CLASP is enriched at EMT-cortex anchor points.

<p><b>A.</b> Surface view of a root tip epidermal cell co-expressing pCLASP::GFP-CLASP (cyan) and pUBQ1::RFP-TUB6 (gray). EMTs often extend into the cortex, and often link to GFP-CLASP at cell edges. Dotted lines indicate cell outline. Cell edge localization is indicated by asterisk. Bar, 5 μm. <b>B.</b> Fluorescence Intensity profile plot corresponding to the EMT-cortex anchor point from panel A. Dotted arrow is drawn next to the region for the plot for reference. Grey line = MTs; Cyan line = CLASP. <b>C.</b> Confocal sections through the cell in A, starting at the surface and moving in at 0.35 μm increments. Arrowhead traces an EMT into the cell interior. The position of each section is indicated by dot blue line in the illustration below. The square box, black line, and orange line illustrate root cell, EMT, and CLASP, respectively. <b>D.</b> Quantification of GFP-CLASP/RFP-TUB6 association. (n = 5 roots, 25 cells, and 316 CLASP spots). p < 0.01, Student’s t test. <b>E.</b> Confocal sections through the cell mid-planes of root tip epidermal cells co-expressing <i>CLASP</i>::<i>TagRFP-CLASP</i> and <i>UBQ1</i>::<i>GFP-MBD</i>, showing enrichment of RFP-CLASP (orange) at sites corresponding to EMT-cortex attachment points (arrows). MTs are visualized by GFP-MBD (grey). Bar, 5 μm. <b>F.</b> GFP-CLASP localization in an elongating root epidermal cell. GFP-CLASP decorates CMTs along their lengths. Cell edge and EMT-anchor enrichment is absent. Bar, 5 μm. <b>G.</b> Confocal sections through cell mid-planes of division stage cells with strong GFP-CLASP accumulation along EMT bundles (arrows). Bar, 5 μm. <b>H.</b> Time series montage showing GFP-CLASP enrichment at sites of stable EMT-cortex attachment (arrow). Intervals between frames is 32 sec, and total time is 16 minutes. Bar, 2.5 μm. <b>I.</b> Root tip epidermal cells expressing GFP-CLASP treated with oryzalin (50 μm, 45min) or mock (45min, 0.5% DMSO). Bar, 5 μm.</p

Globular vacuole appearance and cytoplasmic instability in wild-type plants treated with oryzalin.

<p><b>A.</b> Single view (top) and 3D view (bottom) of vacuoles in wild-type transition stage cells. Vacuoles were visualized following staining with 10μm BCECF staining (10 μm) of mock (0.5% DMSO) and 50μm oryzalin-treated roots. Red is propidium iodide to visualize cell walls. Bars, 10μm. <b>B.</b> Cotyledon epidermal cells from wild-type plants expressing 35S::GFP-ɣTIP, treated for 30min with 50μm oryzalin (right), or mock 0.5% DMSO (left). Bars, 10μm. <b>C.</b> Single time-point images of root epidermal division zone cells from wild-type plants expressing <i>UBQ</i>::<i>GFP-TUB6</i> treated with mock 0.5% DMSO or 50μm Oryzalin. Images were taken after 45 minutes of treatment. <b>D.</b> Time projection (standard deviation method) of cells in A showing the enhanced vacuole/cytoplasm movement in oryzalin-treated plants. White areas indicate regions that underwent positional/morphological change over the course of observation. Images correspond to 120 second time-lapses, 4 second intervals. <b>E.</b> Color merge of two time points from same time series. Green is t = 1 and magenta is t = 120. White areas indicate no movement between time points. <b>F.</b> Kymographs corresponding to yellow lines in the two-colour merged images. Enhanced dynamicity of vacuoles mirrors that observed in <i>clasp-1</i> cells. Total time is120 seconds. Bars, 10μm. <b>G.</b> Life histories of individual vacuoles from a transition stage cell over the course of 160 seconds (4 second intervals). Different coloured traces correspond to individual vacuoles. Left image shows all time-points projected onto a single image frame for reference. Right images are tilted 3D projections. The rear and side walls are kymographs for spatial and temporal reference. Bars, 10μm. <b>H.</b> Coefficients of variation (co.var.) for vacuole area in control (0.5% DMSO) and roots treated with 50 μm oryzalin. Time between compared time points 80 second. n = 30 vacuoles for each genotype. Bars indicate Standard Error.</p

Coefficient of variation of vacuole area.

