JCS007302 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. CAP1 associates with and depletes actin monomers from the supernatant of pulldown assays (A). Actin monomer was added to pulldown buffer to form a final solution of 2.5 μM immediately prior to the addition of glutathione beads coated with GST-CAP1 or GST alone (the final concentration of bead-bound proteins was 9 μM). Actin (either ATP-actin or actin incubated with hexokinase) was incubated with the glutathione bead preparations for 5 minutes. GST-CAP-coated beads depleted the actin monomer solution greater than control preparations, and analysis of protein associated with the glutathione beads after actin depletion confirms that monomeric actin associates specifically with CAP1. ADP-actin monomer binding was confirmed using native gel electrophoresis (B). The electrophoretic mobility of CAP1-bound actin is severely retarded as CAP1 is unable to migrate into the native gel in yeast, CAP and actin form a multimeric complex of approximately 600 kDa (Balcer et al., 2003). Arabidopsis CAP1 is capable of interacting with actin in vivo (C). Haploid yeast containing CAP1 fused to the GAL4 DNA binding domain were mated with yeast carrying actin fused to the GAL4 transcription activating domain. Both strains were also mated with yeast carrying empty GAL4 fusion vectors. All diploid strains grew on non-selective media lacking tryptophan and leucine (−W-L). Interactions between fusion proteins were assayed using the his2 reporter gene on triple dropout medium lacking supplemented histidine. Only the diploid yeast containing both CAP1 and actin were capable of growing on selective medium without histidine (−W-L-H). Mating combinations clockwise from top: CAP1+Actin, pAS2-1+Actin, pAS2-1+pACT2, CAP1+pACT2.

• Supplemental Figure S2 -

  Fig. S2. The sensitivity of the cap1 phenotype to the actin depolymerising drug latrunculin B was tested by exposing seedlings from 7 DAG to half MS growth media supplemented with 0.1% DMSO or one of two concentrations of latrunculin B dissolved in 0.1% DMSO for 48 hours (Mathur et al., 1999). After treatment plants were allowed to recover on standard half MS growth medium for 7 days. After recovery the proportion of trichomes exhibiting growth defects was recorded (using the first four leaves of six example plants for each treatment). A t-test revealed the differences between cap1-1 and Columbia wild-type to be insignificant for all treatments tested (P=0.17, 0.49 and 0.35 for 0, 0.5 and 1 μM latrunculin B, respectively). 0.35% of cap1-1 trichomes (n=619) from DMSO-only control plants morphologically resemble trichomes severely inhibited by drug-induced actin depolymerization (see Fig. 6) compared to 0% (n=704) for wild-type (this difference is reflected in the relatively low t-test P-value of 0.17). Error bars represent the standard error.

• Movie 1
• Movie 2 -

  Movies 1 and 2. Mitochondria of wild-type (Movie 1) and cap1 (Movie 2) root hairs were labelled using mitotracker Red CMXRos. Mitochondria in wild-type root hairs follow the reverse-fountain pattern of cytoplasmic streaming, moving rapidly along vectors parallel to the axis of root hair expansion. Mitochondria within cap1 root hairs appear to move in random patterns with little evidence of reverse-fountain streaming. Frames of Movie 1 were captured at 600 millisecond intervals and frames of Movie 2 were captured at 1.6 second intervals. Both movies are presented at 20 frames per second.

• Movie 3
• Movie 4 -

  Movies 3 and 4. Movies were taken over a 5-day period and are presented at 10,000× normal speed. Movie 3 shows the nastic circumnutation movements of developing wild-type Arabidopsis inflorescences. Movie 4 shows plants homozygous for cap1-2 with suppressed cirumnutation movements (cap1-1 homozygotes show a similar inhibition). In Movie 4, two secondary cap1 inflorescence-heads to the left of the frame have achieved a complete 360° rotation relative to their own stem. Approximately 3 seconds after the start of the movie the inflorescences undergo a rapid uncurling process. During the course of Movie 4 multiple cap1 inflorescences oscillate between pointing towards and then away from the gravity vector.