Primary and secondary chick heart fibroblasts: fast and slow-moving cells show no significant difference in...

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.