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First published online 27 November 2007
doi: 10.1242/jcs.024950

Journal of Cell Science 120, 4416-4425 (2007)
Published by The Company of Biologists 2007
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CLASP localizes in two discrete patterns on cortical microtubules and is required for cell morphogenesis and cell division in Arabidopsis

Viktor Kirik1,2,*,{ddagger}, Ullrich Herrmann1,*, Chaithanyarani Parupalli3, John C. Sedbrook3, David W. Ehrhardt2,§ and Martin Hülskamp1,§

1 University of Cologne, Botanical Institute III, Gyrhofstr. 15, 50931 Cologne, Germany
2 Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, CA 94305, USA
3 Department of Biological Sciences, Illinois State University, Campus Box 4120, Normal, IL 61790, USA


Figure 1
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  		Fig. 1. Molecular characterization of the Arabidopsis CLASP gene. (A) The Arabidopsis CLASP gene consists of 20 exons and 19 introns. The positions of T-DNA insertions of the three alleles are indicated with arrowheads. (B) RT-PCR analysis of AtCLASP expression in the clasp mutant alleles. Elongation Factor 1{alpha} (EF1) was used as a control for RNA extraction and amplification. (C) AtCLASP:GUS expression in the shoot.

 

Figure 2
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  		Fig. 2. Effect of the clasp mutation on shoot development. (A) Flowering wild-type plant. (B) Flowering clasp plant of the same age as that in A. (C) Comparison of wild-type (left) and clasp mutant (right) 5-day-old seedlings. Note the shortened hypocotyl and root in the mutant. (D) Scanning electron micrograph (SEM) of hypocotyl epidermis of a 5-day-old wild-type seedling. (E) SEM of a comparable clasp hypocotyl epidermis. Note the shortened and bulged epidermal cells. (F) Rosette of a wild-type plant. (G) Rosette of a clasp plant the same age as in F. Scale bar: 100 µm for D and E.

 

Figure 3
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  		Fig. 3. Root growth defects in clasp mutants. (A) Ten-day-old wild-type Columbia (Col) and clasp seedlings. Right panel shows a magnified image of the clasp seedlings. Note increased formation of lateral roots. (B) CYCB1;1:GUS expression in wild-type and clasp root tips. Brackets indicate a zone of active cell division as highlighted by CYCB1;1:GUS expression. Note the smaller number of stained cells and the reduction of the zone in which GUS-stained cells are observed. (C) Comparison of the cell elongation zone in the wild type and the clasp mutant root. Brackets span the position of the first elongated cell in the epidermis and the first cell initiating root hair formation. (D) Quantification of cell number in the root elongation zones of wild-type and mutant seedlings. n=12 roots for clasp seedlings and 17 roots for the wild type. Error bars indicate s.d. The differences between means for both epidermal and cortical cells is significant by Student's t-test, P<0.001. (E) Quantification of mature epidermal cell length in wild-type and clasp roots. n=28 cells for wild type and n=51 cells for clasp mutants. The difference in means is significant by Student's t-test, P<0.001. Scale bars: 100 µm (B), 150 µm (C).

 

Figure 4
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  		Fig. 4. Cell morphogenesis defects in clasp mutants. (A) Wild-type (Col) and clasp mutant trichomes. The graph depicts the fraction of trichomes with different numbers of branch-points. (B) Comparison of trichome branch alignment on the an mutant and an clasp double mutant rosette leaf. Right panel shows the distribution of trichome branch alignment angles (degree) relative to the leaf longitudinal axis. 290 an and 160 an clasp trichomes were measured in total. (C) Wild-type and clasp mutant pavement cells. As a measurement of puzzle cell shape, cell shape factor (4{pi} x area/perimeter2) of the clasp mutant and wild type is compared in the right panel. Scale bar: 200 µm (A), 50 µm (C).

 

Figure 5
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  		Fig. 5. MT organization in the upper hypocotyl cells of the clasp mutant. (A) Upper hypocotyl region of a 7-day-old wild-type (Col) seedling. (B) Upper hypocotyl region of a 7-day-old clasp mutant seedling. Microtubules are labeled with CFP:TUA4. Images are montages of projected confocal image stacks. Scale bar: 30 µm.

 

Figure 6
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  		Fig. 6. Overexpression of AtCLASP and {Delta}C-AtCLASP induces spiral arrangement of cortical MTs. (A) Spiral arrangement of pavement cell MTs (CFP:TUA4) in a transgenic line with high expression of YFP:AtCLASP. (B) Enhanced spiral MT phenotype and clear MT bundling in pavement cells of a transgenic line overexpressing YFP: {Delta}-AtCLASP. (C) Pavement cells expressing CFP:TUA4 only. Images are projections of confocal image stacks. Scale bar: 20 µm.

 

Figure 7
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  		Fig. 7. YFP:AtCLASP colocalization with MTs in interphase cells. (A) Colocalization of YFP:AtCLASP driven from the native promoter and CFP:TUA4. (B) Magnification of a subregion of the cell shown in A. (C) Co-localization of YFP:AtCLASP driven from the 35S promoter at a relatively low level of expression and CFP:TUA4. Note spots of the AtCLASP accumulation on the MTs and prominent MT lattice labeling. An oryzalin-treated cell is depicted in the two right-hand images. All images were acquired in hypocotyl cells of 3-day-old seedlings. Scale bars: 5 µm (A,C), 2 µm (B).

 

Figure 8
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  		Fig. 8. Localization of AtCLASP to mitotic arrays. (A) YFP:AtCLASP expressed from the native promoter in dividing root cells, MTs are labeled by expression of CFP:TUA4. On the right is the merged image with YFP:AtCLASP in the red channel and CFP:TUA4 in the green. Note prominent colocalization of YFP:AtCLASP and MTs at the PPB and relatively low signal intensity of YFP:AtCLASP on the spindle MTs. (B) Cells labeled as in A undergoing cytokinesis. Note that phragmoplast MTs are prominently labeled by CFP:TUA4 and almost undetectable by YFP:CLASP. Arrowheads indicate the preprophase band; asterisks depict an anaphase cell with spindle; arrows point to phragmoplast MTs. Scale bars: 10 µm.

 

Figure 9
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  		Fig. 9. YFP:AtCLASP is enriched at the MT plus end. (A) Time-series images acquired at the cortex of a hypocotyl cell expressing CFP:EB1 and YFP:AtCLASP proteins, both expressed from the 35S promoter. Both proteins show localization to the growing ends of cortical MTs (arrowheads). (B) The relative position of EB1 and CLASP was determined by plotting the normalized fluorescent intensity (NFI) along the MT ends in a 5-pixel-wide lane. Normalized fluorescent intensity (NFI) of CFP:EB1 is in red and YFP:AtCLASP in green. The graph shows the mean values of 12 measurements. The measured difference in peak maxima position is significant by Student's t-test, P<0.001. A magnified image of representative MT end is shown below. Scale bar: 2 µm.

 

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© The Company of Biologists Ltd 2007