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. Author manuscript; available in PMC: 2011 Aug 16.
Published in final edited form as: Dev Cell. 2010 Aug 17;19(2):245–258. doi: 10.1016/j.devcel.2010.07.016

Figure 4. Cls1p-GFP molecules bind non-uniformly along GMPCPP-stabilized MTs and recruit soluble tubulin dimers.

Figure 4

A) Top panel, TIRF experiment scheme to detect Cls1p-GFP binding to Texas-red GMPCPP MTs (red). Middle panel, TIRF image of 50 nM Cls1p-GFP (green) binding non-uniformly along GMPCPP MTs (red). Lower panel, kymographs of Cls1p-GFP (green, left) on GMPCPP-MT (red, middle) in isolated channels and overlaid (right). Non-uniform patches of densely bound Cls1p-GFP remain stationary along GMPCPP MTs.

B) Top panel, TIRF experiment scheme to detect untagged Cls1p molecules (grey) recruiting Alexa-Fluor-488 labeled tubulin (green) while bound to GMPCPP MTs (red). Middle panel, TIRF image of Alexa-Fluor-488 labeled tubulin dimers (green) recruited to the GMPCPP MTs (red) by bound untagged-Cls1p. Lower panel Kymographs of Alexa-Fluor-488 labeled tubulin dimers (green, left) recruited by untagged Cls1p along GMPCPP-MTs (red, middle) in isolated channels and overlaid (right).

C) Top panel, Schematic of TIRF experiment to simultaneously detect recruitment of Cls1p-GFP (green) and Texas-red tubulin (red) along non-labeled GMPCPP MTs (grey). Middle panel, TIRF image of 50 nM Cls1p-GFP (green) and 50 nM Texas red tubulin (red) binding non-uniformly along GMPCPP MTs (red). Inset panels II and I show raw images of Cls1-GFP and Texas-red tubulin bound along GMPCPP MTs in the isolated channels above their overlaid image (broken lines). Lower panel, Kymographs of GMPCPP-MT (red) in isolated channels and overlaid. Non-uniform patches of densely bound Cls1p-GFP remain stationary along GMPCPP MTs. Images in top panels were adapted from Brouhard et al (2008).

D) Tracking of Cls1p puncta along dynamic MTs to determine the average diffusion coefficient (see supplementary materials and methods).

E) Experimental images of 10 nM (upper panel) and 50 nM (lower panel) of Cls1p-GFP puncta bound along GMPCPP-MTs.

F) Simulation of Cls1p-GFP binding along linear MT lattices comparing different degrees of self-association. Panel 1: a random association with MT lattice without diffusion. Panel 2: weak self-association among molecules while binding to the MT lattice binding. Panel 3: shows moderate self-association among Cls1p-GFP molecules. Panel 4; a high degree of self-association. Note that the degree of speckling and sharpness of puncta in experimental Cls1p-GFP images is similar to simulations in either panels 1 or 2, and different from simulations in panels 3 and 4