<p>Coefficient of variation of vacuole area.</p

EMT phenotypes in <i>clasp-1</i> plants.

<p><b>A.</b> Anti-tubulin immunofluorescence images of root tip epidermal cells of wild type (left) and <i>clasp-1</i> (right). EMT abnormalities are seen in all three developmental stages. Cellular mid-planes are shown for each stage, using average Z-projections. Division stage shows average projections of 4 Z-slices, corresponding to 2 μm. Transition and elongation stages show average projections of 6 Z-slices corresponding to 3 μm. Bars, 5 μm. <b>B.</b> Anti-tubulin immunofluorescence images at the outer epidermal surface of root tip epidermal cells of wild type (left) and <i>clasp-1</i> (right). Images are average projections of 4 Z-slices, corresponding to 2 μm. Bar, 5 μm. <b>C.</b> Total EMT numbers per cell in wild-type and <i>clasp-1</i> root tip epidermal cells. EMT numbers are reduced in all three developmental stages in <i>clasp-1</i> compared to wild type. n = 24 cells for each genotype; 800 ~ 1700 total EMTs wild type, 600 ~ 1000 EMTs in <i>clasp-1</i>; 3 roots in wild type and 5 roots in <i>clasp-1</i>. <b>D.</b> EMT numbers normalized for cell volumes (EMT number/cubic μm) in each developmental zone of wild type and <i>clasp-1</i>. Taking into account the larger cell volumes of <i>clasp-1</i>, the EMT reductions are more severe. <b>E.</b> Percentage of attached EMTs in <i>clasp-1</i> compared to wild type in each developmental zone. n = 24 cells for each genotype; n = ~600 EMTs <i>clasp-1</i> and ~1000 EMTs WT for each cell stage. <b>F.</b> The EMT attachment angles in <i>clasp-1</i> are smaller than in wild type in each zone of the root tip. n = 12 cells each genotype, n = 60~100 EMTs <i>clasp-1</i> and 300~400 EMTs WT for each cell stage.</p

CLASP promotes stable tethering of endoplasmic microtubules to the cell cortex to maintain cytoplasmic stability in <i>Arabidopsis</i> meristematic cells

<div><p>Following cytokinesis in plants, Endoplasmic MTs (EMTs) assemble on the nuclear surface, forming a radial network that extends out to the cell cortex, where they attach and incorporate into the cortical microtubule (CMT) array. We found that in these post-cytokinetic cells, the MT-associated protein CLASP is enriched at sites of EMT-cortex attachment, and is required for stable EMT tethering and growth into the cell cortex. Loss of EMT-cortex anchoring in <i>clasp-1</i> mutants results in destabilized EMT arrays, and is accompanied by enhanced mobility of the cytoplasm, premature vacuolation, and precocious entry into cell elongation phase. Thus, EMTs appear to maintain cells in a meristematic state by providing a structural scaffold that stabilizes the cytoplasm to counteract actomyosin-based cytoplasmic streaming forces, thereby preventing premature establishment of a central vacuole and rapid cell elongation.</p></div

Globular vacuole appearance and cytoplasmic instability in <i>clasp-1</i> mutant.

<p><b>A.</b> Vacuole morphology of transition stage cells were visualized by BCECF (100μM) and presented in single view (top) and 3D view (bottom) in both wild type and <i>clasp-1</i>. Red is propidium iodide. Bars, 10μm. <b>B.</b> Tonoplast morphology in expanding cotyledon cells of wild-type and <i>clasp-1</i> plants expressing <i>35S</i>::<i>GFP-ɣTIP</i>. Arrowheads indicate spherical dilations of vacuolar membranes in <i>clasp-1</i>. Bars, 10μm. <b>C.</b> Single time-point image of epidermal root division zone cells from wild-type and <i>clasp-1</i> plants expressing <i>UBQ1</i>::<i>eGFP</i>. Bar, 5 μm. <b>D.</b> Time projection (standard deviation method) of cells in A showing the enhanced vacuole/cytoplasm movement in <i>clasp-1</i>. White areas indicate regions that underwent positional/morphological change over the course of observation. Images correspond to 120 second time-lapses, 4 second intervals. <b>E.</b> Color merge of two time points from same time series. Green is start and magenta is t = 120 seconds later. White areas indicate no movement between time points. <b>F.</b> Kymographs corresponding to the black line in the two-colour merged images in C. The enhanced cytoplasmic movements of <i>clasp-1</i> appear as large dark areas of variable size, position, and duration. Total time is 120 seconds. <b>G.</b> Life histories of several vacuoles from transitioning cells of wild type and <i>clasp-1</i>. Different coloured traces correspond to individual vacuoles. Left image shows all time-points projected onto a single image frame for reference. Right images are tilted 3D projections. The rear and side walls are kymographs for spatial and temporal reference. Total time for each series is 160 second (4 second intervals). Bar, 5 μm. <b>H.</b> Variations in vacuolar area over time in wild-type and <i>clasp-1</i> root epidermal cells. Time series were acquired at 4 second intervals for 80 seconds, and vacuolar coefficients of variation (co. var.) were calculated for each vacuole. The bars show the means ± SE of 30 vacuoles for each genotype in each cell stage.</p

