TY - JOUR
AU - Wasteneys, Geoffrey O.
AB - Abstract Numerous forms of cytochalasins have been identified and, although they share common biological activity, they may differ considerably in potency. We investigated the effects of cytochalasins A, B, C, D, E, H and J and dihydrocytochalasin B in an ideal experimental system for cell motility, the giant internodal cells of the characean alga Nitella pseudoflabellata. Cytochalasins D (60 μM) and H (30 μM) were found to be most suited for fast and reversible inhibition of actin-based motility, while cytochalasins A and E arrested streaming at lower concentrations but irreversibly. We observed no clear correlation between the ability of cytochalasins to inhibit motility and the actual disruption of the subcortical actin bundle tracks on which myosin-dependent motility occurs. Indeed, the actin bundles remained intact at the time of streaming cessation and disassembled only after one to several days’ treatment. Even when applied at concentrations lower than that required to inhibit cytoplasmic streaming, all of the cytochalasins induced reorganization of the more labile cortical actin filaments into actin patches, swirling clusters or short rods. Latrunculins A and B arrested streaming only after disrupting the subcortical actin bundles, a process requiring relatively high concentrations (200 μM) and very long treatment periods of >1 d. Latrunculins, however, worked synergistically with cytochalasins. A 1 h treatment with 15 nM latrunculin A and 4 μM cytochalasin D induced reversible fragmentation of subcortical actin bundles and arrested cytoplasmic streaming. Our findings provide insights into the mechanisms by which cytochalasins and latrunculins interfere with characean actin to inhibit motility. Introduction Actin-targeted fungal metabolites known as cytochalasins have been used extensively to understand the role of F-actin in different aspects of cellular function (Petersen and Mitchison 2002). It is generally acknowledged that these compounds cap the plus ends, and thereby alter the dynamic properties of actin filaments. Intracellular motility in plant cells is predominantly generated by myosin-dependent movement of organelles along actin filaments (Staiger et al. 2000). In the giant internodal cells of characean algae, cytochalasin B (CB) and cytochalasin D (CD) rapidly inhibit cytoplasmic streaming without disassembling the prominent and highly stable actin bundles located at the interface of the stationary cortex and the motile endoplasm (Chen 1973, Bradley 1973, Williamson 1975, Foissner and Wasteneys 2000b). In contrast, the delicate cortical actin filaments near the plasma membrane are readily reorganized into short stable rods upon application of CB and CD (Collings et al. 1995), and mechanical disruption of the subcortical actin bundles by wounding allows CD to reorganize them extensively into thick networks (Foissner and Wasteneys 1999). These and other findings indicate that cytochalasins have greater nucleating and bundling properties when actin networks are relatively labile (e.g. Goddette and Frieden 1986). Nevertheless, it is still a mystery how cytochalasins inhibit and arrest organelle motility without affecting the structure or polymer status of the long, continuous bundles of actin filaments along which these organelles move. Several different cytochalasins are commercially available, and structure–activity relationships have shown that subtle changes in molecular structure can result in dramatic differences in specific activities (Walling et al. 1988). One aim of the present study was to exploit the highly organized and reproducible cytoplasmic features of characean giant internodal cells to compare and quantify the effects of different cytochalasins on cytoplasmic streaming and on the arrangement of the actin cytoskeleton. Specifically, we wanted to determine if the ability to arrest cytoplasmic streaming is correlated with the ability of a given cytochalasin to alter the structural organization of actin filament arrays in an attempt to clarify the role of actin filament reorganization in streaming inhibition. We therefore undertook a rigorous evaluation of the effects of eight different cytochalasins in a broad concentration range, on both actin filament architecture and the inhibition and recovery of cytoplasmic streaming. With the discovery of latrunculins isolated from sea sponges, other actin-perturbing drugs with a more defined and less complex action became available (Spector et al. 1983). Latrunculins are monomer sequestering agents and disassemble the actin cytoskeleton of rapidly growing cells such as pollen tubes at nanomolar concentrations (e.g. Gibbon et al. 1999, Vidali et al. 2001, Foissner et al. 2002). During the course of this study, we also investigated the effects of latrunculins A (LatA) and B (LatB) and found that, unlike studies with higher plants, they are not able rapidly and reversibly to inhibit cytoplasmic streaming in characean internodal cells. Latrunculins, however, were found to work synergistically with cytochalasins, providing greater insight into the mechanisms by which cytochalasins and latrunculins interact with actin filament networks. Results The cytoplasm of characean internodal cells consists of a stationary cortex including files of helically arranged chloroplasts and a streaming endoplasm (Fig. 1A). The plasma membrane-associated, or cortical, actin cytoskeleton of untreated, non-elongating characean internodes consists of randomly oriented, delicate, often bent actin strands, and thicker, straight bundles which are usually extensions of the subcortical actin bundles located along the inner side of the stationary chloroplast files (Fig. 1A, B). These bundles consist of up to 100 single actin filaments and generate endoplasmic mass streaming through the interaction with myosin associated with the membranes of endoplasmic reticulum cisternae (Fig. 1A, C; recent reviews by Foissner and Wasteneys 2000a, Grolig and Pierson 2000, Shimmen and Yokota 2004). Fig. 1 View largeDownload slide Cytoplasmic and cytoskeletal organization in characean internodal cells. (A) Schematic longitudinal section showing the cell wall (W), the stationary ectoplasm (EC) including files of helically arranged chloroplasts (C) and the streaming endoplasm (EN). The cortical cytoskeleton (left arrow) consists of actin filaments (thin lines and circles) and microtubules (thick lines and circles) near the plasma membrane. Subcortical actin bundles (right arrow) are present along the inner side of the chloroplasts. (B) Randomly oriented actin strands and thicker bundles in the cortex of a non-elongating internode. (C) Continuous subcortical actin bundles along the chloroplast files. F-actin was visualized by perfusion of cells with fluorescent phalloidin. Scale bar for b and c = 10 μm. Fig. 1 View largeDownload slide Cytoplasmic and cytoskeletal organization