FIGURE 4:

MT-encountering events between single or bundled MTs. (A) (a) Experimental setup. MT seeds are introduced in a flow chamber coated with silane-PEG-biotin on a glass coverslip and silane-PEG on the glass. MT seeds are attached by biotin–NeutrAvidin link to the glass coverslip. They are further elongated by the addition of Alexa 561–labeled tubulin in the absence or the presence of MAP65. (b) Angles as defined in the text. (B) Time lapses of single elongating MTs in the presence of GFP-MAP65-1 (50 nM) and Alexa 568–labeled tubulin (22 μM) that coalign after collision in order to generate a bundle. MTs are in red; MAP65-1 are in green. Arrowheads indicate the growing ends of colliding MTs that encounter resident MTs. (C, D) Histograms representing the frequencies of bundling events between two individual MTs (C) or two bundles (D) as a function of their angle of interaction and of MT polarity in the presence of 50 nM MAP65-1, Ase1, MAP65-4, and Chimera 1-4 (with range of 10°, from 0° to 90°). Encountering frequencies are the ratio of coaligned MTs over the total number of encountering events. Red and blue bars correspond, respectively, to interactions between antiparallel and parallel MTs.