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First published online 30 September 2008
doi: 10.1242/jcs.030940

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G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers

¹ Department of Pharmacology, Kyoto University Faculty of Medicine, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
² Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyoku, Tokyo 113-0032, Japan
³ Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Honcho, Kawaguchi-shi, Saitama 332-0012, Japan

View larger version (35K): [in this window] [in a new window]   Fig. 1. The behavior of wild-type mDia1 in XTC cells. (A) Single-molecule imaging of EGFP-mDia1Full speckles reveals different types of motility. Red lines show trajectories of mDia1Full speckles observed within a 6 second time window. Blue dotted lines indicate the contour of the cell edge (left). Representative trajectories of random (middle) and processive (right) mDia1Full speckles are shown. (B) mDia1Full speckles found within a 6 second time window are classified into the indicated groups (n=11 cells) as described within the text. (C) Motility of mDia1Full speckles in the random group is independent of actin polymerization. Random speckles were still observed 1 minute after 1 µM CytD or 1 µM LatB treatment. Trajectories of mDia1Full speckles found within a 6 second time window are shown.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Latrunculin B at low dose induces processive movement of mDia1. (A) Processive movement of EGFP-mDia1Full was induced after 100 nM LatB perfusion. Arrowheads indicate mDia1Full speckles moving processively at indicated time points. Marked speckles moved in one direction for at least five consecutive frames. Scale bar: 5 µm. (B) Number of speckles moving processively after perfusion with 100 nM LatB. Each colored line indicates data from an individual cell. Dotted black line indicates the average speed of processive speckles in five cells. (C) Normalized frequency of processive mDia1 speckles in cells treated with various concentrations of LatB for 100 seconds. Normalized frequency was calculated by dividing the number of speckles by total intensity of EGFP fluorescence in the imaged region. (D) F-actin structures were well preserved 90 seconds after 100 nM LatB treatment except for a gradual loss in the EGFP-actin signal (12%) in the lamellipodial actin meshwork. Scale bar: 5 µm. (E) The density of capping protein speckles (EGFP-CPβ1) did not change before and 80 seconds after 100 nM LatB treatment. Scale bar, 2 µm. (F) Treatment of EGFP-mDia1Full expressing cells with 500 nM SwinA. Arrowheads indicate mDia1Full speckles moving processively. Scale bar: 5 µm. (G) The number of processive mDia1Full speckles before and 11 minutes after 500 nM SwinA perfusion. Data were analyzed using a two-tailed Student's t-test. *P<0.02. (H) Flag-tagged actins, WT (wild-type), R62D or G13R, were coexpressed with EGFP-mDia1Full. Normalized frequency of processive mDia1 speckles in cells expressing actin WT, R62D or G13R is shown. Data were normalized by dividing the number of processive speckles by total intensity of EGFP fluorescence in the imaged region (mean ± s.d., n=5 cells for WT; n=12 cells for G13R; n=16 cells for R62D) and analyzed using a two-tailed Student's t-test. *P<0.02; **P<0.01.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Rho is required, but the FH2 region is sufficient for processive movement induced by LatB. (A) Cells were electroporated in the presence (right) or absence (left) of C3-exoenzyme and allowed to spread on the PLL-coated glass coverslips for 30 minutes. Phalloidin staining shows the loss of actin stress fibers in C3-treated cells indicating the efficient incorporation of C3-exoenzyme. Scale bar: 20 µm. (B) Cells expressing EGFP-mDia1Full were electroporated in the presence (lower panels) or absence (upper panels) of C3. Then time-lapse images were taken before and 80 seconds after treatment with 100 nM LatB. Arrowheads indicate processively moving mDia1Full speckles. Scale bar: 5 µm. (C) The number of processive mDia1Full speckles in the electroporated cells was counted before and 100 seconds after 100 nM LatB perfusion. The same area within each cell was used for measurement before and after the LatB treatment. C3 inhibited the induction of processive speckles by LatB. (n=5 cells; **P<0.01, two-tailed paired t-test.) (D) A low dose of LatB increases the number of processive speckles of the FH2 region mutant (mDia1F2) and the FH1-FH2 region mutant (mDia1N3) in a C3-insensitive manner. Cells expressing EGFP-mDia1F2 or EGFP-mDia1N3 were electroporated in the presence or absence of C3. For mDia1F2, speckles were counted before and 50 seconds after 50 nM LatB perfusion. For EGFP-mDia1N3, speckles were counted before and 80 seconds after 100 nM LatB perfusion. The same area within each cell was used for the measurement. (*P<0.03 and **P<0.005, two-tailed paired t-test.) Since the measured cell areas are different, the number of processive speckles is not comparable between different constructs.

