Figure 8.

MT nucleation along existing MT bundles. (A and B) Wild-type interphase cells (PT546). Imaging: single plane time-lapse wide field (5-s time interval). (A) The boxed area is shown at successive time intervals. A region of MT overlap (yellow arrowhead), or iMTOC, becomes small and invisible from the fifth image. GFP fluorescence appears (red arrowhead) and moves at a speed of 8.1 μm/min toward the iMTOC region, where motion stops. (B) Kymograph of a similar event. MT growth occurs (green arrowhead) while the MT moves to the iMTOC region (red winding line). Initial rapid motion (blue line) slows down as the new MT grows. A bundle catastrophe (yellow arrowhead) is followed by a catastrophe of the new MT, starting on the left (red arrowhead). (C) Nucleation in cells expressing alp4-GFP and mRFP-tubulin (PT574). Initially, two MTs grow out on the left and one on the right side of a bright iMTOC (yellow bracket). One left MTs has a catastrophe (green arrowhead), after which two dots of alp4-GFP demarcate the region of MT overlap. An MT is nucleated (white arrowhead) from a dim alp4-GFP dot (red arrowhead and red outlined area) that moves toward the iMTOC. The dot associates with the left side of the nucleated MT (yellow arrowhead). Imaging: single plane two-color confocal (6.4-s time interval). Bars, 5 μm.