in characean internodal cells. (A) Schematic longitudinal section showing the cell wall (W), the stationary ectoplasm (EC) including files of helically arranged chloroplasts (C) and the streaming endoplasm (EN). The cortical cytoskeleton (left arrow) consists of actin filaments (thin lines and circles) and microtubules (thick lines and circles) near the plasma membrane. Subcortical actin bundles (right arrow) are present along the inner side of the chloroplasts. (B) Randomly oriented actin strands and thicker bundles in the cortex of a non-elongating internode. (C) Continuous subcortical actin bundles along the chloroplast files. F-actin was visualized by perfusion of cells with fluorescent phalloidin. Scale bar for b and c = 10 μm. Inhibition of cytoplasmic streaming and recovery The effect of cytochalasins on cytoplasmic streaming in characean internodes is age dependent. Younger cells are more susceptible and require lower inhibitor concentrations for streaming arrest than older cells (Collings et al. 1995). In this study, we used non-elongating internodes of approximately the same age, which were collected from the same container in order to avoid any differences between culture batches. We use the terminology from a previous paper (Foissner and Wasteneys 2000b), in which cytochalasin concentrations were defined as follows: streaming-arresting concentration = cytochalasin concentration at or above the level required to arrest streaming within 1 h; streaming-inhibiting concentration = cytochalasin concentration at which streaming velocity is reduced by 15–80% within 1 h but not arrested. The streaming velocity in untreated control cells was about 80 μm s−1 at room temperature. Fig. 2 shows the time course of streaming inhibition and recovery of the various cytochalasins used in this study, and Fig. 3 illustrates the mean relative velocities of cytoplasmic streaming after 1 h recovery from a 1 h treatment at the predetermined streaming-arresting concentration for each cytochalasin, maximum recovery velocities and times required for maximal recovery. The streaming-arresting concentrations of cytochalasins A, B, D, E, H and dihydrocytochalasin B (CA, CB, CD, CE, CH and DHCB) varied between 1 and 200 μM (Fig. 2). Recovery to control rates of cytoplasmic streaming after 1 h treatment with streaming-arresting concentrations took about 1 h with CD and CH, and several hours with CB and DHCB. Cells treated with CA or CE recovered to ≤80% of the original velocities, and these values were reached only after 3 and 5 d, respectively (Fig. 3). Treatment with 200 μM cytochalasin C (CC) or 200 μM cytochalasin J (CJ) for 1 h had no significant effect on the velocity of cytoplasmic streaming (Fig. 2). Fig. 2 View largeDownload slide Time course of cytochalasin-induced streaming inhibition and recovery in internodal cells of N. pseudoflabellata. Cytochalasins were added immediately after assessing the control rate at 0 min. After 60 min treatment, the inhibitor-containing solution was replaced by artificial fresh water. Data are means ± SD. Fig. 2 View largeDownload slide Time course of cytochalasin-induced streaming inhibition and recovery in internodal cells of N. pseudoflabellata. Cytochalasins were added immediately after assessing the control rate at 0 min. After 60 min treatment, the inhibitor-containing solution was replaced by artificial fresh water. Data are means ± SD. Fig. 3 View largeDownload slide Cytochalasins vary in potency to arrest cytoplasmic streaming in N. pseudoflabellata internodal cells and in the rate of recovery after their removal. Concentrations required to arrest cytoplasmic streaming within 1 h are listed on the x-axis. Mean relative velocities (left axis) of cytoplasmic streaming after 1 h recovery from 1 h treatment with streaming-arresting concentrations are shown by white bars, maximum recovery velocities are shown by gray bars, and times (right axis) required for maximum recovery are shown by black bars. Data are means ± SD. Fig. 3 View largeDownload slide Cytochalasins vary in potency to arrest cytoplasmic streaming in N. pseudoflabellata internodal cells and in the rate of recovery after their removal. Concentrations required to arrest cytoplasmic streaming within 1 h are listed on the x-axis. Mean relative velocities (left axis) of cytoplasmic streaming after 1 h recovery from 1 h treatment with streaming-arresting concentrations are shown by white bars, maximum recovery velocities are shown by gray bars, and times (right axis) required for maximum recovery are shown by black bars. Data are means ± SD. All cytochalasins induce the formation of cortical actin rods with variable length and density All cytochalasins caused reversible reorganization of cortical actin filaments at streaming-arresting concentrations, streaming-inhibiting concentrations and even, in the case of CC and CJ, at concentrations that had no effect on cytoplasmic streaming. The minimum concentrations required for cortical actin reorganization were between 1 and 20% of the concentration required to arrest streaming. The extent of reorganization varied considerably between the cytochalasins used, their concentration and the length of treatment. At streaming-inhibiting concentrations, actin reorganization took longer and changes were often less pronounced than at streaming-arresting concentrations. CA induced a distinctive swirling pattern of locally parallel but variably oriented clusters of actin filaments that frequently appeared frayed or branched (Fig. 4A). The length of the actin strands or bundles could not be determined because of their high degree of overlap. Their density varied along the cell surface (see CE treatments for more details and figures), but the narrow strips between the two opposing flows of streaming endoplasm known as neutral lines were always devoid of F-actin. This curving actin pattern formed within one to several hours at the streaming-arresting concentration and persisted for several hours. The curving pattern was later replaced by short, isolated actin rods (Fig. 4B) and these were also found at the neutral line. The cortical rods had a random orientation typical for non-growing internodes as previously shown for CD (Collings et al. 1995). Reorganization of cortical F-actin was reversible even after prolonged incubation in CA (Fig. 4C). Fig. 4 View largeDownload slide Cortical F-actin can be reorganized in internodal cells of N. pseudoflabellata treated with cytochalasins at concentrations that inhibit (SIC) or arrest (SAC) cytoplasmic streaming. (A–C) Cytochalasin A: 1 μM, 3 h (A), 1 μM, 1 d (B), 1 μM, 1 d and 2 d recovery in AFW (C). (D and E) Cytochalasin B: 200 μM, 1 h (D) and 200 μM, 2 h (E). (F and G) Dihydrocytochalasin B: 100 μM, 1 d (F) and 150 μM, 2 d (G). (H–J) Cytochalasin D: 70 μM, 1.5 h (H), 10 μM, 1.5 h (I) and 10 μM, 1 d (J). (K and L) Cytochalasin E: 3 μM, 3 h (K) and 3 μM, 1 d (L). Note the