The WD40 repeat protein NEDD1 functions in microtubule organization during cell division in Arabidopsis thaliana - PubMed (original) (raw)
The WD40 repeat protein NEDD1 functions in microtubule organization during cell division in Arabidopsis thaliana
C J Tracy Zeng et al. Plant Cell. 2009 Apr.
Abstract
Although cells of flowering plants lack a structurally defined microtubule-organizing center like the centrosome, organization of the spindles and phragmoplasts in mitosis is known to involve the evolutionarily conserved gamma-tubulin complex. We have investigated the function of Arabidopsis thaliana NEDD1, a WD40 repeat protein related to the animal NEDD1/GCP-WD protein, which interacts with the gamma-tubulin complex. The NEDD1 protein decorates spindle microtubules (MTs) preferentially toward spindle poles and phragmoplast MTs toward their minus ends. A T-DNA insertional allele of the single NEDD1 gene was isolated and maintained in heterozygous sporophytes, and NEDD1's function in cell division was analyzed in haploid microspores produced by the heterozygote. In approximately half of the dividing microspores exhibiting aberrant MT organization, spindles were no longer restricted to the cell periphery and became abnormally elongated. After mitosis, MTs aggregated between reforming nuclei but failed to appear in a bipolar configuration. Consequently, defective microspores did not form a continuous cell plate, and two identical nuclei were produced with no differentiation into generative and vegetative cells. Our results support the notion that the plant NEDD1 homolog plays a critical role in MT organization during mitosis, and its function is likely linked to that of the gamma-tubulin complex.
Figures
Figure 1.
Arabidopsis NEDD1 Is Related to the NEDD1/GCP-WD Proteins in Animals. (A) Diagrammatic representation of the At NEDD1 protein. The six WD40 repeats toward the N terminus and the coiled-coil domain toward the C terminus are highlighted. (B) Phylogenetic relationship among At NEDD1, other plant NEDD1 homologs, and animal NEDD1/GCP-WD proteins. The plant proteins analyzed are from rice (Os NEDD1), the moss P. patens (Pp NEDD1), and the lycophyte S. moellendorffii (Sm NEDD1). The animal proteins include Hs GCP-WD, Mm NEDD1, Xl NEDD1, and Dgp71WD. The Arabidopsis WD40 protein COP1 is used as an outgroup in the phylogenetic analysis. Bootstrap values at the branches represent the percentages obtained in 1000 replications. Only values >50% are presented. The values presented in parentheses reflect identities and similarities, respectively, of the amino acid sequences when compared with At NEDD1. N.A., not applicable. (C) The native At NEDD1 protein is detected as a band with a molecular mass of 98 kD by immunoblotting. The molecular mass markers (kD) are shown on the right.
Figure 2.
Immunolocalization of Arabidopsis NEDD1 in Dividing Meristematic Cells. All cells were stained for NEDD1 (first column), MTs (second column), and DNA (third column). The merged color images have NEDD1 in green, MTs in red, and DNA in blue. Bar = 5 μm. (A) to (E) At prophase, NEDD1 exhibits a cap-like localization pattern in the future spindle poles on the nuclear envelope (arrowheads). (E) to (H) At late prophase, NEDD1 is prominently present in the poles of the prophase spindle. (I) to (L) At metaphase, NEDD1 appears along the kinetochore MT fibers. (M) to (P) At anaphase, NEDD1 colocalizes with shortening kinetochore fibers (arrowheads), but not obviously with interzonal MTs. (Q) to (T) NEDD1 is detected in the phragmoplast, with the signal more pronounced toward the minus ends, as indicated by a wider gap of anti-AtNEDD1 fluorescence (arrows) than that of antitubulin.
Figure 3.
Colocalization of Arabidopsis NEDD1 with γ-Tubulin. In a late anaphase cell, the NEDD1 signal (A) appears to overlap with that of γ-tubulin (B). In the merged image (C), the yellow signal indicates that NEDD1 (in green) and γ-tubulin (in red) colocalized. DNA was pseudocolored in blue. Bar = 5 μm.
Figure 4.
