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. 2003 Dec;133(4):1926-34.
doi: 10.1104/pp.103.027367. Epub 2003 Nov 6.

Expression of Arabidopsis gamma-tubulin in fission yeast reveals conserved and novel functions of gamma-tubulin

Tetsuya Horio  1 , Berl R Oakley
Affiliations

Expression of Arabidopsis gamma-tubulin in fission yeast reveals conserved and novel functions of gamma-tubulin

Tetsuya Horio et al. Plant Physiol. 2003 Dec.

Abstract

gamma-Tubulin localizes to microtubule-organizing centers in animal and fungal cells where it is important for microtubule nucleation. Plant cells do not have morphologically defined microtubule organizing centers, however, and gamma-tubulin is distributed in small, discrete structures along microtubules. The great difference in distribution has prompted speculation that plant gamma-tubulins function differently from animal and fungal gamma-tubulins. We tested this possibility by expressing Arabidopsis gamma-tubulin in the fission yeast Schizosaccharomyces pombe. At high temperatures, the plant gamma-tubulin was able to bind to microtubule-organizing centers, nucleate microtubule assembly, and support the growth and replication of S. pombe cells lacking endogenous gamma-tubulin. However, the distribution of microtubules was abnormal as was cell morphology, and at low temperatures, cells were arrested in mitosis. These results reveal that Arabidopsis gamma-tubulin can carry out essential functions in S. pombe and is, thus, functionally conserved. The morphological abnormalities reveal that it cannot carry out some nonessential functions, however, and they underscore the importance of gamma-tubulin in morphogenesis of fission yeast cells and in maintaining normal interphase microtubule arrays.

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Figures

Figure 1.
Figure 1.
Schematic diagram of the plasmid-shuffling procedure.
Figure 2.
Figure 2.
Growth of Arabidopsis γ-tubulin-expressing strains. The strains indicated in A were streaked onto 0.5% [w/v] yeast extract and 3% [w/v] Glc (YE) medium and incubated at 36°C (B), 32°C (C), and 20°C (D). All strains carry a disruption of the chromosomal γ-tubulin gene. AH001 carries pCT134 (the wild-type S. pombe γ-tubulin gene on a multicopy vector). Strain AH125 [p(Sp-γ)], carries a plasmid (pTH1203) with the S. pombe γ-tubulin cDNA under control of the cytomegalovirus (CMV) promoter. Strain AH119 [p(Sp-γ), p(At-γ)], carries pCT134 and a plasmid (pTH1197) with the Arabidopsis γ-tubulin cDNA under control of the CMV promoter. Strain AH120 [p(At-γ)] carries pTH1197 only [p(At-γ)].
Figure 3.
Figure 3.
Expression of Arabidopsis γ-tubulin detected by western blotting and RT-PCR. Crude extracts of S. pombe strains, wild type 972 (lane 1), AH001 (lane 2), AH125 (lane 3), AH119 (lane 4), and AH120 (lane 5) were run on a SDS gel and probed with the anti-γ-tubulin antibody G9 (A) or anti-S. pombe γ-tubulin antibody G58 (B). Types of γ-tubulin genes carried by each strain are indicated on top of each lane. The positions of Arabidopsis γ-tubulin (At-γ) and S. pombe γ-tubulin (Sp-γ) are indicated by arrows. The lower bands detected by each antibody are presumably products of proteolytic degradation due to the overproduction of the γ-tubulin. Bars in the middle indicate the positions of Mr markers (94, 67, 43, and 20 kD from top to bottom). C, cDNA fragments amplified from mRNA of AH120 (lanes 1-3) or wild-type HM123 (lanes 4-6). Oligonucleotide primers corresponding to the coding sequence of S. pombe actin gene (lanes 1 and 4), the Arabidopsis γ-tubulin gene (lanes 2 and 5), or the S. pombe γ-tubulin gene (lanes 3 and 6) were used for amplification. Arrows on the left indicate migrated positions of size markers (2.3, 2.0, and 0.56 kb from top to bottom).
Figure 4.
Figure 4.
Aberrant cellular morphology observed in the Arabidopsis γ-tubulin-expressing cells. Phase contrast micrographs of the wild-type strain 972 (A), a strain expressing S. pombe γ-tubulin and Arabidopsis γ-tubulin AH119 [p(Sp-γ), p(At-γ)] (B), and strain AH120 (which expresses only Arabidopsis γ-tubulin) incubated at 36°C [p(At-γ) 36°C] (C) or 20°C [p(At-γ) 20°C] (D). Bar = 10 μm. The length and shape of the cells in each culture was determined (E).
Figure 5.
Figure 5.
Abnormal microtubule arrays observed in the Arabidopsis γ-tubulin-expressing cells at a permissive temperature. Microtubules of S. pombe wild type (A-C) and AH120 (D--L) were stained using the anti-α-tubulin antibody TAT1. Immunofluorescent staining of α-tubulin (A, D, G, J, and M), corresponding 4′6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) staining (B, E, H, K, and N), and phase contrast images (C, F, I, L, and O) are shown. In A through C, wild-type cells in mitosis (arrows) and postmitotic stage (arrowheads) are indicated. Cells with curved microtubules and a mitotic cell in which condensed chromosomes are left behind on the stretched spindle are indicated by arrowheads and an arrow in G and H, respectively. In M through O, an interphase cell with extensively stained microtubules along the outside edge of the cell and a few microtubules in other locations of cytoplasm is shown. Bar = 5 μm.
Figure 6.
Figure 6.
Arabidopsis γ-tubulin localizes to MTOCs. Immunofluorescent staining of Arabidopsis γ-tubulin-expressing strains unextracted (A and C) and partially extracted (B) cells and wild-type green fluorescent protein (GFP)-α-tubulin expressing cells (D). Localization of the γ-tubulin was detected by using G9 antibody (A, a, c, e, and g; B, a, d, and g; C, a, d, g, and j; and D, d, g, and j). GFP-α-tubulin was detected by anti-GFP antibodies (C, b, e, h, and k, and D, b, e, h, and k). Corresponding DAPI staining (A, b, d, f, and h; B, b, e, and h; C,c, f, i, and l; and D, c, f, i, and l) and phase contrast (B, c, f, and i,) images are shown. Positions of G9-stained bodies and corresponding locations in DAPI and microtubule panels are indicated by arrows. G9-stained bodies are SPBs except in Ag, Cg, and Dg, where they are cytoplasmic MTOCs. Bar = 5 μm.
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