Augmin plays a critical role in organizing the spindle and phragmoplast microtubule arrays in Arabidopsis

Abstract

In higher plant cells, microtubules (MTs) are nucleated and organized in a centrosome-independent manner. It is unclear whether augmin-dependent mechanisms underlie spindle MT organization in plant cells as they do in animal cells. When AUGMIN subunit3 (AUG3), which encodes a homolog of animal dim γ-tubulin 3/human augmin-like complex, subunit 3, was disrupted in Arabidopsis thaliana, gametogenesis frequently failed due to defects in cell division. Compared with the control microspores, which formed bipolar spindles at the cell periphery, the mutant cells often formed peripheral half spindles that only attached to condensed chromosomes or formed elongated spindles with unfocused interior poles. In addition, defective cells exhibited disorganized phragmoplast MT arrays, which caused aborted cytokinesis. The resulting pollen grains were either shrunken or contained two nuclei in an undivided cytoplasm. AUG3 was localized along MTs in the spindle and phragmoplast, and its signal was pronounced in anaphase spindle poles. An AUG3-green fluorescent protein fusion exhibited a dynamic distribution pattern, similar to that of the γ-tubulin complex protein2. When AUG3 was enriched from seedlings by affinity chromatography, AUG1 was detected by immunoblotting, suggesting an augmin-like complex was present in vivo. We conclude that augmin plays a critical role in MT organization during plant cell division.

Figure 1.

Defects Caused by the aug3-1 Mutation. (A) AUG3 and the aug3-1 insertional mutation. Exons are shown as open boxes and introns as lines. (B) The aug3-1 mutation causes defects in developing fruits/siliques. Compared with the siliques produced by the +/+ (wild type [wt]) control plant, those of the heterozygous +/aug3-1 plant often contain white unfertilized ovules (white arrows) and brown aborted seeds (red arrows). (C) Scores of normal seeds, unfertilized ovules (white), and aborted seeds (brown) in the control +/+ (wild type) and +/aug3-1 plants. Error bars represent the standard deviations among examined siliques (n = 10). (D) Normal and defective pollen grains shown by DNA staining. A normal pollen grain contains two sperm cells with brightly stained nuclei (blue arrows) and a faintly stained vegetative nucleus (red arrow), as classified as the 2+1 type. The defective 1+1 type pollen grain contains a loosely packed vegetative nucleus (red arrow) and a condensed nucleus (blue arrow). The 1 type pollen grain contains only one nucleus containing loosely packed chromatin (red arrow). (E) Scores of the three types of pollen grains represented in (D) and shrunken ones produced by the +/aug3-1 plant when compared with the wild-type control. (F) Attached tetrad pollen grains produced by the +/aug3-1 qrt/qrt plant. Green asterisks indicate the tetrads containing fully enlarged pollen grains. Red arrows point to shrunken pollen grains. Bars = 5 μm in (D) and 50 μm in (F).

Figure 2.

Defects in Ovule Development and Embryogenesis Caused by aug3-1. (A) An ovule produced by a control plant. Inside, an embryo sac contains the egg cell (EC), two synergids (Sy), and a large central cell with a fused nucleus (Ccn). (B) A defective aug3-1 embryo sac contains a single abnormally large nucleus (arrowhead). (C) After fertilization, a proembryo and cellularized endosperm are formed inside the ovule. (D) and (E) Defective embryos (arrows) have not been cellularized, and cells cannot be discerned in areas surrounding the defective embryos. Instead, a few freely suspended nuclei (arrowheads) can be found.

Figure 3.

Spindle Defects Caused by the aug3-1 Mutation. Spindles of pollen mitosis I are shown for the control ([A] to [C]) and aug3-1 ([D] to [L]) cells. In the merged images, MTs are pseudocolored in green and DNA in red. (A) to (C) A metaphase spindle of the control cell contains converged spindle poles pointing toward the center of the cell. The spindle is placed toward the cell periphery. (D) to (F) MTs formed at the cell periphery and chromosomes are attached to the half spindle. (G) to (I) While the peripheral half spindle contains abundant MTs, only a few MT bundles (arrow) formed in the interior side. (J) to (L) A bipolar spindle contains kinetochore fiber MTs that failed to converge to a common pole (arrow). Bar = 5 μm.

Figure 4.

