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. 2018 Jan;176(1):307-325.
doi: 10.1104/pp.17.01112. Epub 2017 Sep 11.

Microtubule Array Patterns Have a Common Underlying Architecture in Hypocotyl Cells

Andrew Elliott  1 , Sidney L Shaw  2
Affiliations

Microtubule Array Patterns Have a Common Underlying Architecture in Hypocotyl Cells

Andrew Elliott et al. Plant Physiol. 2018 Jan.

Abstract

Microtubules at the plant cell cortex influence cell shape by patterning the deposition of cell wall materials. The elongated cells of the hypocotyl create a variety of microtubule array patterns with differing degrees of polymer coalignment and orientation to the cell's growth axis. To gain insight into the mechanisms driving array organization, we investigated the underlying microtubule array architecture in light-grown epidermal cells with explicit reference to array pattern. We discovered that all nontransverse patterns share a common underlying array architecture, having a core unimodal peak of coaligned microtubules in a split bipolarized arrangement. The growing microtubule plus ends extend toward the cell's apex and base with a region of antiparallel microtubule overlap at the cell's midzone. This core coalignment continuously shifts between ±30° from the cell's longitudinal growth axis, forming a continuum of longitudinal and oblique arrays. Transverse arrays exhibit the same unimodal core coalignment but form local domains of microtubules polymerizing in the same direction rather than a split bipolarized architecture. Quantitative imaging experiments and analysis of katanin mutants showed that the longitudinal arrays are created from microtubules originating on the outer periclinal cell face, pointing to a cell-directed, rather than self-organizing, mechanism for specifying the major array pattern classes in the hypocotyl cell.