View larger version (33K): [in this window] [in a new window]   Fig. 4. LatB treatment paradoxically increases free G-actin concentration in the cell. (A) Effects of LatB on actin polymerization mediated by mDia1F2. Representative results showing no obvious promoting effect of LatB on mDia1F2-induced actin assembly. Actin (1 µM, left panel; 3.3 µM, right panel; 20% pyrene labeled) was polymerized in the presence of various concentrations of LatB with 21.8 nM (left) or 10 nM (right) mDia1F2. (B) Simulation analysis reveals a paradoxical increase in free G-actin upon low-dose LatB treatment. This kinetic model (bottom) concerns changes in the concentration of G-actin (G), either free or bound to thymosin-β4 (T), profilin (P) and LatB (L). The graphs show simulation at 0.1 µM LatB, 0.5 µM free G-actin at t=0, 115.5 µM total G-actin at t=0, 240 µM total actin, 140 µM total thymosin-β4 (40 µM free Tb4 at t=0) and 18 µM total profilin (3 µM free profilin at t=0). On the right, curves for five parameters are enlarged. Note the rapid increase in free G-actin despite the accumulation of LatB to 5 µM in the cytoplasm. (C) Estimation of the equilibrium state also predicts an increase in free G-actin. Relative F-actin contents were quantified by phalloidin binding in cells treated with 100 nM LatB for 0, 1 and 2 minutes (line, mean ± s.d. of three separate experiments; n=275 cells, n=245 cells and n=201 cells, respectively). Dashed line and columns show changes in total G-actin and free G-actin, respectively, estimated by calculating the equilibrium state between G-actin and its interacting molecules based on the observed F-actin decrease.

View larger version (42K): [in this window] [in a new window]   Fig. 5. Overexpression of mDia1Full prevents F-actin disassembly induced by low-dose LatB treatment. (A) F-actin structures visualized by fluorescent phalloidin before and after 100 nM LatB treatment. Note the well preserved thin actin fiber architecture in cells overexpressing EGFP-mDia1Full (arrowheads), whereas F-actin structures were gradually disrupted in nontransfected cells. Scale bar: 10 µm. (B) Cells transfected with pEGFP-mDia1Full were fixed and stained with fluorescent phalloidin before and 8 minutes after 50 nM LatB treatment. Total intensity of the phalloidin signal was measured in each transfected and nontransfected cell. F-actin did not decrease significantly in EGFP-mDia1-expressing cells [mean ± s.d.; non-transfected cells, n=143 (before), n=265 (after); EGFP-mDia1 positive cells, n=42 (before), n=53 (after)]. Data were analyzed using a two-tailed Student's t-test; ***P<0.001.

View larger version (81K): [in this window] [in a new window]   Fig. 6. Frequent actin nucleation by mDia1 around sites of vigorous actin disassembly. (A) Images of EGFP-mDia1F2 (left) and mPlum-AIP1 (middle) were acquired in a live cell. Cells were then fixed, and stained with phalloidin in order to visualize F-actin (right). Note the clusters of mDia1F2 speckles (arrowheads) around the area labeled strongly with AIP1, a cofactor of cofilin. (B,C) Frequency of mDia1 speckle appearance is biased toward the region of high AIP1 concentration. The images show the position of mDia1F2 appearance (B, diamonds) or mDia1Full speckle appearance (C, white dots), mDia1Full trajectories (C, pink circles) and the intensity of mPlum-AIP1 fluorescence (pseudocolor). In the graphs, the x, y position of individual pixels within an mPlum-AIP1 image was binned into ten equal areas according to their fluorescence intensity values. Intensity-sorted positions were then used to correlate the appearance of mDia1 speckles with the local intensity of mPlum-AIP1 fluorescence (B, n=158, 1 cell; C, n=405, 11 cells). mDia1F2 speckles frequently emerged in areas strongly labeled with mPlum-AIP1. Frequency of mDia1Full speckle appearance shows positive correlation with the intensity of mPlum-AIP1 (P<0.01). Scale bars: 5 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 7. Effects of low-dose LatB on the activity of other actin nucleators. (A) Processive movement of EGFP-FRL1 was induced 40 seconds after 100 nM LatB perfusion. Arrowheads indicate FRL1 speckles moving processively. Owing to the decrease in speed, the speckles that moved in one direction for at least seven consecutive frames are labeled as processive after LatB treatment. Dotted lines indicate cell contour. The graph shows the number of EGFP-FRL1 speckles moving processively before and 50 seconds after treatment. Each colored line represents data from an individual cell. Data were analyzed using a two-tailed paired t-test; **P<0.01. (B) No obvious change in the density of speckles of the Arp2/3 complex was observed after 100 nM LatB treatment. Images were taken before and 90 seconds after treatment. The graph shows the number of single-molecule EGFP-p40 speckles before and 90 seconds after treatment (mean ± s.d., n=5 cells). The areas devoid of densely packed speckles were used for analysis. The loss of EGFP-p40 signal between the two conditions as a result of photobleaching is less than 10%. Scale bars: 2 µm.

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