Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable.

Abstract

Abstract.To study the possible mechanism of microtubule turnover in interphase cells, we have used the 266-nm wavelength of a short-pulsed Nd/YAG laser to transect microtubules in situ in PtK2 cells at predefined regions. The regrowth and shrinkage of the transected microtubules have been examined by staining the treated cells with antitubulin mAb at various time points after laser irradiation. The results demonstrate that microtubules grow back into the transected zones individually; neither simultaneous growth nor shrinkage of all microtubules has been observed. The half-time of replacement of laser-dissociated microtubules is observed to be '~10 min. On the other hand, exposure of the core of the microtubule, which is expected to consist almost completely of GDP-tubulin, by transecting the internal regions of the microtubule does not render the remaining polymer catastrophically disassembled, and most transected microtubules with free minus ends do not quickly disappear. Taken together, these results suggest that most microtubules in cultured interphase cells exhibit some properties of dynamic instability (individual regrowth or shrinkage); however, other factors in addition to the hydrolysis of GTP-tubulin need to be involved in modulating the dynamics and the stability of these cytoplasmic microtubules.M ICROTUBULES (MTs) t are one of the three major fibrillar systems of the cytoskeleton and play an important role in cell movement, determination of cell shape, organization of the internal architecture of the cell, and segregation of chromosomes in mitosis (10, 34). For a better understanding of these fundamental cellular processes it is essential to understand the mechanism of assembly of the MT polymer.MTs were initially postulated to be polymers in a simple equilibrium with the free tubulin subunits (18, 30). Subsequently, evidence indicated that this view was overly simple. Experimental as well as theoretical explorations of the role of nucleotide hydrolysis in assembly led to the treadmilling model which proposes that, at steady state, there may be a net tubulin addition at one end of the polymer and a net loss from the other end, resulting in a unidirectional flux of tubulin through MTs (9, 25; reviewed in reference 26). More recently, based on observations of individual MT behavior under conditions in which the free monomer concentration is equal to or below the steady-state concentration and on modeling of dynamics of theoretical MTs using hypothetical values for the rate constants, an alternative "dynamic instability" model was proposed. This model asserts that over a wide tubulin concentration range, a slow growing phase and rapid shrinking phase may coexist in a population of MTs;1. Abbreviation used in this paper: MT, microtubule. growing MTs are postulated to have tubulin subunits with bound GTP at the polymer ends, while the terminal subunits in shrinking MTs are thought to have bound GDP. The two phases interconvert stochastically and infrequently (15,16,23,27,28).At present, it appears tha...

Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

“…Based on our results, we can consider at least two mechanistic models to account for the force production required for poleward flux (Figure 9). Both of these views postulate a mechanism for increasing the lability of microtubule minus ends, which are typically quite stable both in vitro and in vivo (Inoue, 1964;Leslie and Pickett-Heaps, 1984;Tao et al, 1987;Walker et al, 1989;Wilson and Forer, 1988). Poleward flux requires that minus ends depolymerize at defined K.E.…”

Generation of noncentrosomal microtubule arrays

“…In fact, many studies show that newly formed minus ends are relatively stable. For example, generation of free minus ends by UV microbeam severing of MTs does not rapidly lead to depolymerization in vitro or in vivo (Tao et al, 1988;Walker et al, 1989). The minus ends might therefore persist for some time and only need additional factors for long-term stabilization.…”

La nanochirurgie laser en biologie cellulaire

“…[30]. La césure des microtubules par le laser [31] représente une catastrophe artificiellement induite qui ne modifie pas les principes de l'instabilité dynamique, comme nous l'avons récem-ment démontré (Figure 3). L'avantage de la nanochirurgie laser est de permettre d'induire cette catastrophe à n'importe quel endroit de la cellule, en contraste avec les études classiques uniquement possibles via l'utilisation de drogues (nocodazole) qui perturbent de manière globale l'organisation cellulaire, ou avec les études en microscopie à fluorescence possibles seulement dans des régions cellulaires limitées.…”