heterogeneous distribution of F-actin in K. (M–O) Cytochalasin H: 75 μM, 3 h (M), 10 μM, 2 h (N) and 50 μM, 2 d (O). Note local accumulation of actin spears. (P) Cytochalasin C: 100 μM, 2 h. (Q) Cytochalasin J: 100 μM, 2 h. F-actin was stained by perfusion of cells with fluorescent phalloidin. Scale bar = 10 μm (O) and 5 μm (all other figures). Fig. 4 View largeDownload slide Cortical F-actin can be reorganized in internodal cells of N. pseudoflabellata treated with cytochalasins at concentrations that inhibit (SIC) or arrest (SAC) cytoplasmic streaming. (A–C) Cytochalasin A: 1 μM, 3 h (A), 1 μM, 1 d (B), 1 μM, 1 d and 2 d recovery in AFW (C). (D and E) Cytochalasin B: 200 μM, 1 h (D) and 200 μM, 2 h (E). (F and G) Dihydrocytochalasin B: 100 μM, 1 d (F) and 150 μM, 2 d (G). (H–J) Cytochalasin D: 70 μM, 1.5 h (H), 10 μM, 1.5 h (I) and 10 μM, 1 d (J). (K and L) Cytochalasin E: 3 μM, 3 h (K) and 3 μM, 1 d (L). Note the heterogeneous distribution of F-actin in K. (M–O) Cytochalasin H: 75 μM, 3 h (M), 10 μM, 2 h (N) and 50 μM, 2 d (O). Note local accumulation of actin spears. (P) Cytochalasin C: 100 μM, 2 h. (Q) Cytochalasin J: 100 μM, 2 h. F-actin was stained by perfusion of cells with fluorescent phalloidin. Scale bar = 10 μm (O) and 5 μm (all other figures). A similar, transient increase in cortical F-actin was observed with CB (Fig. 4D, E), but the newly formed F-actin appeared as short rods from the very beginning and these were not locally aligned, probably because they were less dense. DHCB had the smallest effect on the cortical F-actin, and delicate strands were still present after 1 h treatment at the streaming-arresting concentration. Only after one to several days treatment at the streaming-arresting concentration or streaming-inhibiting concentration were actin patches and short isolated actin rods found in the cortex (Fig. 4F, G). CD caused the formation of variably oriented but locally aligned short actin filaments (Fig. 4H, I), the density of which differed along the cell surface (not shown). The initial swirling pattern was, after longer incubation times, replaced by actin patches and scattered actin rods (Fig. 4J). CE had similar effects to CA and CD, and the cells exhibited a characteristic cortical zonation, where regions with a high density of F-actin abruptly merged with regions of lower F-actin density (Fig. 4K), both extending over several hundred micrometers. F-actin was absent not only from the neutral line but also from circular areas 10–20 μm across, that were randomly scattered over the cell surface. The dense swirling pattern was later replaced by isolated short rods (Fig. 4L). The effects of CH (Fig. 4M, N) were similar to those observed with CD and CE. However, after several days’ treatment at streaming-inhibiting concentrations, the cortex contained not only isolated short actin rods but also huge clusters of thick, randomly arranged branching networks of actin spears that extended into the subcortex (Fig. 4O). CC (Fig. 4P) and CJ (Fig. 4Q), which both caused only a slight streaming inhibition after several hours or days of treatment, induced the formation of isolated, short actin rods and longer spears without a previous increase in cortical F-actin, just like the streaming-inhibiting concentrations of the more potent cytochalasins. With all cytochalasins tested, cells recovered cortical actin strands after transfer to inhibitor-free artificial fresh water (e.g. Fig. 4C). The recovery times were below those required for recovery of cytoplasmic streaming (not shown). Cytochalasin-induced reorganization of subcortical actin bundles requires several days treatment The effects of cytochalasins on the subcortical actin bundles were less immediate than those observed with cortical actin, and from one to several days’ treatment was necessary to induce reorganization, if it occurred, even if cytoplasmic streaming had been arrested. Up to 2 d treatment with CA at streaming-arresting concentrations had no significant effect on the morphology of the subcortical actin bundles, and 4–7 continuous actin bundles ran parallel to each cortical chloroplast file, just like in untreated cells (Fig. 5A). The small percentage of fragmented subcortical actin bundles, i.e. subcortical actin bundles not continuous over 50 μm in the area investigated, was due to those bundles extending through the chloroplast files into the cortical regions, as in control cells (Fig. 6). Fragmentation of subcortical actin bundles significantly increased after 2 d treatment at streaming-inhibiting concentrations (Figs. 5B, 6) or following recovery from treatment with streaming-arresting concentrations that left the subcortical actin bundles intact (Fig. 5C). Subcortical actin bundles first disappeared from regions between the chloroplasts, whereas those parts that had close contact with the chloroplast surface persisted longer. Some of the subcortical actin bundles were oblique to the chloroplast files (Fig. 5B), an orientation rarely found in untreated cells. CB caused a slight decrease in the mean number of subcortical actin bundles per chloroplast file after two to several days treatment at its streaming-inhibiting concentration, but most of the subcortical actin bundles remained intact (Figs. 5D, 6). DHCB at its streaming-arresting concentration significantly reduced the mean number of subcortical actin bundles after 2 d incubation and, interestingly, this effect was even more pronounced at its lower streaming-inhibiting concentration (Figs. 5E, 6). With the latter treatment, only about 60% of the subcortical actin bundles remained continuous over at least 50 μm, and these subcortical actin bundles were often much thicker than the short remnants of the fragmented subcortical actin bundles. CD, CE and CH were most effective in subcortical actin bundle fragmentation at both their streaming-arresting concentrations and streaming-inhibiting concentrations (Figs. 5F–I, 6). With CD and CH, the subcortical actin bundle fragments were further degraded and, consequently, their number per chloroplast file declined significantly (Figs. 5F, I, 6). The thickness of the subcortical actin bundle fragments varied considerably, and some of them were oriented obliquely to the chloroplast files. Both fragmentation and a decline in subcortical actin bundle number by CD and CH were reversible, and cells formed continuous, nearly evenly thick subcortical actin bundles parallel to the chloroplast files after 2 d recovery in artificial fresh water (Figs. 5G, 6). With CE, however, all subcortical actin bundles became irreversibly fragmented after 2 d treatment at a streaming-arresting or streaming-inhibiting concentration, and the fragments persisted along the inner chloroplast surface (Figs. 5H, 6). Subcortical actin bundles also became fragmented after 2 d recovery from 1 h treatment at the streaming-arresting concentration although the subcortical actin bundles were