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Figures

Figure 1.
Figure 1.
Cortical microtubule arrays in light-grown hypocotyl cells show a unimodal core of microtubule coalignment. Summation images of 3D time lapse confocal microscopy (5–8 optical sections, 3-s interval, 10 frames) showing the trajectory of growing microtubule plus ends labeled with EB1-GFP in 6-d-old Arabidopsis hypocotyl cells representing longitudinal (A), oblique (B), transverse (C), and basket (D) array patterns. Bar = 10 µm. EB1-GFP tracks extracted from each time lapse series (E–H) for quantitative evaluation of microtubule position, orientation, and growth direction. Microtubule coalignment was assayed in 100 randomly selected cells by creating histograms of the microtubule orientation angles displayed as a combined heat map (I). Each array histogram (row) is recentered on the dominant orientation angle (column 0°), and all arrays are sorted from most coaligned to least coaligned (I, top to bottom) using a ±20° sliding window. Color key indicates number of plus ends per 3° bin; red arrows indicate ±17° SD for cumulative data. The fraction of microtubules in the ±20° sliding window (J), for all 100 cells in (I), color coded for the visually determined array pattern (color key in K). The dominant microtubule orientation angle is plotted against the fraction of microtubule plus ends (MTs) found within a ±20° sliding window to evaluate the relationship between array pattern and the relative degree of microtubule coalignment (K). Markers represent the visually determined array pattern. Blue lines represent array classification boundaries.
Figure 2.
Figure 2.
Cortical microtubules exhibit a longitudinal split bipolar array architecture. Vector plots showing the color-coded growth trajectories of microtubule plus ends on the outer periclinal cell face for all array patterns; longitudinal (A), left-oblique (B), right-oblique (C), transverse (D), basket with split bipolarity (E), and basket with patched regions of organization (F). Color coding (key in A) for 90° quadrants representing up (cyan), down (blue), left (green), and right (red). Scale bar = 5 µm. Interleaved histograms showing the spatial distribution of microtubule trajectories as a function of the cell’s long axis in 90° quadrants (G–N). Cumulative histogram (G) of lateral (left side of ordinate) and longitudinal (right side of ordinate) trajectories (n = 24,369 tracks, 100 cells, 18 seedlings) showing spatial separation of apically and basally directed microtubule polymerization. Cumulative cell width (black lines) scaled for presentation. Trajectories for cells with longitudinal (H), left-oblique (I), and right-oblique (J) patterns exhibited a split bipolar arrangement not observed for transverse array patterns (K). Basket patterns (L) were separated by cells showing a split bipolarity (M) or only patches of organization (N). Percent of each pattern in the population in lower corner of frame.
Figure 3.
Figure 3.
Time course observations of array pattern changes. Summation images of EB1-GFP (10 frames at 3-s intervals) taken every 10 to 12 min for 2.5 h (A). Color-coding for the dominant array orientation angle (yellow) using a ±20° window (B) indicates a gradual shift in array pattern coordinated across the cell face. Black arrows indicate orientation angle in B. Trajectory maps of microtubule growth direction color-coded for up (cyan), down (blue), left (green), and right (red) in 90° quadrants indicating a sustained bias for the direction of microtubule polymerization across that long axis of the cell (C). Bar = 5 µm.
Figure 4.
Figure 4.
Quantitative evaluation of time course data shows gradual and sustained shifting between oblique and longitudinal array patterns. Time course data for five cells imaged at 10- to 12-min intervals for >2 h Microtubule trajectory maps for the first and last time point of each series (A–E) with color coding for up, down, left, and right (key in E). The distribution of microtubule orientation angles (5° bins) for each time point assembled into a heat map (A′–E′) with time running from top to bottom. The heat maps show a gradual shifting of the dominant polymer orientation without a substantial broadening of the microtubule coalignment. The dominant orientation angle was plotted against the fraction of coaligned microtubules using a ±20° window for each cell and color coded green-to-red (A′′–E′′) to show the temporal sequence. The cumulative distribution of all microtubule orientation angles from the 100 cell fixed time point data (blue) plotted with the cumulative distribution of all orientation angles from the five time course experiments (F). Blue lines represent array classification boundaries and green trace represents Gaussian fit to fixed time point data set.
Figure 5.
Figure 5.
Newly appearing EB1-GFP foci are at a higher density at the cell’s midzone. Trajectory map (A) for microtubule plus ends in five longitudinally oriented arrays imaged for 5 min at 3-s intervals (n = 500 frames, 5 cells, 11,512 trajectories). Color coding represents 90° bins with up (cyan), down (blue), left (green), and right (red) per legend. The positions of all newly appearing EB1-GFP foci (i.e. microtubule contributions from nucleation, rescue, and internal cell positions) were identified by hand (n = 2,139 events) and plotted with the same color coding for direction (B). Black traces represent cell perimeters. Histograms representing the total trajectory count (C) and nascent EB1-GFP events (D) as a function of the cells’ long axes (bin = 1 µm). The accumulated cell width, scaled for appearance (C, black trace), indicates a relatively uniform trajectory distribution with a depression at the midzone. Interleaved histograms representing the direction of mapped trajectories (E) and nascent EB1-GFP events (F) as a function of the cells’ long axis (bin = 1 µm) indicate a split bipolar arrangement of longitudinal events for both populations. Lateral events plotted to the left of the ordinate and longitudinal events to the right for E and F with black traces indicating the accumulated cell width. The accumulated orientation angles for the five cells (G) showing a symmetric, peaked distribution centered at longitudinal (µ = 87° ± 26°).
Figure 6.
Figure 6.
Gamma-tubulin complexes are distributed uniformly across the outer periclinal cell face. The distribution of residency times (A) for GCP2-GFP foci at the cell cortex of Arabidopsis hypocotyl cells (B) imaged every 6 s for >90 s using mCherry-TuA5 to label microtubules. Scale bar = 5 µm in (B); green is GCP2-GFP and red is mCherry-TuA5. Combined positions of all GCP2-GFP localized foci from 10 cells (n = 8,179 foci, 990 s total time) color coded for residency times (C) according to A. Histogram of the accumulated GCP2-GFP foci as a function of position on the cells’ long axis (D) color coded as in A (1-µm bins). Histogram of GCP2-GFP residency times as a function of normalized cell length (E) for 10 cells with events on a log scale to compare distributions of less frequent events. Cumulative totals for 6 s (6,649), 12 to 24 s (951), 30 to 42 s (429), and >48 s (150) for 10 cells with an average projected outer face surface area of 995 ± 292 µm2.
Figure 7.
Figure 7.
The katanin p60 mutant maintains a split bipolar array architecture. Trajectory map (A) for microtubule plus ends in five longitudinally oriented arrays from katanin p60 mutant seedlings imaged for 5 min at 3 s intervals (n = 500 frames, 5 cells, 6810 trajectories). Color coding represents 90° bins with up (cyan), down (blue), left (green), and right (red) per legend. The position of all newly appearing EB1-GFP foci (n = 2,506 events) were plotted with the same color coding for direction (B). Black traces represent cell perimeters. Histograms representing the total trajectory count (C) and nascent EB1-GFP events (D) as a function of the cells’ long axes (bin = 1 µm). Interleaved histograms representing the direction of mapped trajectories (E) and nascent EB1-GFP events (F) as a function of the cells’ long axis (bin = 1 µm) show a split bipolar arrangement of longitudinal events for both populations. The accumulated orientation angles for the five cells (G) showing a symmetric, peaked distribution centered at longitudinal (µ = 90° ± 33°).
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References

    1. Abe T, Hashimoto T (2005) Altered microtubule dynamics by expression of modified alpha-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants. Plant J 43: 191–204 - PubMed
    1. Allard JF, Ambrose JC, Wasteneys GO, Cytrynbaum EN (2010a) A mechanochemical model explains interactions between cortical microtubules in plants. Biophys J 99: 1082–1090 - PMC - PubMed
    1. Allard JF, Wasteneys GO, Cytrynbaum EN (2010b) Mechanisms of self-organization of cortical microtubules in plants revealed by computational simulations. Mol Biol Cell 21: 278–286 - PMC - PubMed
    1. Ambrose C, Allard JF, Cytrynbaum EN, Wasteneys GO (2011) A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis. Nat Commun 2: 430. - PMC - PubMed
    1. Ambrose C, Wasteneys GO (2014) Microtubule initiation from the nuclear surface controls cortical microtubule growth polarity and orientation in Arabidopsis thaliana. Plant Cell Physiol 55: 1636–1645 - PMC - PubMed

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