Primary and secondary chick heart fibroblasts: Fast and slow‐moving cells show no significant difference in microtubule dynamics

Abstract

Highly motile chick heart fibroblasts in primary culture (1 degree CHFs) gradually convert into much slower-moving secondary (2 degrees) cells. The polarized movement of the latter, but not the former, cell type has been found to be dependent on an intact microtubule (MT) network [Middleton et al., 1989, J. Cell Sci. 94:25-32]. To investigate the comparative stability of the MT networks of 1 degree s and 2 degrees s, turnover was investigated by microinjection of biotin-labeled brain tubulin to act as a reporter. MTs in both cell types were found to be very dynamic, with the MT networks effectively disassembled by about 30 min in 1 degree CHFs and 60 min in 2 degrees CHFs, with mainly MT fragments remaining beyond these times. All MTs and fragments were found to have turned over by 1 h in 1 degree CHFs and 80 min in 2 degrees s. Because 2 degrees CHFs were found to be on average six times larger than 1 degree s, the difference in MT turnover time was considered largely due to the size difference. For both 1 degree and 2 degrees cells, the more slowly turning over MTs were generally curly and perinuclear in distribution, resembling stable MTs in other systems, but they appeared significantly earlier in CHFs. However, no discrete subpopulations of slower turning over MTs were found to be associated with either the leading edges or the processes of either cell type. In addition, no major differences were identified in the patterns of modified alpha-tubulin along the MTs or of MT cold or drug stability. It is concluded that MTs do not have a direct structural or skeletal function in maintaining a polarized 2 degrees CHF cell shape, but rather play an ancillary role.

Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications

“…Nontyrosinatable and glycylated tubulins are thought to occur in the most stable microtubules; thus, the glycylated tubulin isoform could represent a marker for cold-stable microtubules. Whereas acetylated tubulin may contribute to stability, results from other cell types argue against this (Thompson et al 1984;Maruta et al 1986;Piperno et al 1987;Morales and Fifkova 1991;Brown and Warn 1993).…”

“…Cold-stable microtubules are found in many model systems used to study the cytoskeleton, including epithelial cells and fibroblasts (Brown and Warn 1993;Lieuvin et al 1994;Bershadsky et al 1979), flagella (L'Hernault and Rosenbaum 1985), axons (Jones et al 1980;Sahenk and Brady 1983;Black et al 1984;Brady et al 1984) and mitotic spindles (Brinkley and Cartwright 1975). The subset of cold-stable microtubules in supporting cells is labelled by antibodies to α-, nontyrosinatable, acetylated and glycylated tubulins, although staining is weaker and more patchy than in controls, but not by antibodies to detyrosinated and polyglutamylated tubulin.…”