Characterization of the Arabidopsis augmin complex uncovers its critical function in the assembly of the acentrosomal spindle and phragmoplast microtubule arrays

Abstract

Plant cells assemble the bipolar spindle and phragmoplast microtubule (MT) arrays in the absence of the centrosome structure. Our recent findings in Arabidopsis thaliana indicated that AUGMIN subunit3 (AUG3), a homolog of animal dim γ-tubulin 3, plays a critical role in γ-tubulin-dependent MT nucleation and amplification during mitosis. Here, we report the isolation of the entire plant augmin complex that contains eight subunits. Among them, AUG1 to AUG6 share low sequence similarity with their animal counterparts, but AUG7 and AUG8 share homology only with proteins of plant origin. Genetic analyses indicate that the AUG1, AUG2, AUG4, and AUG5 genes are essential, as stable mutations in these genes could only be transmitted to heterozygous plants. The sterile aug7-1 homozygous mutant in which AUG7 expression is significantly reduced exhibited pleiotropic phenotypes of seriously retarded vegetative and reproductive growth. The aug7-1 mutation caused delocalization of γ-tubulin in the mitotic spindle and phragmoplast. Consequently, spindles were abnormally elongated, and their poles failed to converge, as MTs were splayed to discrete positions rendering deformed arrays. In addition, the mutant phragmoplasts often had disorganized MT bundles with uneven edges. We conclude that assembly of MT arrays during plant mitosis depends on the augmin complex, which includes two plant-specific subunits.

Figure 1.

Purification of the Arabidopsis Augmin Complex and Identification of Six Augmin Subunits. (A) Proteins copurified with AUG3-c-myc using anti-c-myc antibody were subjected to SDS-PAGE and immunoblotting analysis. Gel was stained with silver nitrate. Asterisks indicate the bait, AUG3-4×c-myc. The numbers next to the gel image indicate the position of the gel regions analyzed in the following LC-MS/MS analysis described in (B). AUG3-myc, transgenic line expressing AUG3-4×c-myc; M, molecular weight markers; WT, wild-type negative control. (B) MS results obtained from the samples shown in (A). Six augmin subunits, including two previously reported augmin subunits (AUG1 and AUG3), were identified. Gene identification name according to TAIR database (Gene ID), name, number of unique peptides (# peptides), and sequence coverage (Coverage) are listed. The bait AUG3-c-myc is highlighted in gray. (C) Properties of Arabidopsis augmin subunits. Calculated molecular mass of the proteins (MW [kD]) and theoretical pI are shown.

Figure 2.

Phenotypic Analysis of Mutations in AUG Genes. (A) Schematic illustration of the AUG1, AUG2, AUG4, and AUG5 genes and the positions of corresponding T-DNA insertions. Exons and introns are displayed as open boxes and lines, respectively. (B) Genetic segregation patterns of offspring derived from self-fertilization of the heterozygous aug mutants. All segregation ratios are significantly different from the expected ratio (1 +/+: 2 +/aug: 1 aug/aug) based on the χ² test (P < 0.5). (C) to (E) Defects in MT organization caused by aug1-1, aug 4-1, and aug5-1 mutations at metaphase (C), telophase (D), and cytokinesis (E) during pollen mitosis I visualized by immunofluorescence. Bars = 5 μm. (C) A wild-type (WT) metaphase spindle exhibits a bipolar configuration with converged spindle poles (top panel). In aug1-1 and aug5-1 mutants, metaphase spindles only contain randomly packed MTs in the peripheral side (middle panel) or are elongated and disorganized (bottom panel). (D) During telophase, compared with the laterally expanding phragmoplast MT array positioned near the cell periphery in the wild type (top panel), elongated MT arrays are observed in aug4-1 and aug5-1 mutants that show no sign of lateral expansion (middle and bottom panels). (E) During cytokinesis, a curved phragmoplast can be seen in the wild-type cell (top panel). In the aug4-1 and aug5-1 mutants, phragmoplast MTs are disorganized (middle and bottom panels).

Figure 3.

Localization of AUG5 and AUG6 in Mitotic Root Meristematic Cells. (A) to (L) Immunolocalization of AUG5-c-myc. In merged images, AUG5 is pseudocolored in green, MTs in red, and DNA in blue. (A) to (C) In a prophase cell, AUG5-c-myc signal is observed along MTs on the nuclear envelope. (D) to (F) In a metaphase cell, AUG5-c-myc signal appears along kinetochore fiber MTs in a punctate manner. (G) to (I) During anaphase, AUG5-c-myc particularly decorates the shortening kinetochore fibers at the poles. (J) to (L) AUG5-c-myc signal decorates phragmoplast MTs. (M) Live-cell imaging of the AUG6-GFP. Snapshots were extracted from a time-lapse movie of a mitotic root cell expressing AUG6-GFP and mCherry-TUB6 (see Supplemental Movie 1 online). AUG6 and MTs are shown in green and red, respectively. The starting time is set at 0, and snapshot images are taken at times (hour:minutes:seconds) as shown on the bottom left. Like AUG5-c-myc, AUG6-GFP prominently decorates spindle and phragmoplast MT arrays from prophase to cytokinesis. Bars = 5 μm.

Figure 4.

