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. Author manuscript; available in PMC: 2009 Jul 2.
Published in final edited form as: Dev Cell. 2007 Jun;12(6):917–930. doi: 10.1016/j.devcel.2007.04.002

Fig. 7. CLASPs dissolved in the cytosol support MT nucleation but not minus end anchoring.

Fig. 7

A-D. Mixed culture of GCC185-positive (pos) and GCC185-depleted (neg) cells 45″ after nocodazole washout. Boxes from B enlarged in C and D. GCC185 (green, A,C,D). Tubulin (red, B-D). GM130, (Blue B-D). C, In a control cell, MTs grow from the Golgi stacks. D, In GCC185-depleted cell, Golgi stacks (hollow arrows)are not associated with random single MTs (asterisks). E. In brefeldin A, GFP-CLASP2 (green) dissociates from the Golgi (7′) prior to Golgi-ER fusion (8′). Fusion is visualized by acute loss of mCherry-GT signal (red). Arrow hue illustrates presence of proteins. Time in brefeldin A is shown. F-H, In brefeldin A, CLASPs coat non-centrosomal MTs formed 45″ after nocodazole washout. F, tubulin. G, CLASPs. H, merge. Immunostaining. I, MTs formed in control (red), GCC185-depleted (GCCkd, blue) and Brefeldin A-treated (BFA, green) cells 45″ after nocodazole washout. For each set, 30 cells immunostained for tubulin were analyzed. J, Number of MTs nucleated singly and in groups after nocodazole washout with (BFA, green) and without (red) Brefeldin A. Live recordings of 7 mCherry-EB3-expressing cells for each set were analyzed. K-L. Non-centrosomal MTs in GFP-EB3-expressing RPE cells. Inverted images. Left, EB3 tracks within 3′. Right, enlarged inset video frames. Time after nocodazole removal is shown. K, In a control cell, MTs grow from distinct centers (arrows). L, In brefeldin A treated cell, minus ends of single MTs (asterisks) do not remain at the nucleation sites (arrows). M. Model for mechanisms of CLASP-dependent MT formation. γ-tubulin nucleates MT seeds in cytosol or at the Golgi. Cytoplasmic CLASPs associate with the MT plus end and support polymerization while minus end is unstable and depolymerizes resulting in MT treadmilling (top). CLASPs bound to TGN via GCC185 coat MT portion proximal to the minus end, anchor it to the membrane and prevent it's depolymerization, while CLASP-associated plus end steadily grows (middle). In the absence of CLASPs, MT seeds are unstable (bottom). GC, Golgi complex. N, Potential roles of Golgi-originated MTs in cell migration (model). Front-oriented MTs likely support post-Golgi vesicular trafficking (red), actin polymerization (blue) and focal adhesion turnover behind the leading edge (yellow).