CLASP Facilitates Transitions between Cortical Microtubule Array Patterns

Abstract

Acentrosomal plant microtubule arrays form patterns at the cell cortex that influence cellular morphogenesis by templating the deposition of cell wall materials, but the molecular basis by which the microtubules form the cortical array patterns remains largely unknown. Loss of the Arabidopsis (Arabidopsis thaliana) microtubule-associated protein, CYTOPLASMIC LINKER ASSOCIATED PROTEIN (AtCLASP), results in cellular growth anisotropy defects in hypocotyl cells. We examined the microtubule array patterning in atclasp-1 null mutants and discovered a significant defect in the timing of transitions between array patterns but no substantive defect in the array patterns per se. Detailed analysis and computational modeling of the microtubule dynamics in two atclasp-1 fluorescent tubulin marker lines revealed marker-dependent effects on depolymerization and catastrophe frequency predicted to alter the steady-state microtubule population. Quantitative in vivo analysis of the underlying microtubule array architecture showed that AtCLASP is required to maintain the number of growing microtubule plus ends during transitions between array patterns. We propose that AtCLASP plays a critical role in cellular morphogenesis through actions on new microtubules that facilitate array transitions.

Figure 1.

The cell expansion phenotype of the atclasp-1 mutant is altered differently by different microtubule marker transgenes. A, Average hypocotyl lengths ± sd for 6-d-old, light-grown, wild-type and atclasp-1 seedlings in the Col-0, GFP-TUB1, YFP-TUA5, and EB1-GFP backgrounds. Differences between the wild type and atclasp-1 are significant in all backgrounds (P < 0.01 [n > 40 in all cases], Student’s t test used for all growth assays), and differences between all transgenic wild-type backgrounds and the Col-0 parent line are significant in all cases (P < 0.01). B, Mean cell length (L) and width (W) ± sd for fluorescent tubulin-expressing lines in the wild-type and atclasp-1 backgrounds (n = 14 plants, n > 530 cells for tubulin lines; n = 6 plants, n > 230 cells for EB1-GFP lines). Cell lengths between the wild type and the mutant in both fluorescent tubulin backgrounds are not statistically different (P > 0.05 in both cases), while the atclasp-1 cells are wider on average compared with the wild type in both backgrounds (P < 0.001). Cell lengths and widths are significantly different (P < 0.001) between the wild type and the mutant in the EB1-GFP background. C, Cellular anisotropy expressed as cell length divided by cell width ± sd for fluorescent tubulin-expressing lines.

Figure 2.

atclasp-1 has only small effects on the distribution of microtubule array pattern types in light-grown hypocotyl cells. A and B, Maximum projection of tiled confocal microscopy images from wild-type (A) and atclasp-1 mutant (B) 6-d-old light-grown seedlings expressing a GFP-TUB1 microtubule marker. Bars = 30 µm. C to F, Examples of cells showing the four dominant microtubule array patterns. Bars = 10 µm. G and H, Stacked bar graphs showing the accumulated percentage of cells with each array pattern from 14 untreated hypocotyls (n > 530 cells for each line) in GFP-TUB1 and YFP-TUA5 lines, with the color key given in C to F. I and J, Dot plots showing the percentage of transverse patterned cells per hypocotyl induced by mock treatment or combined IAA and GA₄ treatment after 2 h in GFP-TUB1 and YFP-TUA5 backgrounds. Bars represent mean values.

Figure 3.

Microtubule arrays transition between patterns less frequently in atclasp-1 cells. A and B, Hypocotyl cells from projected confocal image stacks displayed with inverted contrast showing the number of transitions between pattern types over a 2-h period for the wild type (A) and atclasp-1 (B). The color code is shown in the key, and images were taken at 15-min intervals. C, A single wild-type cell imaged at 15-min intervals with color coding showing four transitions over 120 min. D, Interleaved histograms of pattern transition frequencies for the wild type and atclasp-1 mutants in both GFP-TUB1 and YFP-TUA5 lines with the corresponding mean number of observed transitions ± sd per 120 min. B, basket; L, longitudinal; O, oblique.

Figure 4.

