regions of cellular MT assembly ( Chabin-Brion et al ., 2001 ; Grimaldi et al ., 2013 ). In live mammalian cells, it is extremely difficult, if not impossible, to measure the total number of MTs and thus the concentration of free MT ends.…”

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

“…Microtubules grow mainly from the centrosome. Relatively recently it was found that Golgi membranes are also directly involved in the nucleation and organization of cellular microtubules [72,73]. By gliding along micro tubules growing from them, Golgi membranes can con centrate in a sufficiently compact area within the cell even in the absence of a centrosome, e.g.…”

“…Microtubule nucleation at the Golgi membranes can be reproduced in vitro using isolat ed membranes and purified tubulin: under suitable condi tions the tubulin is polymerized into microtubules extending from the membrane [72]. If the microtubule system in the cells is destroyed by nocodazole and cool ing, then at early stages of recovery (nocodazole wash out and warming) the microtubules can be seen growing directly from vesicles containing Golgi markers [73]. Several proteins are involved in the process of γ tubulin recruitment to the Golgi: GMAP210 -golgin of cis Golgi [78]; CG NAP (AKAP450, yotiao) -a long fibril lar protein, acceptor of protein kinase A [79,80]; p115 [81]; myomegalin -a protein similar to AKAP450 [82]; CDK5RAP2 protein [83].…”