Abstract
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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Data availability statement
All original, raw data obtained are either included in the main body of the manuscript tables and figures, in the supplementary online resources provided with the submission, or are stored in the equipment and archives of the laboratories where the work was undertaken and available upon request to the authors.
Abbreviations
- BY-2:
-
Bright-Yellow 2
- CA3:
-
Chromomycin A3
- CCP:
-
Clathrin-Coated Pit
- CCV:
-
Clathrin-Coated Vesicle
- CHC:
-
Clathrin heavy chain
- CLC:
-
Clathrin light chain
- CME:
-
Clathrin-mediated endocytosis
- CS:
-
Cell suspension
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- PI:
-
Propidium iodide
- PM:
-
Plasma membrane
- PMT:
-
Photomultiplier
- SC:
-
Shape coefficient
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NLC and SO conceived and designed research. CE, CD, CP, CR, JF, NLC and SO conducted experiments. CE, CD, NLC and SO analyzed data. SO and NLC wrote the manuscript. All authors read and approved the manuscript.
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Supplementary Information
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Supplementary Figure S1.
Experimental design applied. CL, cell length; CSA, cell surface area; CSAD, cell surface area distribution; CW, cell width; FCM, flow cytometry assessments; MCA, metaphasic chromosome alignment; MI, mitotic index; NSA, nucleus surface area; NSAD, nucleus surface area distribution; SC, shape coefficient. (PPTX 43.8 kb)
Supplementary Figure S2.
Relative hub expression in leaves (a) and Xanthi and BY-2 cells (b and c) of trangenic lines. Expression values are means ± SEM relative to genes EF-1α (a, c ; n=5) or EF-1α and L25 (b) (n = 6). Bars with different letters indicate significant differences at P<0.05 between transgenic lines of a same cultivar. Controls not exhibiting any hub expression, letters were not assigned to them. (PPTX 51.7 kb)
Supplementary Figure S3.
Length (µm), width (µm) and surface area (µm²) of cells from callus of Xanthi (a,b) and BY-2 (c,d) tobacco lines and their corresponding representative images. Data are the mean ± SD from n = 15 to 30 cells per line and trait measured and from two independent measurements for all lines. Bars with different letters were significantly different (P<0.05). Scale bars = 100 μm. (PPTX 679.0 kb)
Supplementary Figure S4.
Length (µm), width (µm) and surface area (µm²) of cells from cell suspensions of Xanthi (a,b) and BY-2 (c,d) tobacco lines and their corresponding representative images. Data are the mean ± SD from n = 15 to 30 cells per line and trait measured and from two independent measurements for all lines. Bars with different letters were significantly different (P<0.05). Scale bars = 100 μm. (PPTX 1116.1 kb)
Supplementary Figure S5.
Surface area distribution (µm²) for cells from suspension cultures of lines Xanthi, hub 1.5 and hub 14.11. The scale bar in the micrographs corresponds to 50 μm. (PPTX 2793.4 kb)
Supplementary Figure S6.
Surface area distribution (µm²) for cells from suspension cultures of tobacco lines BY-2, B1A (a), CLC6 and C20 ( b), and mean ± SD of surface areas (µm²) of the various cell lines (c) that were all statistically similar (at P< 0.05). (PPTX 194.9 kb)
Supplementary Figure S7.
Nucleus changes in Hub Xanthi cells. (a) Measurement of metaphasic chromosome alignment in µm. Box plot from n = 41 (Xanthi) and n=23 (Hubs) measurements that were all statistically different (at P< 0.01), with representative images of metaphases below. (b) Image of interphasic nuclei with nucleoli in cells of lines Xanthi, Hub 1.5 and Hub 14.11. (PPTX 638.6 kb)
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Eicher, C., Der, C., Pfister, C. et al. Flow cytometry and morphometry of tobacco cells expressing the C-terminal domain of the clathrin heavy chain. Plant Cell Tiss Organ Cult 148, 247–258 (2022). https://doi.org/10.1007/s11240-021-02179-z
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DOI: https://doi.org/10.1007/s11240-021-02179-z