Nuclear gamma-tubulin during acentriolar plant mitosis

Abstract

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that gamma-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of gamma-tubulin in nuclei increased during the G(2) phase, when cells are synchronized or sorted for particular phases of the cell cycle. gamma-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear gamma-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of gamma-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

Figure 1.

Immunolocalization of γ-Tubulin in Fava Bean Cells, Isolated Nuclei, and Isolated Chromosomes. γ-Tubulin staining ([A], [C], [E], [G], and [J]) and corresponding chromatin labeling with DAPI ([B], [D], [F], [H], and [I]). (A) and (B) Group of cells in different stages of the cell cycle. Staining of γ-tubulin in prophase nuclei (long arrow in [A]), in metaphase spindle (arrowhead in [A]), and in telophase phragmoplast (short arrow in [A]). (C) and (D) Cells during the G₁ stage of the cell cycle. (E) and (F) Cells in the late G₂ stage of the cell cycle with staining of γ-tubulin in nuclei. (G) and (H) Isolated G₂ nuclei with γ-tubulin staining (arrow in [G]). (I) and (J) Isolated chromosomes with staining of γ-tubulin in the kinetochore region (arrow in [J]). ; ; .

Figure 2.

Confocal Scanning Laser Microscopy of DNA, γ-Tubulin, and MPM2 Staining of Fava Bean Meristem Cells. Optical sections 1.0 μm thick exhibit γ-tubulin labeling (red) and DNA staining (blue). (A) Stereopair of a cell in early G₂ stage of the cell cycle; stereoimages were obtained by progression through a three-dimensional reconstruction program. (B) Gallery of 12 optical sections of a cell in late G₂ stage of the cell cycle. Note that some of the γ-tubulin–labeled dots are located at the chromosomes near the nuclear envelope, and perinuclear γ-tubulin forms caps that are focused to the poles. (C) Optical section through a roscovitine-treated cell showing the distribution of γ-tubulin. (D) Optical section through a roscovitine-treated cell showing the distribution of the MPM2 antigen. Note that the MPM2 signal is associated with the kinetochore/centromeric region and is also seen in the nucleoplasm, whereas γ-tubulin is restricted to the kinetochore/centromeric region. (E) Cell with prophase spindle and short kinetochore fibers decorated with anti–γ-tubulin antibody. .

Figure 3.

γ-Tubulin in Cellular Fractions of Asynchronous Meristem Root Tips of Fava Bean. (A) Protein gel blot analysis of supernatant (lanes 1, 3, and 5) and pellet (lanes 2, 4, and 6) of centrifuged cell extracts. Shown are Coomassie blue staining (lanes 1 and 2) and immunostaining with antibodies raised against α-tubulin (lanes 3 and 4) or against γ-tubulin (lanes 5 and 6). Positions of molecular mass standards are indicated at left by horizontal lines (from top to bottom: 205, 116, 97, 66, and 45 kD). Each lane was loaded with 10 μg of protein. (B) Solubilization of nuclear γ-tubulin under various buffer conditions. Isolated nuclei were treated in suspension and then pelleted by centrifugation. Pelleted nuclei (lanes 1, 3, 5, and 7) and supernatants (lanes 2, 4, 6, and 8) were used for protein gel blot analysis. Nuclei and the supernatant of the control were treated with buffer only (lanes 1 and 2), 75 mM NaCl (lanes 3 and 4), 200 mM NaCl (lanes 5 and 6), or 0.5 M KI (lanes 7 and 8). (C) Treatment of isolated nuclei with DNase. Shown are the results of protein gel blot analysis of untreated nuclei (lane 1), pelleted nuclei after DNase treatment (lane 2), supernatant after DNase treatment (lane 3), control pelleted nuclei (lane 4), and control supernatant (lane 5). Proteins from 200,000 nuclei were loaded per lane.

Figure 4.

Flow Cytometric Sorting of Nuclei and Detection of γ-Tubulin during G₁ and G₂ Stages of the Cell Cycle. (A) Example of flow sorting of synchronized G₂ nuclei. At top is a histogram of relative fluorescence (fluorescence pulse area) derived after analysis of isolated nuclei, which shows a high degree of synchronization (high number of nuclei in G₂ stage). At bottom is a dot plot of fluorescence pulse area versus fluorescence pulse width used for the sorting of nuclei. The population of G₂ nuclei was sorted from the marked sort region (R1). Similarly, synchronized G₁ nuclei were flow sorted. (B) DNA labeling of sorted nuclei during G₁ and G₂ stages of the cell cycle. (C) Protein gel blot analysis of γ-tubulin in nuclei during G₁ (lanes 1 and 3) and G₂ (lanes 2 and 4) stages of the cell cycle. Coomassie blue staining of total nuclear proteins (lanes 1 and 2) is shown as well as immunostaining of γ-tubulin (lanes 3 and 4). Proteins from 200,000 nuclei were loaded per lane. Positions of molecular mass standards are marked at left with horizontal lines (from top to bottom: 94, 67, 43, 30, and 20 kD). (D) Protein gel blot analysis of γ-tubulin in supernatants of cell extracts from a synchronous cell population in G₁ (lanes 1 and 3) and G₂ (lanes 2 and 4) stages of the cell cycle. Lanes 1 and 2 show Coomassie blue staining; immunostaining is shown in lanes 3 and 4. Ten micrograms of protein was loaded per lane. Positions of molecular mass standards are marked at left with horizontal lines (from top to bottom: 205, 116, 97, 66, and 45 kD).