TONNEAU2/FASS regulates the geometry of microtubule nucleation and cortical array organization in interphase Arabidopsis cells

Abstract

Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B'' subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from γ-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation.

Figure 1.

Cell Morphogenesis Defects in ton2 Epidermal Cells. (A) to (D) Leaf trichomes ([A] and [B]) and pavement cells ([C] and [D]) in the wild type ([A] and [C]) and in the ton2 mutant ([B] and [D]) imaged with a scanning electron microscope. Bars = 100 μm in (A), 50 μm in (B), and 25 μm in (C) and (D). (E) Reduced number of lobes in ton2 pavement cells. Average number of lobes was 8.9 ± 2.6 in wild-type (WT) (n = 95) and 2.0 ± 1.5 in ton2 (n = 120) leaf pavement cells. P value calculated by the Student’s t test indicates a significant difference. Error bars show sd.

Figure 2.

Comparison of Microtubule Array Density in ton2 and Wild-Type Cells. (A) Confocal reconstructions of upper hypocotyl cells of dark-grown seedlings expressing YFP:TUA5. Leaf epidermal pavement cells are shown below. Bar = 10 μm. wt, wild type. (B) The percentage of the image area occupied by signal above a threshold value was measured as a rough estimate of microtubule array density (see Methods). The difference between paired means by cell type is significant at P < 0.01 (two-tailed Student’s t test, n = 10 cell for the wild type and ton2). Bars indicate se. [See online article for color version of this figure.]

Figure 3.

TON2 Is Required to Regulate the Geometry of Microtubule-Associated Microtubule Nucleation. Confocal time series at the cortex of ton2 pavement cells expressing 35S:mCherry:TUB5 (red channel) and GCP2:GFP (green channel) constructs. (A) Recruitment of γ-tubulin complexes (arrowheads) to a cortical microtubule that results in a branching nucleation event (white arrowhead and carat) and a parallel nucleation event (yellow arrowhead and carat). GCP2:GFP also appears on the branching daughter microtubule but without evidence for nucleation from this location. (B) Recruitment of a labeled nucleation complex (arrowhead) to a cortical site free of detectable microtubules (open arrowhead), resulting in nucleation of a new microtubule (carat). The green label at top right is autofluorescent background. Bars = 2 μm for (A) and (B). (C) Probability of recruited nucleation (nucl.) complexes giving rise to nucleation events. Observation of stable localization of GCP2:GFP over at least three consecutive acquisition frames (~8 s) was defined as a cortical recruitment event. A total of 245 stabilized GCP2:GFP-labeled nucleation complexes were analyzed for wild-type (wt) cells and 163 for ton2 cells. (D) Proportion of all observed nucleation events that were branched, parallel, or de novo. The proportion of branching nucleation dropped approximately fourfold in ton2 cells (n = 79 nucleation events in the wild type and 58 events in ton2; 10 cells from 10 plants for each). (E) Distribution of branching nucleation angles in the wild type and ton2. The distributions are not significantly different from each other by either a Wilcoxon rank sum test or a two-tailed Student’s t test. (F) Mean time from recruitment of GCP2:GFP-labeled γ-TURC complexes at cortical microtubules to the first detected nucleation as visualized by mCherry-TUA5. Measurements were acquired from 10 ton2-15 mutant cells and 11 wild-type cells. The numbers of measured events are indicated next to the bars.

Figure 4.

Reorganization of Cortical Microtubule Arrays in Response to Light. (A) Confocal reconstructions of YFP:TUA5-labeled microtubules acquired before and after exposure to light for 60 min. Bar = 5 μm. (B) Distribution histogram of observed microtubule angles in relation to the longitudinal cell axis. Microtubules angles were grouped into bins of 10°. Microtubules perpendicular to the cell longitudinal axis were assigned the 90° angle. Measurements were taken from the same cells before and after light treatment. Note a change of microtubule orientation from predominantly transverse to longitudinal in the wild type but not in the ton2 mutant.

Figure 5.

Ectopic Targeting of TON2 to the Plasma Membrane Causes Organ and Tissue Morphogenesis Defects. (A) Leaf shape of the wild type (WT) and two different PM-TON2 plants. Average leaf width was reduced twofold in the PM-TON2 plants (P < 0.001) with no significant change in the leaf length (P = 0.4). The third and fourth mature rosette leaves were measured from 10 wild-type plants and nine PM-TON2 plants in the T1 generation. Error bars show sd. (B) Scanning electron microscopy images of trichomes on wild-type and PM-TON2 leaves. Trichome branching data were collected from 10 wild-type plants and nine PM-TON2 plants in T1 generation (n = 676 for the wild type; n = 381 for PM-TON2). (C) Pavement cells expressing PM-TON2 displayed less curved cell outlines with reduced number and length of lobes and necks. Data are presented as means ± se. P values in the diagrams show that differences in lobe length (n = 211 for the wild type; n = 123 for PM-TON2), neck width (n = 210 for the wild type; n = 123 for PM-TON2), and number of lobes (n = 80 for the wild type; n = 100 for PM-TON2) were significant. Bars = 100 μm in (B) and 30 μm in (C). [See online article for color version of this figure.]

Figure 6.

Plasma Membrane–Targeted TON2 Disrupts Microtubule Organization in Pavement Cells. Confocal reconstructions of PM-TON2 and wild-type pavement cells expressing YFP:TUA5. Arrowheads point to cell indentations with dense microtubules in a wild-type pavement cell. Note that pavement cells of the PM-TON2 plants have a more uniform distribution of microtubules along the lateral cell outlines. Bar = 10 μm. [See online article for color version of this figure.]

Figure 7.

te130 and te500 Mutations Enhance the ton2-15 Phenotype. (A) Comparison of 7-d-old seedlings of the ton2-15 mutant with the te130 ton2-15 and te500 ton2-15 double mutants. (B) Confocal sections of propidium iodide-stained root tips of the wild type (WT), ton2-15, te130, ton2-15 heterozygote (ton2-15/+), te500, and ton2-15 heterozygote homozygous for the te500 mutation (te500 ton2-13/+). Bar = 10 μm. [See online article for color version of this figure.]