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Files in this Data Supplement:
Fig. S5. See figure file for legend.
Movie 1. F-actin does not flow at a similar rate to mDia1ΔN3 movement in cells. Images of tropomyosin (TPM) speckles (top; used as a marker for the F-actin flow) were taken at the intervals of 200 msecond in cells expressing mPlum-mDia1ΔN3 (bottom). There was no apparent fast movement of TPM speckles even in the region where mDia1ΔN3 accumulated at the periphery. The rate of retrograde flow (0.014 µm/second; measured by TPM-EGFP acquired at 5 second intervals in the same cell) was 63 times slower than the speed of fast moving processive mPlum-mDia1ΔN3 speckles (0.88 µm/second). Time is in seconds. Scale bar, 5 µm.
Fig. S1. Characterization of mDia1Full behavior in XTC Cells. (A) Single-molecule imaging of EGFP-mDia1Full speckles revealed different types of the motility. mDia1Full speckles found within a 6 second time window are classified into the indicated groups. Each color indicates data from the same cell (n=11 cells). Average percentages for each behavior are shown in Fig. 1B. (B) Classification of mDia1Full speckles in LatB-treated cells (Fig. 2B). The numbers of speckles that existed in one frame at the indicated time points was measured and classified into four groups. Most randomly moving speckles after perfusion might not be detected because of altered acquisition intervals for detecting processive mDia1Full speckles moving at the slower rate.
Fig. S2. Free barbed ends visualized by Rhodamine-labelled actin incorporation did not increase upon low-dose LatB treatment. Cells were permeabilized with 0.1% Triton X-100 for 10 seconds, and labelled with Rhodamine-actin (10%, 0.5 µM) for 10 seconds. Upper panels show distribution of incorporated Rhodamine-actin in cells with or without LatB treatment (100 nM, 1 minute) before permeabilization. The graph shows total cellular Rhodamine-actin fluorescence (mean ± s.d.; n=62 cells for untreated control; n=41 cells for cells treated with LatB for 1 minute). Note the redistribution of free barbed ends from the periphery to the central region of cells induced by low-dose LatB treatment. These results are in agreement with the negative response of capping protein speckles to low-dose LatB (Fig. 2E).
Fig. S3. Expression of nonpolymerizable actin mutants induce processive movement of mDia1 without changing F-actin structures. (A) Flag-tagged actins, WT (wild-type), R62D or G13R, were coexpressed with EGFP-mDia1Full. Arrowheads indicate mDia1Full speckles moving processively (top panels). Cells were fixed and stained for Flag-tags after time-lapse observations (bottom panels). Boxed regions indicate the area of the time-lapse images. Note that diffuse cytoplasmic staining of R62 and G13R actins, indicating the deficient polymerization of these mutants. See also Movie 5. Scale bars, 5 µm (top) and 20 µm (bottom). (B) Flag-tagged actins, WT, R62D or G13R, were coexpressed with EGFP-mDia1Full. Arrowheads indicate mDia1Full speckles moving processively (top panels). Cells were fixed and stained by fluorescent phalloidin (bottom panels). Boxed regions indicate the area of the time-lapse images.
Fig. S4. Effects of LatB on actin nucleation mediated by mDia1F2. (A) To eliminate the effect of LatB on the reduction of the actin elongation rate, the number of filaments polymerized with mDia1F2 and with or without LatB was examined. Actin (2 µM) was polymerized for 4 minutes in the presence or absence of 100 nM LatB with 30 nM mDia1F2. The efficiency of filament formation was monitored by diluting 10 times with 2 µM actin (10% pyrene-labeled). For the control, a diluted concentration of mDia1F2 (column 4) or mDia1F2 and LatB (column 5) were added to monitor the effect on polymerization. Note that there was no significant acceleration in the polymerization rates between mDia1F2 (column 2) and mDia1F2 plus LatB (column 3). Experiments were performed as described previously (Suetsugu et al., 2001) and pyrene fluorescence (excitation 365 nm, emission 407 nm) was monitored by FP-6500 (JASCO). Data are average slope of the first 5 minutes of six experiments. Error bars show s.d. (B) Representative results showing no obvious promoting effect of LatB on mDia1F2-induced actin assembly in the presence of profilin. Actin (4 µM, 7.9% pyrene labeled) was polymerized in the presence of profilin (left panel; 1 µM, right panel; 2 µM), 20 nM mDia1F2 and with various concentrations of LatB. Pyrene fluorescence (excitation 365 nm, emission 407 nm) was monitored by Envision 2103 Multilabel Reader (Perkin Elmer).
