Dynamics of microtubule depolymerization in monocytes.

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.

Sputter shadowing improved by using a tungsten target.

This work builds upon a previous paper (W. Colquhoun, 1984, J. Ultrastruct. Res. 87, 97) in which a sputter shadowing device was briefly described. The device allowed TEM specimens to be shadowed in a conventional sputter coater. Images obtained by sputter shadowing with a standard Au/Pd target were of good quality but were slightly inferior to the best that could be obtained by e--beam evaporation of tungsten. Here we show that construction and use of a tungsten target greatly improves the quality of the sputter shadowed deposit. Images of DNA and ribosomal subunits contrasted by sputter shadowing with tungsten are shown. The DNA images indicate that sputter shadowing with tungsten is a gentle contrasting technique. The sputter shadowed images of the 30 S ribosomal subunits show the major features of the particle revealed by evaporation shadowing using the most sophisticated of methods in that technology. Advantages of sputter shadowing are discussed and a rationale for the improved grain obtained by sputtering tungsten is suggested.