Defects in the Development of the Phragmoplast MT Array Caused by the aug3-1 Mutation. MTs in the spindle midzone and phragmoplast are shown in developing pollen grains of the control ([A] to [C] and [G] to [I]) and aug3-1 ([D] to [F] and [J] to [L]) mutant. In the merged images, MTs are pseudocolored in green and DNA in red. (A) to (C) Upon completion of anaphase in a control cell, a relatively short spindle (green bidirectional arrow) is placed toward the cell periphery and contains ambient MTs in the spindle midzone. (D) to (F) An aug3-1 cell exhibits an elongated spindle (red bidirectional arrow), which contains MT bundles in the spindle midzone. (G) to (I) A control cytokinetic cell exhibits a curved phragmoplast with a clear midline (green arrows). (J) to (L) An aug3-1 cell contains disorganized MTs between two reforming nuclei. Bar = 5 μm.

Figure 5.

Localization of AUG3 in Mitotic Cells in Root Meristematic Cells. In merged images, AUG3 is pseudocolored in green, MTs in red, and DNA in blue. (A) to (C) In a prophase cell, the AUG3-4xc-myc signal decorates MTs on the nuclear envelope. (D) to (F) AUG3-4xc-myc appears along kinetochore MT fibers as a punctate signal in a metaphase cell. (G) to (I) During anaphase, AUG3-4xc-myc conspicuously decorates the remnants of kinetochore fibers (green arrows). (J) to (L) At telophase/early cytokinesis, spindle midzone MTs are organized into an antiparallel array. Punctate AUG3-4xc-myc signal can be detected along the MT bundles. (M) to (O) Phragmoplast MTs are decorated with the AUG3-4xc-myc signal. The dark gap in the middle (green arrowheads) left by the AUG3-4xc-myc signal appears to be wider than in the MT fluorescent image. Bar = 5 μm.

Figure 6.

Localization of AUG3 and the γ-Tubulin Complex Protein GCP2 in Live Cells. Snapshots were taken from time-lapse movies covering metaphase to cytokinesis in root meristematic cells undergoing mitosis. The starting time is set at 0, and individual images are at time (in seconds) after the starting time and are shown on the bottom left. (A) to (E) AUG3-GFP in the spindle and phragmoplast. At metaphase (Meta), the GFP signal appears in the spindle. The signal becomes more prominent at spindle poles during anaphase (Ana). At late anaphase and telophase (Telo), conspicuous AUG3-GFP remains associated with the spindle poles (arrows), while weaker signals can be discerned toward the spindle midzone. The appearance of AUG3-GFP in the phragmoplast leaves a wide gap in the middle region (arrowheads) during cytokinesis (Cyto). (F) to (J) GCP2-GFP gives a similar dynamic localization pattern as AUG3-GFP. Conspicuous signal can be seen at the spindle poles (arrows) during late anaphase and telophase. A wide dark midzone can be detected in the phragmoplast during cytokinesis (arrowheads). Bar = 5 μm.

Figure 7.

Diminished γ-Tubulin Localization in the Phragmoplast of the aug3-1 Mutant. Triple localizations of γ-tubulin, MTs, and DNA in developing microspores of the control ([A] to [C]) and aug3-1 ([D] to [F]) mutant. (A) to (C) In the control cell, conspicuous γ-tubulin signal (A) can be detected in the developing phragmoplast. The dark zone in the anti-γ-tubulin fluorescent image (arrowheads) is much wider than that in the MT fluorescent image. (D) to (F) Disorganized MT bundles (E) can be detected between two sets of segregated chromosomes (F). γ-Tubulin is not obviously detected among MTs. Bar = 5 μm.

Figure 8.

Interaction of AUG3 and AUG1 in Vivo. Immunoblotting with anti-c-myc (α-myc) and anti-AUG1 (α-AUG1) antibodies. Protein samples were prepared from extracts of wild-type (WT) seedlings and those expressing AUG3-4xc-myc (A). In the negative control, the AUG3-4xc-myc–enriched proteins were probed with an anti-AUG1 depleted preparation (Dα). The AUG3-4xc-myc band is highlighted by an arrow and AUG1 by an asterisk. The AUG band is no longer detected after the specific anti-AUG1 antibodies were depleted. Molecular masses in kilodaltons are shown on the left.

Figure 9.

AUG1-GFP in a Root Cell Undergoing Mitosis. Snapshots of AUG1-GFP at late anaphase to telophase. Conspicuous GFP signal can be seen at spindle poles and in the midzone (arrows). A wide dark zone (arrowheads) can be seen in the middle of the phragmoplast. Bar = 5 μm.