Dynamic properties of the cortical microtubules in the wild-type and atclasp-1 mutant lines in GFP-TUB1 and YFP-TUA5 backgrounds. A to D, Interleaved histograms of growth and shortening velocities from wild-type and atclasp-1 cells (normalized for total counts) in the GFP-TUB1 (A and B) and YFP-TUA5 (C and D) backgrounds. Insets show distributions of observed growth run times (A and C) or shortening run times (B and D) used for the calculation of catastrophe and rescue frequencies, respectively. Tabular data are presented in Table 1. E, Distribution of growth and shortening velocities taken for each growth or shortening run for the wild type and mutant in GFP-TUB1 and YFP-TUA5 backgrounds. F, Monte Carlo simulation showing the loss of microtubules from an initial population of 100 (average of 25 trials for each curve) using mean microtubule dynamics parameters from wild-type and atclasp-1 plants in the GFP-TUB1 and YFP-TUA5 backgrounds. Time in log scale for a 60-min simulation was taken at 1-s intervals.

Figure 5.

AtCLASP increases the density of longitudinally oriented microtubule plus ends across the cell face. A, Scatterplot relating the mean ± sd number of EB1-GFP foci per cell face (from five consecutive images) to cell face area for the wild type and atclasp-1 mutants. The inset histogram shows EB1-GFP comet densities per cell for wild-type and mutant lines. Trend lines represent mean density plotted from the origin (n > 80 cells). B and C, Interleaved histograms showing cumulative distributions of growing microtubule (MT) plus ends on the cell’s long axis by growth direction, taken in 90° quadrants (up, down, left, and right), for wild-type and atclasp-1 seedlings (n = 25/25 cells). Longitudinal directions are to the right of the ordinate and transverse directions to the left. Solid black lines indicate the scaled cumulative cell area as a function of the long axis. D, Spatial distribution of growing plus ends as a function of the normalized long axis (21 bins) scaled for total counts. Trend lines represent fourth-order polynomial fits to the wild-type and mutant data showing no obvious difference in the spatial distribution. E, Rose plot showing the number of microtubule trajectories per angle relative to the cell axis in the wild type and the mutant. F, Dot plots showing the density of EB1-GFP foci per cell for each of four defined microtubule orientations (i.e. transverse, 0°–15°/165°–180°; left oblique, 16°–74°; longitudinal, 75°–105°; and right oblique, 106°–164°). Data in B to F are from the same 25 cells for the wild type and the atclasp-1 mutant.

Figure 6.

Microtubule arrays in atclasp-1 show comparatively slow changes in array orientation and coalignment over time. A and B, Time-course analysis of a single wild-type (A) and atclasp-1 mutant (B) cell showing EB1-GFP trajectories color coded by growth direction at ∼15-min intervals over a 150-min period. The key in B shows the color coding trajectory. C to H, Histograms of microtubule orientation (1°–180°) taken at ∼15-min intervals determined from EB1-GFP growth trajectories plotted as heat maps with time running from top to bottom for three wild-type (C–E) and three atclasp-1 (F–H) cells starting in the same pattern. C′ to H′, Plots of the relative degree of array coalignment over the dominant microtubule orientation angle, color coded green to red for time, for the wild type (C′–E′) and atclasp-1 (F′–H′) using a ±21° window.

Figure 7.

The atclasp-1 mutant shows background-dependent defects in microtubule severing at crossover sites. The schematic indicates microtubule crossover as a black arrow resolved through depolymerization (reversed arrow) or through severing at the crossover site. A, Spatial distribution of crossover events normalized to the cell’s long axis showing events resolved by depolymerization (gray, left of ordinate) and severing (black, right of ordinate) in GFP-TUB1 and YFP-TUA5 backgrounds for wild-type and atclasp-1 cells. B, Angle of microtubule crossover for all backgrounds in stacked histograms showing depolymerization (gray) and severing (black) events. C to F, Histograms showing the cumulative number of severing events for microtubules over a range of microtubule orientation angles relative to the long axis of the cell. Gray lines represent expected distributions of orientation angles estimated from EB1-GFP wild-type and mutant lines (Fig. 5) and scaled to the number of severing events in each histogram. The key in F gives the spatial orientation of histogram bins. G, Stacked bar graphs showing the fraction of crossover sites that were resolved by either severing (black) or depolymerization (gray) for wild-type and atclasp-1 mutant cells in GFP-TUB1 (n = 256/258) and YFP-TUA5 (n = 237/236) backgrounds. The difference between the wild type and the mutant in the GFP-TUB1 background is significant at P < 0.001 and not significant in the YFP-TUA5 background at P = 0.05 (Student’s t test). H, Box-and-whisker plots showing the amount of time in each crossover recorded prior to a severing or depolymerization event. Boxes represent quartiles, whiskers indicate ranges, and circles represent arithmetic means.