Flow cytometry and morphometry of tobacco cells expressing the C-terminal domain of the clathrin heavy chain
Eicher, C. | Der, C. | Pfister, C. | Conreux, C. | Fromentin, J. | Leborgne-Castel, N. | Ochatt, Sergio
Transgenic Nicotiana tabacum cells constitutively expressing the C-terminal domain (hub domain) of clathrin heavy chain, as a dominant negative mutation for the onset of endocytosis, were examined. We first analyzed the morphometry parameters (length, width, surface area) of cells from Xanthi and BY-2 callus and suspensions and we calculated their shape coefficient. Cells expressing the hub domain tended to be more elongated than the wild-type cells. Then, we performed flow cytometry characterization after staining with the A–T specific dye 4,6-diamidino-2-phenylindole dihydrochloride to assess their relative DNA content, with propidium iodide to determine their genome size, and with chromomycin A3 to assess their G–C content. The A–T level was reduced in callus, but variable in cell suspensions. On the other hand, genome size was variable for callus and cell suspensions of both Xanthi and BY-2 cultivars. Xanthi cells being more affected than BY-2 cells in general, this prompted a detailed study with cell suspensions of Xanthi lines which showed that DNA content, genome size, nucleus and cell morphometry are correlated. These results suggest that disturbing clathrin process(es) impacts flow cytometry and morphometry traits of cells in culture.
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