Figure 1.

CLIP-170 comets serve as docking sites for p150^Glued vesicles in cells. (A) Deconvolved max projection STED image of microtubules in the neuronal growth cone with the cell border outlined in white (left). Schematic of the recruitment of dynein-dynactin organelles in the neuronal growth cone (right). (B) Schematic of p150^Glued (above) showing the CAP-Gly, basic (+), and coiled-coil domains. Schematic of CLIP-170 with relevant CAP-Gly, Ser rich, coiled-coil, and Zinc knuckle (Zn) domains. The phosphorylation sites S-309, -311, -313, -319, -320 and phosphomutant constructs are shown below. (C) Confocal slices of COS-7 cells co-transfected with HT-CLIP-A and either full-length myc-p150^Glued WT or G71R (top row). Upsampled images of individual comets show punctate p150^Glued associated with CLIP-A comets (below). The dashed white lines denote where line profiles were performed in D. (D) Line intensity profiles along upsampled images in C. (E) 3D-STORM super-resolution was used to image microtubules in fixed COS-7 cells (Movie S1). Shown are cropped microtubule plus ends from the two different conditions. CLIP-A (magenta) fully decorates the plus end as comet-like structures and discrete p150^Glued clusters associate with the CLIP-A comet (tubulin is unlabeled in these images). This shows that p150^Glued forms discrete punctate structures (white arrowhead) on CLIP-A comets; the morphology of these punctae are consistent with a vesicular population. In addition, there is a separate population of point-localized individual p150^Glued molecules (gray arrowhead). (F) Histogram of the maximum equatorial diameter for ellipses manually fit around clusters of p150^Glued WT puncta in STORM images (n = 133 puncta total from 2 cells, N= 1).