intact when cells were transferred to artificial fresh water (not shown, compare CA). CC did not cause reorganization of subcortical actin bundles (Figs. 5J, 6) and CJ had a weak effect (Fig. 6). Fig. 5 View largeDownload slide Subcortical actin bundles in internodal cells of N. pseudoflabellata treated with or recovering from cytochalasins applied at concentrations that inhibit (SIC) or arrest (SAC) cytoplasmic streaming. (A–C) Cytochalasin A: 1 μM, 1 d (A), 0.05 μM, 3 d (B), 1 μM, 3 h and several days recovery in artificial fresh water (C). (E) Cytochalasin B: 60 μM, 2 d. (E) Dihydrocytochalasin B: 30 μM, 2 d. (F and G) Cytochalasin D: 50 μM, 1 d (F), 50 μM, 1 d and 2 d recovery in artificial fresh water (G). (H) Cytochalasin E: 1 μM, 2 d and 2 d recovery in artificial fresh water. Subcortical actin bundles are still fragmented. (I) Cytochalasin H: 50 μM, 2 d. (J) Cytochalasin C: 200 μM, 2 d. F-actin was stained with fluorescent phalloidin. Scale bar = 10 μm (C) and 5 μm (all other figures). Fig. 5 View largeDownload slide Subcortical actin bundles in internodal cells of N. pseudoflabellata treated with or recovering from cytochalasins applied at concentrations that inhibit (SIC) or arrest (SAC) cytoplasmic streaming. (A–C) Cytochalasin A: 1 μM, 1 d (A), 0.05 μM, 3 d (B), 1 μM, 3 h and several days recovery in artificial fresh water (C). (E) Cytochalasin B: 60 μM, 2 d. (E) Dihydrocytochalasin B: 30 μM, 2 d. (F and G) Cytochalasin D: 50 μM, 1 d (F), 50 μM, 1 d and 2 d recovery in artificial fresh water (G). (H) Cytochalasin E: 1 μM, 2 d and 2 d recovery in artificial fresh water. Subcortical actin bundles are still fragmented. (I) Cytochalasin H: 50 μM, 2 d. (J) Cytochalasin C: 200 μM, 2 d. F-actin was stained with fluorescent phalloidin. Scale bar = 10 μm (C) and 5 μm (all other figures). Fig. 6 View largeDownload slide Effects of cytochalasins on the arrangement of subcortical actin bundles of N. pseudoflabellata internodal cells after 2 d treatment with streaming-arresting concentrations (SAC 2 d), 2 d treatment with streaming-arresting concentrations followed by 2 d recovery in artificial fresh water (SAC 2d, AFW 2d; CD, CE and CH only) and after 2 d treatment with streaming-inhibiting concentrations (SIC 2d). White bars indicate the number of subcortical actin bundles per chloroplast file (left axis); gray bars show the extent of fragmentation (right axis). Streaming-arresting concentrations for CA, CB, DHCB, CD, CE and CH were 1, 220, 150, 60, 1 and 30 μM. Streaming-inhibiting concentrations for CA, CB, DHCB, CD, CE, CH, CC and CJ were 0.5, 40, 75, 30, 0.5, 25, 200 and 200 μM. Data are means ± SD. Fig. 6 View largeDownload slide Effects of cytochalasins on the arrangement of subcortical actin bundles of N. pseudoflabellata internodal cells after 2 d treatment with streaming-arresting concentrations (SAC 2 d), 2 d treatment with streaming-arresting concentrations followed by 2 d recovery in artificial fresh water (SAC 2d, AFW 2d; CD, CE and CH only) and after 2 d treatment with streaming-inhibiting concentrations (SIC 2d). White bars indicate the number of subcortical actin bundles per chloroplast file (left axis); gray bars show the extent of fragmentation (right axis). Streaming-arresting concentrations for CA, CB, DHCB, CD, CE and CH were 1, 220, 150, 60, 1 and 30 μM. Streaming-inhibiting concentrations for CA, CB, DHCB, CD, CE, CH, CC and CJ were 0.5, 40, 75, 30, 0.5, 25, 200 and 200 μM. Data are means ± SD. With all cytochalasins tested, the decrease in subcortical actin bundle number and extent of fragmentation was more pronounced with the lower streaming-inhibiting concentrations than with streaming-arresting concentrations (Fig. 6). In the non-elongating internodes used in this study, additional endoplasmic actin, as described for CD treatment of younger cells (Collings et al. 1995), was not generated. Cytochalasin applied by perfusion does not interact with actin filaments or induce actin reorganization Cytochalasins are readily cell permeant, and it seems unlikely that their mode of action is limited by the ability to enter cells. CD introduced by perfusion, however, had no effect on the organization of actin filaments, even at concentrations up to 70 μM and incubation times up to 1.5 h, a treatment that caused extensive reorganization of cortical F-actin when applied from the outside of intact cells (Fig. 4H). The inability of cytochalasins to reorganize actin filament structures when applied by transcellular perfusion may relate to their inability to bind actin under these semi-in vitro conditions. CD conjugated with a Bodipy fluorophore is non-permeant and was therefore introduced by transcellular perfusion. CD–Bodipy at concentrations of up to 3.5 μM failed to label cortical actin strands or the subcortical bundles but did stain organelles which, according to their dimension and location, corresponded to mitochondria and cisternae of the endoplasmic reticulum (not shown). Similar staining results were obtained with other Bodipy-conjugated proteins, e.g. the microtubule-stabilizing paclitaxel and the actin-stabilizing phalloidin. Therefore, labeling is probably due to non-specific adsorption of the dye molecule. Latrunculins fail on their own but work synergistically with cytochalasins to arrest intracellular motility Latrunculins are generally more effective disruptors of actin filaments than cytochalasins and work at submicromolar concentrations. In the characean internodal cell, however, LatA and LatB proved to be relatively ineffective at inhibiting cytoplasmic streaming. At the highest concentration tested (200 μM) and after 1 h treatment, LatA and LatB decreased the mean streaming rates by only 56 ± 5 and 62 ± 12%, respectively. At these concentrations, cortical F-actin either was no longer visible or consisted of scattered short actin rods. Subcortical actin bundles, although faintly stained, remained intact. Prolonged exposure to LatB failed to reduce streaming rates any further, but streaming could be completely arrested after 1 d treatment with 200 μM LatA. These cells contained faintly stained cortical actin patches (not shown) and fragmented subcortical actin bundles of uneven thickness, which were occasionally obliquely oriented (Fig. 7A), suggesting that latrunculins also have reorganizing properties at concentrations insufficient for complete disassembly of F-actin (compare Gibbon et al. 1999, Vidali et al. 2001, Hörmanseder et al. 2005). Both streaming cessation and staining results (Fig. 7B, C) were reversible, but recovery in artificial fresh water took several days. Fig. 7 View largeDownload slide Actin cytoskeleton of N. pseudoflabellata internodal cells after treatment with LatA and CD. (A) One day treatment with 200 μM LatA is required for streaming cessation and correlates with the disassembly of subcortical actin bundles. (B) Continuous subcortical bundles but only a few cortical actin filaments (C) have regenerated after 3 d recovery in artificial fresh water. (D) Combined treatment of 15 μM LatA with 4 μM CD arrests streaming within 1 h and disassembles the subcortical bundles. (E and F) When LatA (E) or CD (F) are applied separately, cytoplasmic streaming continues and subcortical bundles remain intact. (G–I) The cortex of LatA-treated cells [in combination with CD (G) or alone, (H)] is devoid of F-actin, whereas delicate actin rods are seen in cells treated with CD (I). F-actin was stained with fluorescent phalloidin. Scale bar = 10 μm. Fig. 7 View largeDownload slide Actin cytoskeleton of N. pseudoflabellata internodal cells after treatment with LatA and CD. (A) One day treatment with 200 μM LatA is required for streaming cessation and correlates with the disassembly of subcortical actin bundles. (B) Continuous subcortical bundles but only a few cortical actin filaments (C) have regenerated after 3 d recovery in artificial fresh water. (D) Combined treatment of 15 μM LatA with 4 μM CD arrests streaming within 1 h and disassembles the subcortical bundles. (E and F) When LatA (E) or CD (F) are applied separately, cytoplasmic streaming continues and subcortical bundles remain intact. (G–I) The cortex of LatA-treated cells [in combination with CD (G) or alone, (H)] is devoid of F-actin, whereas delicate actin rods are seen in cells treated with CD (I). F-actin was stained with fluorescent phalloidin. Scale bar = 10 μm. Combined treatment with latrunculins and cytochalasins, however, rapidly arrested cytoplasmic streaming even at concentrations which, when applied separately, had only mild effects on the streaming rate. The combination of 15 μM LatA and 4 μM CD arrested cytoplasmic streaming and disrupted subcortical actin bundles into short fragments within 60 min (Fig. 7D; Table 1). Cells recovered continuous bundles and about 40% of the control streaming rate after 1 h in artificial fresh water (Table 1). Recovery to near control rates took several days. When applied separately, 15 μM LatA and 4 μM CD decreased the streaming rate by about 64 and 26%, respectively, and did not affect the appearance of the subcortical bundles (Figs 7E, F; Table 1). The cortex of cells treated with 15 μM LatA (with or without CD) was completely devoid of F-actin (Fig. 7G, H). Cells treated with 4 μM CD contained delicate actin rods (Fig. 7I). Table 1 Effects of simultaneous and separate treatment with LatA and CD on cytoplasmic streaming and the actin cytoskeleton of N. pseudoflabellata internodal cells   Control  15 μM LatA, 4 μM CD; 1 h  15 μM LatA; 1 h  4 μM CD; 1 h  Velocity of cytoplasmic streaming (% of control)  100 ± 8.6  0 ± 0  36.0 ± 14.4  73.7 ± 17.6  Fragmentation of subcortical actin bundles (%)  3.4 ± 1.4  100 ± 0  4.4 ± 1.1  3.7 ± 3.5  No. of subcortical actin bundles per chloroplast file  5.4 ± 0.8  3.5 ± 1.3  4.6 ± 1.2  5.7 ± 0.6  Velocity of cytoplasmic streaming after 1 h recovery from 1 h treatment (% of control)    40.9 ± 13.3  47.6 ± 15.4  91.3 ± 14.8    Control  15 μM LatA, 4 μM CD; 1 h  15 μM LatA; 1 h  4 μM CD; 1 h  Velocity of cytoplasmic streaming (% of control)  100 ± 8.6  0 ± 0  36.0 ± 14.4  73.7 ± 17.6  Fragmentation of subcortical actin bundles (%)  3.4 ± 1.4  100 ± 0  4.4 ± 1.1  3.7 ± 3.5  No. of subcortical actin bundles per chloroplast file  5.4 ± 0.8  3.5 ± 1.3  4.6 ± 1.2  5.7 ± 0.6  Velocity of cytoplasmic streaming after 1 h recovery from 1 h treatment (% of control)    40.9 ± 13.3  47.6 ± 15.4  91.3 ± 14.8  Data are means ± SD. View Large Discussion Cytochalasins D and H are best suited for reversible streaming inhibition in characean internodal cells Characean internodal cells are one of the best studied model systems for understanding myosin-based motility. In this study, we found that latrunculins, which have previously been shown to be potent disruptors of actin-based processes in higher plants (e.g. Gibbon et al. 1999), have relatively mild effects on streaming in characean internodal cells. Similarly, jasplakinolide (Sawitzky et al. 1999) and the jasplakinolide-related chondramides (own unpublished data; Holzinger and Lütz-Meindl 2001) are unable rapidly and reversibly to arrest streaming in Nitella internodes. It seems plausible that the extraordinary bundling within the actin cables and the protective effect of actin-binding proteins renders them very stable towards the action of these drugs. Cytochalasins thus remain the only actin-perturbing drugs suited for inhibition of cytoplasmic streaming for these cells. Ideal inhibitors are effective at low concentrations, are highly soluble and have actions that are readily reversible. The relative potencies of cytochalasins on cytoplasmic streaming and actin reorganization are summarized in Table 2. Those inhibitors with the lowest streaming-arresting concentration (1 μM) and most potent in rapidly arresting streaming, CA and CE, however, also had the lowest capacity for recovery. These cytochalasins presumably have a strong affinity for characean F-actin that greatly slows recovery. DHCB and CB reversibly arrested cytoplasmic streaming, but the required streaming-arresting concentrations were relatively high (150 and 200 μM, respectively). CC and CJ failed to arrest streaming even at the highest concentrations tested. Table 2 Relative potencies of cytochalasins on cytoplasmic streaming, reorganization of the actin cytoskeleton and recovery in internodal cells of N. pseudoflabellata in decreasing order (based on data presented in Figs. 3 and 6 and starting with the lowest streaming-arresting concentration, most severe actin reorganization, fastest and most effective recovery) Cytoplasmic streaming  CA  =CE  >CH  >CD  >DHCB  >CB  >>CJ  >>CC  Recovery of cytoplasmic streaming after 1 h treatment with streaming-arresting concentration  CD  =CH  >CB  >DHCB  >CA  >>CE      Fragmentation of subcortical actin bundles after 2 d treatment with streaming-arresting concentration  CH  =CD  >CE  >DHCB  >CB  ≥CA      Fragmentation of subcortical actin bundles after 2 d treatment with streaming-inhibiting concentration  CE  =CD  =CH  >CA  >DHCB  >CJ  >CB  ≥CC  Reduction of subcortical actin bundles number after 2 d treatment with streaming-arresting concentration  CD  >CH  ≥DHCB  >CE  ≥CA  ≥CB      Reduction of subcortical actin bundles number after 2 d treatment with streaming-inhibiting concentration  CD  >CH  >DHCB  >CJ  >CB  >>CA  >CE  =CC  Recovery of continuous subcortical actin bundles after 2 d treatment with streaming-arresting concentration  CH  =CD  >>CE            Reorganization of cortical actin  CA  =CD  =CE  =CH  >CB  >DHCB  >>CJ  >>CC  Cytoplasmic streaming  CA  =CE  >CH  >CD  >DHCB  >CB  >>CJ  >>CC  Recovery of cytoplasmic streaming after 1 h treatment with streaming-arresting concentration  CD  =CH  >CB  >DHCB  >CA  >>CE      Fragmentation of subcortical actin bundles after 2 d treatment with streaming-arresting concentration  CH  =CD  >CE  >DHCB  >CB  ≥CA      Fragmentation of subcortical actin bundles after 2 d treatment with streaming-inhibiting concentration  CE  =CD  =CH  >CA  >DHCB  >CJ  >CB  ≥CC  Reduction of subcortical actin bundles number after 2 d treatment with streaming-arresting concentration  CD  >CH  ≥DHCB  >CE  ≥CA  ≥CB      Reduction of subcortical actin bundles number after 2 d treatment with streaming-inhibiting concentration  CD  >CH  >DHCB  >CJ  >CB  >>CA  >CE  =CC  Recovery of continuous subcortical actin bundles after 2 d treatment with streaming-arresting concentration  CH  =CD  >>CE            Reorganization of cortical actin  CA  =CD  =CE  =CH  >CB  >DHCB  >>CJ  >>CC  View Large Complete and rapid recovery within 1 h was only possible with CD and CH, and the streaming-arresting concentrations were 60 and 30 μM, respectively. These cytochalasins are thus best suited for rapid and reversible streaming arrest in internodal cells of N. pseudoflabellata. In Chara corallina, a concentration of 8 μM CD was sufficient to arrest cytoplasmic streaming (Foissner and Wasteneys 2000b), which suggests that sensitivity to cytochalasins may vary between taxa within the Characeae, although relative sensitivities to different cytochalasins are probably conserved. Interestingly, CD and CH are also the cytochalasins that induced the most pronounced and yet most reversible changes in the arrangement of subcortical actin bundles (Table 2, see below). Streaming arrest by latrunculins but not by cytochalasins is due to the disassembly of subcortical actin bundles The subcortical actin bundles of untreated characean internodes consist of up to 100 single filaments (Nagai and Hayama 1979) and are arranged in groups of 3–7 located along and parallel to the helically arranged chloroplast files (Fig. 1A). Some extend into the cortex, but most of them appear continuous over huge distances, eventually encircling the whole cell end to end (Wasteneys et al. 1996). This continuity implies that individual actin filaments are very long, suggesting that the number of exposed actin filament plus ends, the preferred targets of cytochalasins (e.g. Bonder and Mooseker 1986, Cooper 1987), is low. This and the presence of actin bundling protein(s) that may inhibit binding of cytochalasins explains why subcortical actin bundles are less susceptible to reorganization than the shorter cortical actin strands, whose ends are more exposed. A protective effect of actin-binding proteins would also explain the high concentrations of cytochalasins and latrunculins needed for the disassembly of subcortical actin bundles and the ineffectiveness of jasplakinolide (Sawitzky et al. 1999). With LatA, fragmentation of subcortical actin bundles clearly coincided with streaming arrest after 1 d treatment at 200 μM, but the loss of motility achieved with cytochalasins was never correlated with disruption of the subcortical actin bundles. Indeed, subcortical actin bundles remained intact when exposed to the streaming-arresting concentration of CA for several days. This suggests that inhibition of streaming by cytochalasins is not caused by the disassembly of F-actin or even its reorganization through plus end capping, but rather it results from lateral binding, which somehow inhibits interaction with the motor protein myosin. In agreement with this hypothesis, Nothnagel et al. (1981) found that the staining of subcortical actin bundles by fluorescent heavy meromyosin was diminished in the presence of CB. The experiments of Urbanik and Ware (1989) indicate that there is more than one cytochalasin-binding site on the actin molecule, and this is consistent with a mechanism by which cytochalasins may bind along the length of, rather than only at the ends of, actin filaments. CD has been reported to depolymerize F-actin via dephosphorylation of and activation of actin-depolymerizing factor (ADF)/cofilin (Rückschloß and Isenberg 2001). In this case, CD-induced fragmentation of subcortical actin bundles should be prevented by phosphatase inhibitors that cause hyperphosphorylation of ADF/cofilin. We found that the combined treatment with CD and the phosphatase type II inhibitor calyculin A did not ameliorate the fragmentation observed in the presence of CD alone (results not shown). Thus, the effect of cytochalasins on characean F-actin is probably more direct. In agreement with this, Selden et al. (2001) found that CD and profilin bind competitively to Mg-ATP-actin isolated from vertebrate non-muscle cells. Comparison of cytochalasin-dependent disruption of subcortical actin bundles with in vitro experiments The subcortical actin bundle fragmentation activities of cytochalasins after 2 d treatment at streaming-arresting concentrations were in the order of CH = CD > CE > DHCB > CB ≥ CA (Table 2). This correlates with the order of activity found in in vitro experiments, where CH, CD and CE were most effective in cleaving filaments and inhibiting filament elongation and steady-state assembly (Walling et al. 1988). CD and CH were also very active in further disassembly and reorganization of subcortical actin bundles, which was reflected in the reduced number of subcortical actin bundle fragments per chloroplast file and their uneven thickness (Fig. 5F, I). In contrast, CE did not degrade further the remnant subcortical actin bundles found in close contact with the chloroplast envelope. The changes to the organization of subcortical actin bundles induced by CH and CD, whose structures differ only by one peripheral oxygen (Walling et al. 1988), were readily reversible, just as was the arrest of cytoplasmic streaming (Table 1). Paradoxically, CE had less effect on the subcortical actin bundles but recovery took up to 2 weeks. The different effects of CD/CH and CE could reflect the fact that, in contrast to CD and CH, CE does not accelerate microfilament assembly under certain in vitro conditions (Walling et al. 1988). The structural integrity of subcortical actin bundles in cells treated with CA or CB was barely disturbed, consistent with the absence of cleavage and a low filament shortening activity (Walling et al. 1988). Also seemingly paradoxical is the fact that the disruption of the subcortical actin cytoskeleton was more pronounced at the lower concentrations that slowed but did not arrest streaming (cf. Foissner and Wasteneys 2000b). Similarly, disruption was observed only after transfer of cells into artificial fresh water to allow the drug levels to drop, and not during treatment with CA and CE. This suggests that repeated binding and detachment of cytochalasins enhances their capacity to reorganize the actin cytoskeleton, whereas treatment at the higher streaming-arresting concentration is more likely to stabilize existing bundles (Williamson 1978, Williamson and Hurley 1986). Unfortunately, fluorescent CD did not label or induce actin reorganization and, therefore, we still do not know whether cytochalasins are