The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Abstract

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.

Figure 1.

At CLASP Protein Domain Organization, Gene Phylogeny, and Expression. (A) Protein domain organization of CLASP family members. HEAT repeats are black, TOG domains are gray, and CLIP-interacting domains of human orthologs are stippled. Arrows at top indicate conserved regions. (B) Phylogenetic tree of chTOG domain proteins. Both XMAP215/MOR1 and CLASP/ORBIT/MAST (circled) families are shown. (C) Expression of At CLASP as determined by RT-PCR. S, stems; FB, floral buds; CL, cauline leaves; RL, rosette leaves; IF, inflorescences; R, roots; C, cotyledons; WS, whole seedlings.

Figure 2.

GFP-CLASP Is a +TIP That Localizes to All Microtubule Arrays and Induces Stable Microtubule Bundles at High Expression Levels. (A) Stable low expression of GFP-CLASP results in microtubule labeling with enrichment at growing microtubule plus ends in leaf guard cells (arrowheads). (B) Kymograph of the boxed region surrounding a single microtubule. The microtubule initially grows and then switches to depolymerization, at which point plus end accumulation is lost. Time is 10 s between frames. (C) Stable moderate (left panel) and high (middle and right panels) expression of GFP-CLASP in a leaf epidermal pavement cell. (D) Transient high expression of 35S:GFP-CLASP in a tobacco leaf epidermal cell. (E) Localization of GFP-CLASP to mitotic microtubule arrays when stably expressed in tobacco BY-2 cells. The arrow indicates spindle midzone accumulation during anaphase. Bars = 5 μm.

Figure 3.

clasp-1 Null Mutant Plants Exhibit Dwarfed Stature. (A) Structure of the CLASP gene and positions of Salk T-DNA insertions. (B) RT-PCR showing lack of CLASP transcript in 7-d-old whole seedlings of clasp-1 mutants. (C) Seven-day-old seedlings. (D) Lengths of hypocotyls in 5-d-old etiolated seedlings. Error bars represent sd. (E) Twenty-three-day-old plants. (F) Forty-four-day-old plants.

Figure 4.

Root Growth Is Retarded in clasp-1 Mutants. (A) Lengths of primary roots in wild-type and clasp-1 seedlings. Diamonds, wild type; squares, clasp-1. (B) Images of typical wild-type and clasp-1 roots. The graph at right illustrates the root growth parameters measured in this study. DZ, division zone; EZ, elongation zone. Width denotes mature root width. (C) Division zone plus elongation zone lengths. (D) Widths of roots at the division zone and mature region (MZ). Error bars represent sd.

Figure 5.

Cell Expansion Is Abnormal in clasp-1 Mutants. (A) Cell lengths in epidermal and cortical tissues of mature roots. Error bars represent sd. (B) Scanning electron micrographs of 5-d-old etiolated hypocotyls. Bar = 100 μm. (C) Scanning electron micrographs of 14-d-old leaf epidermal cells. Bar = 100 μm. (D) Leaf epidermal cell lobing parameters. Arrows depict lobe length (left) and neck width (right). Error bars represent sd.

Figure 6.

Microtubule Organization in Wild-Type and clasp-1 Root Tips. Antitubulin immunofluorescence staining of 7-d-old wild-type (A) and clasp-1 (B) primary root tips. Images are montages of confocal micrographs representing one to three compressed Z images. Bars in insets = 10 μm.

Figure 7.

clasp-1 Null Mutants Are Hypersensitive to Oryzalin. Data are mean root widths ± sd.

Figure 8.

The Organization of Mitotic and Cytokinetic Microtubule Arrays in clasp-1 Is Abnormal. Antitubulin immunofluorescence staining of root squashes of 7-d-old plants is shown. (A) Early prophase cells containing a broad PPB. The nucleolus in the wild type is indicated with an asterisk. (B) Late prophase cells containing narrow PPBs and bipolar prophase spindles. Note the broad and disorganized clasp-1 PPB in the right panel. This cell is beginning nuclear envelope breakdown. (C) Metaphase spindles. (D) Anaphase spindles. (E) Phragmoplast during cytokinesis. Brackets denote PPB width and spindle and phragmoplast length. Arrowheads denote indentations of the prophase spindle and nucleus. Green, microtubules, blue, 4′,6-diamidino-2-phenylindole. Bars = 5 μm.