Reciprocal Purification of Augmin Subunits and Identification of Two Plant-Specific Subunits. (A) Immunoaffinity purification results using transgenic plants expressing AUG3-c-myc, AUG4-c-myc, or AUG5-c-myc. Purified proteins together with those from a wild-type negative control are revealed in a silver-stained gel. Asterisks indicate the bait for each purification. The positions of AUG1, 3, 4, and 5 are indicated at the right side of the gel image. M, molecular weight markers. (B) Immunoblotting analysis using anti-c-myc, anti-AUG1, anti-AUG3, anti-AUG4, and anti-AUG5 antibodies. Proteins purified from AUG3-c-myc (A3), AUG4-c-myc (A4), and AUG5-c-myc (A5) include all subunits of the augmin complex. The baits (asterisks) and endogenous augmin subunits (arrows) are revealed by immunoblotting. WT, wild type. (C) Mass spectrometry results obtained through three independent immunopurifications using AUG3-c-myc, AUG4-c-myc, and AUG5-c-myc as baits. Besides AUG1-6, two additional subunits (AUG7 and AUG8) were identified. Gene ID represents gene identification number by TAIR; # peptides, number of unique peptides; # spectra, number of total spectra; coverage, sequence coverage. The bait of each experiment is highlighted in gray.

Figure 5.

Characterization of the AUG7 Gene. (A) Schematic diagram of the AUG7 protein. A coiled-coil is predicted in the middle of the protein. aa, amino acids. (B) Schematic representation of the AUG7 gene with exons and introns as open boxes and lines, respectively. The aug7-1 mutation has a T-DNA insertion in the first exon. (C) Assessment of the expression level of AUG7 by quantitative RT-PCR. The wild-type (WT) expression level was set at 1. The error bars represent sd of four replicates. (D) Root length test for the offspring derived from self-fertilization of heterozygous aug7-1 mutant. Genotypes of the seedlings were determined based on the detection of the wild-type AUG7 locus (top panel) and/or aug7-1 mutation (middle panel). Among 10-d-old seedlings (bottom panel), the homozygotes have shorter roots (asterisks). Bar = 10 mm. (E) Comparison of root lengths of wild-type, heterozygous (Hetero), and homozygous (Homo) aug7-1 mutant seedlings at 5, 10, and 15 d after germination. The root lengths of homozygous seedlings are significantly shorter than those of the wild type and heterozygotes (asterisks, t test, P < 10⁻⁸). Data are shown as mean ± sd with a minimum of 11 plants. (F) Pronounced growth retardation caused by the aug7-1 mutation. Five weeks after germination, the homozygous aug7-1 mutant (left panel inset) shows very limited vegetative growth compared with a heterozygous plant, which has already produced an inflorescence (left panel). At 21 weeks after germination, the homozygous aug7-1 plant remains dwarf and produces aborted flowers (right panel). Bars = 10 mm. (G) Live-cell imaging of AUG7-GFP in a root meristematic cell undergoing mitosis. Snapshots were extracted from Supplemental Movie 2 online. Time is shown in seconds. In metaphase, the AUG7-GFP signal appears in the spindle. The signal is prominent on the shortening kinetochore fiber MTs during anaphase and then seen in the phragmoplast during cytokinesis. Bar = 5 μm.

Figure 6.

Abnormal MT Organization in the Mitotic Spindle and Phragmoplast of the aug7-1 Mutant. (A) and (D) Immunofluorescence of MTs in metaphase spindles (A) and phragmoplasts (D) in root meristematic cells of the wild-type (WT) control and aug7-1 mutant. In merged images, MTs are pseudocolored in red and DNA in blue. Cell outline is indicated by white frames in the merged images. (A) A metaphase spindle of the wild type shows converged spindle poles. The aug7-1 mutant cells often form long, diagonally oriented and/or disorganized spindles with unconverged poles (arrows). Discrete MTs are also detected in the cytoplasm (arrowheads). (D) Compared with MTs in the wild-type phragmoplast, phragmoplast MTs in aug7-1 tend to be longer and disorganized. Discrete MTs are also detected elsewhere in the cytoplasm (arrowheads). Bar = 5 μm. (B) and (E) Quantification of the spindle length with the cell length as a reference (B) and the phragmoplast length with the cell length as a reference (E) in the wild-type control and aug7-1 mutant. Generally, longer spindles (B) and phragmoplasts (E) are found in aug7-1 cells compared with those of the wild type. (C) and (F) Proportions of spindles (C) and phragmoplasts (F) that show normal and abnormal (long, disorganized, and long and disorganized patterns) configurations in the wild type and aug7-1 mutant.

Figure 7.

Delocalization of γ-Tubulin in the Mitotic Spindle and Phragmoplast of aug7-1 Mutant. (A) to (C) Immunofluorescence of γ-tubulin and MTs in the wild-type and aug7-1 mutant cells. In the merged images, γ-tubulin is pseudocolored in green, MTs in red, and DNA in blue. WT, wild type. Bar = 5 μm. (A) In a wild-type metaphase cell, γ-tubulin decorates kinetochore fiber MTs with biases toward spindle poles (top panel). Such a pattern is barely detected in the unconverged metaphase spindle of the aug7-1 cell at a similar stage (bottom panel). (B) During anaphase, γ-tubulin signal is prominently detected along shortening kinetochore fiber MTs in the wild type (top panel), but the signal is greatly diminished in an anaphase aug7-1 cell (bottom panel). (C) In the wild-type phragmoplast, γ-tubulin localizes on MTs with biases toward minus ends (top panel). In aug7-1, only weak signals are detected on disorganized phragmoplast MTs (bottom panel). (D) and (E) Quantitative assessment of the fluorescent signal intensities of γ-tubulin (D) and α,β-tubulin (E). The ratios of the spindle- or phragmoplast-localized signals to the cytoplasmic ones are shown as mean ± sd. When marked with asterisks, the decreases in the relative intensity are statistically significant (t test, P < 0.0001).