a major component of the actin rods and bundles formed during treatment. All cytochalasins induce spatio-temporal reorganization of cortical F-actin It has been described previously (Collings et al. 1995, Foissner and Wasteneys 2000b) that CB and CD reorganize the delicate cortical actin filaments into short, relatively stable rods. Here we show that actin rod formation occurred with all cytochalasins tested. A hitherto unknown fact is, however, that formation of actin rods is often preceded by a spectacular increase in more delicate, non-rigid cortical actin strands. This effect was most pronounced with CA, CD, CE and CH. All of these drugs transiently increased the number of cortical actin filaments, which became organized into dense, swirling patterns. Transient, dense patterns of shorter, rod-like cortical F-actin were also observed in cells treated with CB. Only with DHCB, CC and CJ were cortical actin strands directly replaced by sparingly distributed actin rods, although we cannot fully exclude the possibility of a very short period with an enhanced cortical F-actin content. As yet, we have no explanation for this transient increase in nucleation and elongation activity of most cytochalasins because, contrary to our expectation, CA and CE are the two cytochalasins that do not accelerate assembly of F-actin in vitro (Walling et al. 1988). The increase in number and length of cortical actin filaments prior to the formation of rods suggests that, similar to pollen tubes, a considerable fraction of the actin pool in the cortex of characean internodes is present in the form of G-actin (Collings et al. 1995, Yokota et al. 2005). The heterogeneous distribution of cytochalasin-induced cortical actin may result from the well-known pH banding phenomenon of characean internodal cells (Shimmen et al. 2003, Babourina et al. 2004, and references therein). One previous study has shown that cortical microtubules are less abundant in the alkaline bands of C. corallina internodal cells than they are in the acid bands where net H+ efflux occurs (Wasteneys and Williamson 1992). In the case of cortical F-actin, the activity of H+-ATPases and the resulting changes in the ion content of the cortical cytoplasm may regulate the G-actin pools and/or the actin dissociation and polymerization rates (Sampath and Pollard 1991). The fact that perfusion of cytochalasins did not cause an increase in cortical F-actin probably reflects their inability to bind actin under these semi-in vitro conditions or the depletion of G-actin in perfused cells. Cytochalasins potentiate the effects of latrunculins Long treatment times and high concentrations of LatA or LatB were required to arrest cytoplasmic streaming in our experiments with Nitella internodal cells, even though cortical F-actin was rapidly affected. Since latrunculins are monomer-sequestering drugs, these findings probably indicate a low turnover of the subcortical actin bundles. At far lower concentrations, however, LatA rapidly arrested cytoplasmic streaming when applied together with low concentrations of CD. As with latrunculins alone, streaming cessation correlated with the fragmentation of subcortical actin bundles, suggesting that CD potentiated the destructive effects of LatA. One possible explanation is that during the combined treatment, CD may bind not only laterally but also to the free filament ends created by LatA by complexing G-actin required for regeneration of actin filaments and bundles. The capping of actin filament plus ends might then enhance the severing activities of CD (Cooper 1987, Urbanik and Ware 1989) and cause further disassembly of actin bundles. Cytochalasins as well as latrunculins affected cortical F-actin at concentrations below those required to arrest cytoplasmic streaming. Low concentrations of these drugs can therefore be used to study F-actin-dependent processes in the cortex without indirect effects caused by cessation of endoplasmic streaming. Latrunculins are better suited for this purpose because they disassembled cortical actin filaments completely, whereas cytochalasins reorganized cortical actin filaments into cortical patches, branching clusters and rods, which may have unpredictable effects on cortical physiology and even morphology. It is possible that the high concentrations of latrunculins required for actin depolymerization in characean algae are due to very low plasma membrane permeability. Variations in plasma membrane permeability could also account for the quantitative differences observed with the different cytochalasins. In order to address these questions and to shed more light on the mechanism of actin-disturbing drugs, we plan to perform perfusion experiments which allow the introduction of inhibitors under controlled conditions and independently of a plasma membrane barrier. Indirect effects of cytochalasins and actin–microtubule interactions The variable responses observed with the different cytochalasins, cytochalasin concentrations and organisms (e.g. Yahara et al. 1982, Zackroff and Hufnagel 1998, Spector et al. 1999) imply that both the impact on cytoplasmic streaming and the morphology of the actin cytoskeleton can be cell specific. Furthermore, some cytochalasin effects may also be indirectly related to the actin cytoskeleton. It has been reported that cytochalasins and other actin-disturbing drugs inhibit membrane transport and ion currents (Bray 1992, Rückschloss and Isenberg 2001). Newly formed actin aggregates in cytochalasin-treated animal cells associate with receptors, markers and proteins, suggesting a disturbance of cell signaling and endocytosis (Mortensen and Larsson 2003). CD leaves characean microtubules intact (Foissner and Wasteneys 1999), but in vitro experiments have shown that CA inhibits the depolymerization not only of muscle actin but also that of brain tubulin (Himes 1976). Therefore, CA may affect actin filaments and cytoplasmic streaming not only directly but also indirectly via depolymerization of microtubules. Earlier studies have indeed shown that depolymerization of microtubules makes cytoplasmic streaming in characean internodal cells more sensitive to cytochalasin treatment (Collings et al. 1995). In another study, we found that subcortical actin bundle disruption by cytochalasin is considerably enhanced in the presence of microtubule-depolymerizing drugs, and speculated that the release of microtubule-bound proteins and their subsequent interaction with the actin cytoskeleton could enhance the effects of cytochalasin (Foissner and Wasteneys 2000b). Recent studies in Arabidopsis thaliana demonstrate that mutation-dependent microtubule disruption can generate hypersensitivity to cytochalasins and latrunculins (Collings et al. 2006). The similar synergistic effect of LatA on cytochalasins suggests that the as yet unidentified microtubule-binding factor implicated in the microtubule disruption studies may have