Fig. S6. Colocalization of AIP1 and the Xenopus cofilin XAC2 in XTC cells. Cells transfected with indicated constructs were seeded on PLL-coated glass coverslips and allowed to spread for 45 minutes. Cells were then fixed and stained either for AIP1 or XAC2 using specific antibodies (A-C). In D, Images of a live cell coexpressing mPlum-AIP1 and XAC2EGFP are shown. Note the nearly identical localization of AIP1 and XAC2 both for endogenous proteins and tagged constructs at the cell periphery. Scale bars, 5 µm.
Movie 2. A low dose of LatB induces processive movement of EGFP-mDia1Full. Images were taken before and 10, 20 and 84 seconds after 100 nM LatB perfusion (the same cell as in Fig. 2A). Time-lapse imaging was carried out intermittently by altering the acquisition intervals in order to track gradually slowing mDia1 movement. Only a few mDia1Full speckles exhibited processive movement before the perfusion. The density of mDia1Full speckles moving processively gradually increased after the perfusion. Time is in minutes:seconds. Scale bar, 5 µm.
Movie 3. LatB did not affect density of capping protein speckles. The density of capping protein speckles did not increase after 100 nM LatB treatment. LatB was perfused at 1:09. Time is in minutes:seconds. Scale bar, 2 µm.
Movie 4. Swinholide A induces processive movement of EGFP-mDia1Full. The left and right movies show time-lapse images of mDia1Full speckles taken before (left) and 14 minutes after treatment (right) with 500 nM swinholide A, respectively (Fig. 2F). Green circles indicate the speckles moving in one direction for at least five consecutive frames. Time is in seconds. Scale bar, 2 µm.
Movie 5. Nonpolymerizable actin mutant, G13R, but not wild-type actin induced processive movement of mDia1Full. FLAG-tagged wild-type (left) or G13R actin (right) was coexpressed with EGFP-mDia1Full. Processively-moving mDia1Full speckles were frequently observed in cells expressing nonpolymerizable actin mutants (Fig. 2H and Fig. S3). Green circles show the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 2 µm.
Movie 6. C3-exoenzyme inhibited processive movement of mDia1Full induced by LatB. Cells were electroporated in the absence (left) or presence (right) of C3-exoenzyme (Fig. 3B). Time-lapse images were taken 60-70 seconds after 100 nM LatB treatment. Green circles show the mDia1Full speckles moving in one direction for at least five consecutive frames. Time is in seconds. Scale bar, 2 µm.
Movie 7. mDia1F2 is activated by LatB. The number of processively moving speckles of mDia1F2 increased after 50 nM LatB treatment. Identical results were observed in the cells treated with C3 (Fig. 3D). The left and right movies show images before and 30 seconds after perfusion of 50 nM LatB, respectively. Time is in minutes:seconds. Scale bar, 5 µm.
Movie 8. mDia1ΔN3 is activated by LatB. The density of processively moving speckles of mDia1ΔN3 increased after 100 nM LatB treatment. Identical results were observed in cells treated with C3 (Fig. 3D). The left and right movies show images before and 60 seconds after treatment, respectively. Time is in minutes:seconds. Scale bar, 5 µm.
Movie 9. mDia1F2 is frequently activated around the sites of vigorous actin disassembly. Images of EGFP-mDia1F2 and mPlum-AIP1 coexpressed in XTC cells were acquired by occasionally alternating the filters (Fig. 6B). The images of mPlum-AIP1 are labelled with ‘AIP1’ on the top right. Note that the areas of frequent mDia1F2 appearance coincide with the moving clusters of AIP1 signals. Time is in seconds. Scale bar, 2.5 µm.
Movie 10. FRL1 is activated by LatB. The number of processively moving speckles of EGFP-FRL1 increased 40 seconds after 100 nM LatB treatment (Fig. 7A). The left and right movies show images before and 40 seconds after the treatment, respectively. Time is in seconds. Scale bar, 2 µm.
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