Phenotypic Characterization of the nedd1 Mutant. (A) Diagrammatical illustration of the NEDD1 gene structure and the position of the T-DNA insertion rendering the nedd1 mutation. Exons and introns are represented as boxes and lines, respectively. (B) In wild-type pollen grains, two brighter sperm nuclei (arrows) and a less bright vegetative nucleus (arrowhead) are revealed by DAPI staining. (C) A defective pollen grain produced by the NEDD1/nedd1 plant contains two identical loosely packed chromatin masses. (D) A defective pollen grain of NEDD1/nedd1 with overlapping DNA masses. (E) In the qrt/qrt background, the control plant produced four attached pollen grains, all of which contain the normal configuration of two sperm nuclei plus one vegetative nucleus. (F) In the qrt/qrt background, the NEDD1/nedd1 plant produces four attached pollen grains: two normal and two (arrowheads) that lack sperm. (G) Pollen viability assessment in tetrads in the qrt/qrt background. Percentages of tetrads containing 4, 3, 2, 1, or 0 viable pollen grains in the NEDD1/NEDD1 versus the NEDD1/nedd1 plants. Bars = 5 μm.
Figure 5.
Defects in MT Organization Caused by the nedd1 Mutation during the First Mitosis in Developing Pollen. Rows represent stages of the division as labeled for metaphase (Met), anaphase (Ana), telophase (Telo), telophase/cytokinesis transition (Telo/Cyto), cytokinesis (Cyto), and two-cell stage (2-Cell). MTs are shown in the first and third columns and DNA in the second and fourth columns. Bar = 5 μm. (A) to (L) Control NEDD1 microspores undergoing mitosis. (A) and (B) A peripherally positioned metaphase spindle. (C) and (D) Interzonal MTs appear upon the arrival of chromotids near the spindle poles at anaphase. (E) and (F) At telophase, interzonal MTs are bundled and begin to be organized in an antiparallel fashion. (G) and (H) A mature phragmoplast is formed. (I) and (J) During cytokinesis, MT signals are prominent at the periphery of an expanding phragmoplast array. At this stage, the DNA mass of the generative cell nucleus (arrowhead) is much more compact than that of the vegetative nucleus. (K) and (L) Upon completion of the microspore mitosis, the generative chromatin is brightly labeled (arrowhead). MTs are nucleated from the surface of the vegetative nuclear envelope. (M) to (X) Mutant (nedd1) microspores undergoing mitosis. (M) and (N) A metaphase spindle has its peripheral pole anchored near the plasma membrane and the other pole beyond the center of the cytoplasm. (O) and (P) The anaphase spindle has elongated interzonal MTs. (Q) and (R) The interzonal MTs become bundled at telophase. (S) to (V) The interzonal MTs remain highly bundled and fail to be organized into a functional phragmoplast array during telophase and cytokinesis. (W) and (X) MT bundles are randomly arranged between two reformed nuclei.
Figure 6.
Spindle Lengths of Wild-Type and Mutant Microspore Cells. The length (in micrometers) was measured for both metaphase and anaphase spindles of wild-type, normal short spindles produced by the heterozygous NEDD1/nedd1 plant (Het-S) and abnormal long ones by NEDD1/nedd1 (Het-L).
Figure 7.
Defects in Cell Plate Formation Caused by the nedd1 Mutation. Callose in the cell plate (green; white arrowheads) and DNA/nuclei (red) are labeled. In the control (NEDD1), the first mitotic cell division of the microspore results in a hemispherical cell plate separating the generative cell and vegetative cell (A). In the mutant (nedd1), an incomplete cell plate (B) or a highly branched one (C) is observed. The blue arrows point at the vegetative nucleus in the control or identical nuclei in the mutant and the purple arrow at the generative nucleus. Scale bar, 5 μm.
References
- Alexander, M.P. (1969). Differential staining of the aborted and non aborted pollen. Stain Technol. 44 117–122. - PubMed
- Brown, R.C., and Lemmon, B.E. (1991. a). Pollen development in orchids 3. A novel generative pole microtubular system predicts unequal pollen mitosis. J. Cell Sci. 99 273–281.
- Brown, R.C., and Lemmon, B.E. (1991. b). Pollen development in orchids 5. A generative cell domain involved in spatial control of the hemispherical cell plate. J. Cell Sci. 100 559–566.
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