G-actin-complexing properties. This interpretation, however, remains speculative in view of the complex interactions of these drugs with G- and F-actin, and their competition with the cell's own actin-binding proteins (Selden et al. 2001). Materials and Methods Plant material and culture conditions Shoot tips of Nitella pseudoflabellata A. Br., em. R.D.W. were planted in a soil–peat–sand mixture covering the bottom of 10 liter aquaria filled with tap water. The temperature in the culture room was about 20°C, and fluorescent lamps (Gro-lux; Silvana, Erlangen, Germany) provided a photoperiod of 16 h light and 8 h dark. The fourth upper internode of each stem was used for this study. These cells were no longer growing or elongating and were harvested at least 2 d prior to experiments, trimmed of neighboring internodal cells and left in artificial fresh water (1 mM NaCl, 0.1 mM KCl, 0.1 mM CaCl2). Perfusion and staining of the actin cytoskeleton Perfusion of internodal cells was as described (Williamson et al. 1989). Briefly, an internodal cell was placed on the cover slip bottom of a perfusion chamber and pressed lightly into vacuum grease lines positioned several millilmeters away from the cell ends. Small reservoirs with grooves were carefully placed over the grease lines and pressed down firmly without damaging the cells. The central portion of the cell between the reservoirs was then covered with silicon fluid (Wacker, Burghausen, Germany) in order to prevent evaporation. The ends of the cells within the reservoirs were bathed in isotonic perfusion solution: 200 mM sucrose, 70 mM KCl, 4.5 mM MgCl2, 5 mM ethyleneglycoltetraacetic acid (EGTA), 1.48 mM CaCl2, 10 mM piperazine-N,N′-bis (2-ethanesulfonic acid) (PIPES, pH 7.0). Following reduction of turgor, cells were cut with small scissors, and a small difference in solution levels between the two reservoirs ensured a gentle flow of perfusion solution through the cell. After 1 min, actin filaments were stained by replacing the perfusion solution with perfusion solution containing Alexa phalloidin (Molecular Probes, Leiden, The Netherlands; 6.6 μM in methanol) at a concentration of 0.16 μM. Cells were ready for microscopic examination after 20 min. Prior stabilization of actin filaments with m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS; Sigma, Deisenhofen, Germany) (Sonobe and Shibaoka 1989) was not required. This method yielded excellent images of the cortical actin filaments but the fluorescent signal of subcortical actin bundles was occasionally attenuated by the intense autofluorescence of the chloroplasts and/or their starch grains. Subcortical actin bundles were therefore also visualized in cells fixed in glutaraldehyde (1% in cytoskeleton-stabilizing buffer: 10 mM EGTA, 5 mM MgSO4, 100 mM PIPES/KOH, pH 6.9; Traas et al. 1987) for 10 min and stained in Alexa phalloidin (0.16 μM in buffer) for at least 20 min after a brief wash in buffer. Cylindrical fragments of these internodes were opened out to produce single layer preparations with the endoplasmic side face up, and mounted in the staining solution. To rule out any possibility that the disassembly and reconstruction of cortical actin filaments was influenced by the preservation and labeling method, cells were also processed for indirect actin immunofluorescence as described (Foissner et al. 1996) using the N350 actin antibody (Amersham, Buckinghamshire, UK) and a fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Sigma). This method produced images equivalent to the more rapid phalloidin staining (not shown). Inhibitor treatments Stock solutions of CA, CB, CC, CD, CE, CH, CJ, DHCB (Sigma; 10 mM) and LatA and LatB (Calbiochem, Darmstadt, Germany; 10 mM) were prepared in dimethylsulfoxide (DMSO) and diluted with artificial fresh water. Controls containing DMSO up to a concentration of 2% affected neither cytoplasmic streaming nor the organization of the actin cytoskeleton. Fluorescent CD (Bodipy FL conjugate) for transcellular perfusion was purchased from Molecular Probes (Leiden, The Netherlands). The 10 mM stock solution in DMSO was diluted with perfusion solution up to a concentration of 3.5 μM. Microscopy The microscope used for visualizing fluorescently labeled actin and autofluorescent chloroplasts was a Zeiss Axiovert 100M inverted microscope equipped with a confocal laser scanner (Zeiss LSM 510, Oberkochen, Germany) including an argon ion and an HeNe laser. Projections of series of optical sections (Z-series) and 3D anaglyphs were generated using the Zeiss LSM 510 software and further processed with Adobe Photoshop. Statistical evaluation Cytoplasmic streaming velocity was determined in at least seven cells by measuring the distance traveled by detached chloroplasts within a given time period. Movement of organelles was also studied by analyzing slow-motion or frame-by-frame playbacks of recorded time series collected in the confocal microscope's transmission mode or from video images. The number of subcortical actin bundles per chloroplast file was determined from Z-series obtained from 50 × 50 μm subcortical regions between the chloroplast-free zones known as neutral lines, which mark the borders between the upward and downward flows of cytoplasm. A minimum of three cells and three Z-series per cell were investigated. The extent of fragmentation was determined from the same regions by calculating the percentage of interrupted subcortical actin bundles as assessed by gaps in the fluorescent signal. Further fragmentation within a discontinuous bundle was not considered. Mean values are given together with their standard deviation. Data were evaluated using one-way analysis of variance (ANOVA) and considered to be significant when P ≤ 0.01. Acknowledgments I.F. gratefully acknowledges financial support from the Stiftungs- und Förderungsgesellschaft der Universität Salzburg. 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Microbiol ,  1998, vol.  45 (pg.  397- 403) Google Scholar CrossRef Search ADS   Abbreviations: Abbreviations: ADF actin-depolymerizing factor CA cytochalasin A CB cytochalasin B CC cytochalasin C CD cytochalasin D CE cytochalasin E CH cytochalasin H CJ cytochalasin J DHCB dihydrocytochalasin B DMSO dimethylsulfoxide FITC fluorescein isothiocyanate EGTA ethyleneglycoltetraacetic acid LatA latrunculin A LatB latrunculin B MBS m-maleimidobenzoyl N-hydroxysuccinimide ester PIPES piperazine-N,N′-bis (2-ethanesulfonic acid) © The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
TI - Wide-Ranging Effects of Eight Cytochalasins and Latrunculin A and B on Intracellular Motility and Actin Filament Reorganization in Characean Internodal Cells
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcm030
DA - 2007-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/wide-ranging-effects-of-eight-cytochalasins-and-latrunculin-a-and-b-on-0bPqU0lrM0
SP - 585
EP - 597
VL - 48
IS - 4
DP - DeepDyve
ER -