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Fig. 8. Localization of human GFP-p150^Glued in HeLa cells. (A) GFP-Hsp150^Glued localizes to MT plus-ends in transiently transfected HeLa cells. (B) At intermediate expression level, p150^Glued decorates the length of MTs as well as localizing to punctate structures that can be seen in time-lapse sequences to translocate along MTs. Bar, 10 µm; arrows in inset highlight two structures that translocate in the direction of the arrows. See supplementary movie 5. (C) Tracking of GFP-p150^Glued in low expressing cells. Time-lapse imaging (see supplementary material Movie 7, upper panel) reveals punctate structures that translocate in a non-linear manner through the cytoplasm. Tracks are shown on the movie sequence that follow three such structures (supplementary material Movie 6, lower panel); one of these is overlayed on the still images taken from this movie (red). The lower panel shows a maximum-intensity projection of all time points from this sequence shows the trajectories taken by these structures as they translocate through the cytoplasm. (D) Density-gradient centrifugation shows that GFP-Hsp150^Glued co-distributes across the gradient with endogenous Hsp150^Glued. GFP and CLIP-170 distribute away from this complex whereas p50^dynamitin co-distributes as expected, with p150^Glued. We consistently observe a population of uncomplexed p50^dynamitin for which we do not have a good explanation. (E) GFP-Hsp150^Glued puncta colocalize with ERGIC-53 (ER-Golgi transport intermediates, top panels), known to localize to dynamic structures, but not with COPII-coated (Sec24Cp-positive, lower panels) ER exit sites, which are mainly immobile. Arrowheads indicate colocalizing structures, arrows indicate structures that are only positive for GFP-Hsp150^Glued.

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