Hypothetical balloon in a box model to predict LTPG distribution patterns.

<div><p><b>A</b> Three different scenarios for inflating a balloon in a box with differentially curved walls. As the balloon inflates, LTPG accumulates passively to spaces, which appear below sharply curved regions of the cell. The box 1 is analogous to a young boxy epidermal cell. Box 2 is analogous to a bulbous mature epidermal cells. Box 3 represents the hypothetical scenario of a perfectly round/walled sphere, which results in perfectly uniform LTPG distribution. Box 4 is analogous to our cover glass flattening scenario, wherein induced flattening of a rounded surface generates small regions of curvature bordering the flattened region. </p> <p><b>B</b> Balloon in a leaky box model, wherein the naked internal walls of the epidermal cells created by the separation of underlying mesophyll cells leak apoplastic LTPG.</p></div

YFP-LTPG is excluded from cell-cell contacts and coverslip contact sites.

<div><p><b>A</b> Cotyledon epidermal cells showing inner face (yellow) and midplane (blue), and overlay of these two planes. Arrowheads show ridges of fluorescence surrounding fluorescence free zones that are in contact with underlying mesophyll cells. </p> <p><b>B</b> Contact with coverslip copies cell-cell contact clearing. 3D reconstruction tilted image from cotyledon petiole epidermal cells. Spaces occupy a plane on the outer periclinal face. Orthogonal views illustrate the planar nature of clearing (brackets), and show ridges as well. Arrow indicates normal anticlinal accumulation. Images in right panel show the top slice from the Z-series in C (top), and a maximum Z-projection of the series (bottom).</p> <p><b>C</b> Clear zones at cell-cell (left panel) and cell-coverslip (right panel) contact sites. In both cases, ridges (arrowheads) surround clear zones. </p> <p><b>D</b> Coverslip contact results in clear zones within the filamentous patterning (brackets) in mature epidermal cells.</p> <p><b>E</b> YFP-LTPG contact clear zones form and enlarge as the coverslip is appressed over time. Images are ~1 minute apart. Yellow arrowheads track leading edge of tip clearing as it spreads to the lateral walls. Red arrowheads show where two clearing from adjacent cells meet and cross over their shared anticlinal wall. See also movie S1.</p> <p>Scale bars, 5 µm.</p></div

Cell Geometry Guides the Dynamic Targeting of Apoplastic GPI-Linked Lipid Transfer Protein to Cell Wall Elements and Cell Borders in <i>Arabidopsis thaliana</i>

<div><p>During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of <i>Arabidopsis thaliana</i>. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.</p> </div

Cell geometry-dependent distribution of apoplastic YFP-LTPG.

<div><p><b>A</b> Orthogonal views showing variable anticlinal and periclinal polarization. 1 - unexpanded leaf; 2 - young hypocotyl; 3 - mature hypocotyl; 4 - Loss of anticlinal enrichment in very bulged cells.</p> <p><b>B</b> Loss of anticlinal enrichment in the bulbous cotyledon epidermal cells of <i>mor1-1</i> and <i>clasp-1</i> mutants.</p> <p><b>C</b> Flattening of the outer periclinal face of bulging epidermal cells with coverslip generates increase in fluorescence over anticlinal walls (arrowheads).</p> <p><b>D</b> Surface view showing increase in external anticlinal fluorescence (arrowhead) upon flattening of cell.</p> <p><b>E</b> Formation of radial strations (arrowheads) along anticlinal walls in periclinally flattened epidermal cells. Left panel shows mid-stage cotyledon petiole cells, right panel shows mature hypocotyl cells. Bar, 5 µm.</p></div