Microtubule Release from the Centrosome T. J. Keating, J. G. Peloquin, V. I. Rodionov, D. Momcilovic, and G.G. Borisy PNAS, 94(10): 5078-5083, 1997

While microtubules (MTs) are generally thought to originate at the centrosome, a number of cell types have significant populations of MTs with no apparent centrosomal connection. The origin of these noncentrosomal MTs has been unclear. Here we demonstrate by direct observation in cultured cells that noncentrosomal MTs arise by constitutive nucleation at and release from the centrosome. The fate of MTs after release was also determined. When MTs were marked by laser photobleaching, it was determined that released MTs were transported with their plus ends leading, suggesting that this movement is driven by a minus-end directed motor. Released MTs were often seen to bend while they were transported, further supporting the idea that they were being acted upon by motor molecules. Released MTs were also dynamic. Plus ends of released MTs shortened, paused, or grew while the minus ends were stable or shortened. Microtubule release and transport are presumably needed to generate ordered noncentrosomal MT arrays in epithelial cells. Release of MTs contributes to polymer turnover by exposing MT minus ends, thereby providing additional sites for loss of subunits. The noncentrosomal population of MTs may reflect a steady-state of centrosomal nucleation, release, and dynamics.

	Figure 1 (44K) - Fluorescently labeled MTs in PtK1 cells.
	Figure 2 (105K) - Microtubule nucleation and release from the centrosome.
	Figure 3 (50K) - Marking of MTs by laser bleaching.

	Movie 1 (671k) - Covers 3.5 min beginning at 2.5 min after injection of fluorescent tubulin. This sequence includes the one case in which we saw breakage of a MT; this seems to have been caused by stress on the MT.
	Movie 2 (624k) - Covers 3.5 min beginning at 2.5 min after injection of fluorescent tubulin.
	Movie 3 (1547k) - Covers 13.5 min beginning at 4 min after injection of fluorescent tubulin. In this sequence one can see released MTs move away from the centrosome, out of the nuclear region, and continue to move about in the cytoplasm.
	Movie 4 (537k) - Covers 5.5 min beginning at 27.5 min after injection of fluorescent tubulin. A laser was used to create a reference mark on the labeled microtubules by photobleaching; the mark moves with the released MTs, indicating that the entire MT moves, rather than the plus end growing and the minus end shortening (i.e. treadmilling). This movement is presumably due to a minus end-directed motor molecule, such as cytoplasmic dynein, attached to the surface of the nucleus or other organelles.
	Movie 5 (1157k) - Covers 14 min beginning at 36 min after injection of fluorescent tubulin. Again, photobleaching was used to create a reference mark on the MTs. Besides the marked MTs, many unmarked MTs (which arose after laser bleaching) are seen to be released and move away from the centrosome.