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The plant Spc98p homologue colocalizes with {gamma}-tubulin at microtubule nucleation sites and is required for microtubule nucleation

Mathieu Erhardt1, Virginie Stoppin-Mellet1,*, Sarah Campagne1, Jean Canaday1, Jérôme Mutterer1, Tanja Fabian2, Margret Sauter1,2, Thierry Muller1, Christine Peter1, Anne-Marie Lambert1 and Anne-Catherine Schmit1,{ddagger}

1 Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique UPR 2357, Université Louis Pasteur, 12 rue du Général Zimmer F-67084, Strasbourg Cedex, France
2 Institut für Allgemeine Botanik, Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany
* Present address: Laboratoire de Physiologie Cellulaire Végétale, UMR 5019 CEA/CNRS/UJF, CEA Grenoble, 17 Avenue des Martyrs 38054 Grenoble cedex 9, France



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  		Fig. 1. Multiple sequence alignment of Spc98p homologues using CLUSTAL W (Thompson et al., 1994Go). Comparison of plant (Os, Oryza sativa and At, Arabidopsis thaliana) Spc98p, Hs, Homo sapiens GCP3/HsSpc98p and Sc, Saccharomyces cerevisiae Spc98p amino-acid sequences in the most conserved part of the protein. Identical and similar amino acids are shadowed, and the consensus sequence is shown on the bottom line.

 


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  		Fig. 2. Immunoblot analysis of tobacco BY-2 cell extracts. (A) Extracts from cells expressing the Spc98p-GFP fusion protein. Lane 1, anti-{alpha}-tubulin (1/5000); lane 2, anti-ß-tubulin (1/5000); lane 3, anti-{gamma}-tubulin (1/1000), which does not crossreact with {alpha}- or with ß-tubulin; lane 4, anti-GFP (1/2000) reveals one band at the expected size of the fusion protein; lane 5, anti-Spc98pB (1/1000) reveals the endogenous Spc98p (95 kDa, double arrow) as well as the fusion protein (125 kDa, arrow). (B) Lanes 6 to 8, affinity-purified antibodies directed against three Spc98 peptides (pA, pB and pC) were tested on control BY-2 extracts; best results were obtained using the pB peptide (DLDSIAKDYTSSLDA), a plant/animal consensus sequence. Antibodies directed against the C-terminal peptide pC did not crossreact with BY-2 extracts.

 


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  		Fig. 3. Plant Spc98p and {gamma}-tubulin localization at the surface of isolated tobacco BY-2 nuclei after in vitro MT nucleation. (A) Mid-section of a confocal z-stack labeled with anti-Spc98pB/Alexa 488. (B) Top section at the surface of the same nucleus labeled with anti-{alpha}-tubulin/Alexa 568. (C) Merged mid-section showing the perinuclear sites of MT nucleation. (D) Midsection of a nucleus revealed with anti-{gamma}-tubulin/Alexa 488. (E) Top-section at the surface of the same nucleus labeled with anti-{alpha}-tubulin/Alexa568. (F) Merged mid-section using a Zeiss LSM 510 microscope. Insets, Nomarski images of the isolated nuclei. (G) Inhibition of MT nucleation by preincubation with anti-{gamma}-tubulin antibodies. Merged projection of an entire nucleus. No MTs are polymerized. (H) Inhibition by anti-Spc98pB antibodies: a few short MT remnants are observed at the surface of the nucleus in this merged projection, but no MT nucleation was observed. (I) Following a competition assay for 1 hour with preincubation using anti-Spc98pB antibodies and Spc98pB peptide, nucleation was not inhibited. Bar, 10 µm.

 


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  		Fig. 4. In vitro MT nucleation activity on BY-2 nuclei in control and inhibition assays. (A) Without the antibody, the relative nucleation activity was 100%, and this was compared with the observed after incubation in the presence of anti-{gamma}-tubulin 1/200 (B1), 1/100 (B2), 1/50 (B3); anti-Spc98pA 1/200 (C1), 1/50 (C2); anti-Spc98pB 1/200 (D1), 1/50 for 20 minutes (D2), 40 minutes (D3), 60 minutes (D4); non-specific anti-Spc98pC 1/200 (E) and anti-GFP 1/200 (F). A competition assay incubating anti-Spc98pA 1/200 with the corresponding peptide 1µM for 1 hour before nucleation is shown (G). 70 to 95% inhibition of MT nucleation was observed using specific antibodies, indicating that both Spc98p and {gamma}-tubulin are directly involved in MT nucleation.

 


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  		Fig. 5. Distribution of plant Spc98p, {gamma}-tubulin and MTs in G2 tobacco BY-2 cells. Immunolocalization with (A) anti-Spc98p/Alexa 488 and (B) anti-{alpha}-tubulin/Alexa 568 in the same cell. In (A), a slightly oblique cut through the z-stack clearly shows the nucleus (n), vacuole (v) and surrounding cytoplasm. In (B), the projection of three confocal views shows the preprophase band in formation. Immunolocalization with (C) anti-{gamma}-tubulin/Alexa 488 and (D) anti-{alpha}-tubulin/Alexa 568 in the same cell. DIC image of the cell (D, inset). Both Spc98p and {gamma}-tubulin accumulate densely at the nuclear surface. Spc98p is not present along MTs, whereas {gamma}-tubulin is detected. Spc98p spots are also present inside the nucleus, suggesting import of the protein. Bar, 10 µm.

 


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  		Fig. 6. GFP and Spc98p-GFP fusion protein in live tobacco BY-2 cells observed in confocal microscopy. (A) GFP, detected by fluorescence, is diffuse in the cytoplasm and the nucleus. (B) DIC microscopy of the same cell. The Spc98p-GFP fusion protein (C) is detected on the nuclear surface and as regularly spaced cortical signals close to the plasma membrane (arrows). (D) DIC microscopy of the same cell. (E,F) Plasmolysis recovery in DIC microscopy. (G-L) The same cell expressing Spc98p-GFP fusion protein. The fluorescent signals observed using time-lapse microscopy in the cortical area detailed in E, close to the plasma membrane shows slight displacements. During recovery (J-L), the fluorescent signals moved according to the plasma membrane movements, indicating a close relationship. (Large dotted line, cell wall; small dotted line, edge of the plasma membrane). (M) Movements of both GFP dots labelled by an arrow and an arrowhead from G to L are drawn. (N,O) Microtubule immunolabeling in a cell expressing Spc98p-GFP. The reconstructed cross-section shows that GFP fluorescence localizes at microtubule bundle ends. n, nucleus. v, vacuole. Bar, 10 µm.

 


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  		Fig. 7. A working model of plant MT nucleation and organization. The soluble cytoplasmic {gamma}-tubulin/Spc98p-containing complexes are recruited to two different MT nucleation sites (arrows): the plasma membrane and the nuclear surface. Recruitment and activation of these soluble complexes would be responsible for nucleation of the cortical MTs involved in cell growth and for the nucleation of the perinuclear and preprophase band MTs, which determine polarity before mitosis. The activation of MT nucleation sites and coordinated regulation of the MT assembly would be controlled by cell cycle and/or developmental signals. Such signals would enter into cell cycle check points in higher plants.

 

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© The Company of Biologists Ltd 2002