The structure of microtubule ends during the elongation and shortening phases of dynamic instability examined by negative-stain electron microscopy
Creators: name: The University of North Carolina at Chapel Hill University Libraries
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 1990

description: Microtubules (MTs) are dynamic polymers that can exist in phases of elongation and rapid-shortening at steady-state. These phases have been observed in vitro and in living cells, and this property of MTs has been termed 'dynamic instability'. The purpose of this study was to use negative-stain electron microscopy (EM) to test if there are structural differences between the ends of MTs in the elongation and shortening phases, which could provide insight into the mechanisms of dynamic instability. MTs in the elongation phase were obtained by seeding either highly purified porcine brain tubulin (PC-tubulin) or tubulin containing microtubule-associated proteins (MTP), from isolated Tetrahymena axonemes. The results are that, in addition to intact cylindrical MTs, a significant fraction of the tubulin polymer in the elongation phase occurred as sheets of parallel protofilaments, as found in previous investigations with self-assembled MTs. Therefore, sheet formation is an intrinsic property of MT assembly that does not depend on the tubulin purity or the method of nucleation. Also, since sheets lack helical symmetry, at least a fraction of tubulin polymers seeded from axonemes did not assemble by helical addition of tubulin dimers to the ends, an assumption often made in mathematical models of dynamic instability. Sheets and intact MTs that were seeded from isolated axonemes, emanated both from the intact MT wall of the axoneme A-subfiber and from the incomplete wall of the B-subfiber. Therefore, axoneme seeds do not provide a homogeneous nucleation site for tubulin growth, or produce a homogeneous population of tubulin polymers under our conditions. Previous evidence has indicated that MT disassembly can occur by a segmental release of tubulin oligomers from the ends and at sites along the length of MTs. However, these studies were performed with MTP, and disassembly was induced by cold depolymerization. We examined MT shortening under conditions that closely represent shortening via dynamic instability, namely isothermal dilution at 37 degrees C of self-assembled MTs. This was compared with the morphology of cold-disassembled MTs. The cold-depolymerization of MTs composed of MTP showed rings and protofilament curls as previously observed using similar methods. Surprisingly, cold-depolymerization of MTs assembled from PC-tubulin induced not only shortening, but also the opening of a large fraction of MTs into sheets, suggesting that the MT lattice contains a cold-labile seam. Under conditions that mimic stochastic shortening, MTs were intact, closed cylinders with ends that were approximately blunt. Therefore, rapid shortening occurs at the ends of the MT, without a long-range disruption of the MT wall. In conclusion, MTs in the elongation phase can have highly irregular ends and need not elongate by a helical assembly process. Conversely, MTs in the shortening phase can have relatively blunt, even ends and can depolymerize in a relatively uniform fashion.