Journal: The New Phytologist
Article Title: Metamorphosis of a unicellular green alga in the presence of acetate and a spatially structured three‐dimensional environment
doi: 10.1111/nph.20299
Figure Lengend Snippet: A spatially structured three‐dimensional environment (S3‐D) causes severe changes in the subcellular structures of Chlamydomonas reinhardtii regarding its cilia, cell size, eyespot and cell wall. (a–l) Algal cells were cultivated in the presence (+GB) or absence (−GB) of small glass beads. Cells were either grown mixotrophically (Tris‐Acetate‐Phosphate (TAP) medium in (a–c) and all parts in (d–l)) or photoautotrophically (minimal medium in (a–c)). (a–c) Chlamydomonas reinhardtii cilia are shorter in S3‐D compared to liquid culture in mixotrophically grown cells and their size is reduced. (a) Brightfield microscopy images of cilia (left panels) are presented as well as immunofluorescence images of cilia stained with anti‐acetylated tubulin antibodies (see the section) (right panels). (b) Ciliary length (c) Vertical cell length. (b, c, e) n = 3 independent biological replicates. In each biological replicate, at least 500 cells were measured for determination of cell size whereby the vertical length of the cell was considered. For the determination of the length of the cilia, 100 cells were used per replicate as well as for the calculation of the eyespot area, shown in (e). (b, c, e, h, i) Error bars indicate SDs. Asterisks indicate significant differences between liquid culture and S3‐D as calculated by Student's t ‐test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (d) Chlamydomonas reinhardtii eyespot in S3‐D compared to liquid culture. Representative brightfield microscopy images of the eyespot are shown. The eyespots are indicated by black arrows. (e) The C. reinhardtii eyespot area increases in S3‐D. (f, g) Electron microscopy (EM) images of the eyespot globules from cells in pure liquid (f) compared to S3‐D (g). White arrows point exemplarily to globules. (h) Significantly more algal cells remain intact after autolysin treatment (see Supporting Information Methods ) in S3‐D compared to pure liquid culture. The experiment represents an average of two independent batches, whereas each batch includes three biological replicates. n = 6 biological replicates. (i) A significant number of cells bear a double‐layered wall in S3‐D. Cells were prepared as described in Methods . n = 3 independent biological replicates. In each biological replicate, 1600 cells per sample were checked. (j) Algal cells with double‐layered walls (indicated by white arrows) are observed in S3‐D compared to liquid culture. The cell wall was stained in green with conA. Brightfield and SIM images were taken by the ZEISS Elyra 7 widefield and structured illumination microscope. See Methods for further details. (k) Exemplary algal cells with double‐layered walls in S3‐D. Cell walls were stained as mentioned in (j). Confocal microscopy images were taken by a Leica Stellaris 8 with or without overlay with the Chl channel assigned as a purple color map. (l) EM images of the cell wall area from cells in pure liquid compared to S3‐D. Arrows indicate visible layers. The outermost layer is speckled in S3‐D.
Article Snippet: We found a strong increase in cells that bear a double‐layered cell wall in S3‐D compared to the pure liquid culture (Fig. ), as evident from structured illumination microscopy (SIM) on a Zeiss Elyra 7 microscope (Fig. ).
Techniques: Microscopy, Immunofluorescence, Staining, Electron Microscopy, Confocal Microscopy
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