Clathrin and pericentrin traffic along a shared route toward late-interphase centrosomes. (A) Asynchronous clone 3.3 cells were transiently transfected with GFP-pericentrin 48 h before labeling SNAP-uLCa with SNAP-TMR-Star. Cells were analyzed over 2 h by time-lapse confocal microscopy, taking 2D images every 10 min (37°C in 5% CO2 at 95% humidity). Colocalization (yellow in merged images) between SNAP-TMR-Star–labeled SNAP-uLCa and GFP-pericentrin occurs at centrosomes (arrowheads) and adjacent submicrometer vesicles (arrows). Top right corners of merged images show time interval in minutes. Bar, 10 µm. Images are 3D maximum projections. (B) Asynchronous clone 3.3 cells transiently transfected with GFP-pericentrin and labeled with SNAP-TMR-Star were imaged by confocal microscopy. Colocalization of pericentrin and SNAP-uLCa is shown (yellow in merged image) at two separated centrosomes of a late-interphase cell and overlayed with the brightfield image. Bar, 10 µm. Images are 2D. (C) The cell shown in B was magnified twofold and analyzed by time-lapse confocal microscopy for 2 min with 10-s imaging intervals. SNAP-uLCa localizes with GFP-pericentrin at the centrosome, and pericentriolar SNAP-uLCa vesicles move toward the centrosome (arrowheads). Top right corners of merged images show the time interval. Bars, 5 µm. (insets) Boxed regions are magnified threefold. Bars, 2 µm. Images are 3D maximal projections.
