Real-time observations of microtubule dynamic instability in living cells

Abstract

Individual microtubule dynamics were observed in real time in primary cultures of new 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 μ/min ± 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 μm/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-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 μm 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.