Real-time observations of microtubule dynamic instability in living cells.

Abstract

Abstract. Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 ~tm/min + 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 Ixm/min + 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s -~ for catastrophe and 0.044 s -~ for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 × 35 ~tm microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations. T HE great majority of cellular microtubules are labile polymers that exist in an equilibrium with a cytoplasmic pool of tubulin subunits (11,23,27). Dynamic microtubule polymers are essential for the rapid rearrangements of the microtubule cytoskeleton which occur during the transitions between the interphase and mitosis portions of the cell cycle. In addition, dynamic remodeling of microtubules is important in cell morphogenesis and directed cell motility (5,6,33).Although several models have been proposed to explain the mechanism of microtubule assembly behavior, studies of highly purified tubulin have identified dynamic instability as the mechanism of microtubule assembly dynamics in vitro (4,9,13,15,16,24,36). In this model microtubules exist in one of two phases: they are either elongating or rapidly shortening, with rare, abrupt, and random transitions between these phases. The entire microtubule population behaves heterogeneously because these transitions occur randomly; at any given time most microtubules will be growing at a relatively slow rate while fewer microtubules will be shortening at a relatively faster rate (15, 16).Recently, Horio and Hotani (9), Walker et al. light microscopic methods. These real-time observations have provided essential information on the dynamic instability behavior of microtubules that could not be obtained from analysis of samples fixed at time points. Detailed analysis of microtubule assembly by Walker et al. (36) has led to the following model of the dynamic history of a microtubule in vitro undergoing dynamic instability: a microtubule is initiated at a...

Microtubule-Stabilizing Activity of Microtubule-Associated Proteins (MAPs) Is Due to Increase in Frequency of Rescue in Dynamic Instability: Shortening Length Decreases with Binding of MAPs onto Microtubules.

“…They play various important roles in cellular activity (7). Individual microtubules have been reported to show an unusual behavior called dynamic instability, both in living cells (5,32,33) and in cell-free system (ll, 12, 24, 25, 40). The term dynamic instability was coined to describe microtubule assembly in which individual microtubules showed alternating phases of growth and rapid shortening (25).…”

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

“…A hallmark of MTs is their dynamic nature, whereby individuals lengthen and shorten rapidly (Mitchison and Kirschner, 1984;Cassimeris et al, 1988), and hundreds may turn over within minutes (Saxton et al, 1984;Schulze and Kirschner, 1986;Sammak et al, 1987). The continual rearrangement of MTs (or MT ends) that arises as a direct consequence of rapid turnover may be particularly useful in accommodating the cell-shape changes associated with morphogenesis and cell motility (Kirschner and Mitchison, 1986;Sammak and Borisy, 1988b).…”

G Protein α Subunits Activate Tubulin GTPase and Modulate Microtubule Polymerization Dynamics

“…Microtubule ends can interconvert between slow elongation and rapid shortening, a process called dynamic instability, because of the presumed gain and loss of a small region of tubulin-liganded GTP at the microtubule end (3)(4)(5). Tubulin dimers bind 2 mol of GTP/mol of tubulin, one exchangeable (the E-site 1 in ␤-tubulin) and the other nonexchangeable (in ␣-tubulin).…”