Villin-Induced Growth of Microvilli Is Reversibly Inhibited by Cytochalasin D

Journal of Cell Science 105, 765-775 (1993) 765 Printed in Great Britain © The Company of Biologists Limited 1993

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D

E. Friederich1,*, T. E. Kreis2 and D. Louvard1 1Unité de Biologie des Membranes, Institut Pasteur URA 1149, CNRS Département de Biologie Moléculaire, 75724 Paris Cédex 15, France 2Department of Cellular Biology, Université de Genève, 30 quai Ernest Ansermet, Genève 4CH 1211, Switzerland *Author for correspondence

SUMMARY

Villin is an actin-binding protein that is associated with rapid disassembly of actin rodlets occurred after a lag the cytoskeleton of brush border microvilli. In vitro, phase. The present data stress the important role of the villin nucleates, caps or severs actin filaments in a Ca2+- plasma membrane in the organization of the actin dependent manner. In the absence of Ca2+, villin orga- cytoskeleton and suggest that the extension of the nizes microfilaments into bundles. Transfection of a microvillar plasma membrane is dependent on the villin-specific cDNA into cultured cells that do not pro- elongation of microfilaments at their fast-growing end. duce this protein results in the growth of long surface Inhibition of microfilament elongation near the plasma microvilli and the reorganization of the underlying actin membrane by cytochalasin D may result in the ‘random’ cytoskeleton. Here we studied the effects of low con- nucleation of actin filaments throughout the cytoplasm. centrations of cytochalasin D on the induction of these On the basis of the present data, we propose that villin plasma membrane-actin cytoskeleton specializations. is involved in the assembly of the microvillar actin Transfected cells were treated with concentrations of bundle by a mechanism that does not prevent monomer cytochalasin D that prevent the association of actin association with the prefered end of microfilaments. For monomers with the fast-growing end of microfilaments instance, villin may stabilize actin filaments by lateral in vitro. In villin-positive cells, cytochalasin D inhibited interactions. The functional importance of the carboxy- the growth of microvilli and promoted the formation of terminal F-actin binding site in such a mechanism is rodlet-like actin structures, which were randomly dis- stressed by the fact that it is required for the formation tributed throughout the cytoplasm. The formation of of F-actin rodlets in cytochalasin D-treated cells. Finally, these structures was dependent on large amounts of our data further emphasize the observations that the villin and on the integrity of an actin-binding site located effects of cytochalain D in living cells can be modulated at the carboxy terminus of villin, which is required for by actin-binding proteins. microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imag- Key words: villin, actin-binding, brush border, membrane ing revealed that when cytochalasin D was removed, protrusion, transfection

INTRODUCTION for human villin into fibroblast-like cells (the CV-1 cell line), which normally do not produce this protein, causes the Brush border microvilli are plasma membrane extensions growth of surface microvilli and the concomitant reorgani- present at the apical surface of some absorptive epithelial zation of the underlying microfilament network (Friederich cells (for review see Arpin and Friederich, 1992; Louvard et et al., 1989, 1992). In vitro, villin modulates the state of actin al., 1992). These finger-like membrane interdigitations, polymerization and organization by its Ca2+ -d e p e n d e n t which are supported by an axial array of microfilaments, are mi c r o fi lament bundling, capping, nucleating or severing a suitable model with which to investigate how plasma mem- activities (Craig and Powell, 1980; Bretscher and Weber, brane-actin cytoskeleton interactions contribute to the mod- 1980; Mooseker et al., 1980; Glenney et al., 1981a; for ulation of cell shape. We have previously provided evidence review see Mooseker, 1985; Friederich et al., 1990). In the that villin, one of the major actin-binding proteins associated present study we have investigated the assembly of the with the axial microfilament bundle of intestinal microvilli microvillar actin cytoskeleton near the plasma membrane and (Bretscher and Weber, 1979; Matsudaira and Burgess, 1979), how villin is involved in this process. Towards this goal CV- actively participates in the assembly of the microvillar struc- 1 transfected with villin cDNA were exposed to the actin- ture (Friederich et al., 1989). Transfection of a cDNA coding binding drug cytochalasin D (CD) during or after microvilli 766 E. Friederich, T. E. Kreis and D. Louvard formation. In order to avoid the numerous effects of this drug periods (see figure legends). The prewarmed CD-containing on actin polymerization by its interaction with actin medium was then added and the cells were further incubated for monomers (Goddette and Frieden, 1986a,b; for review see the time periods indicated in the figure legends. In most cases Cooper, 1987). We used low CD concentrations (2´ 10 - 9 to cells were briefly washed (less then 30 s) with phosphate-buffered 2´ 10 - 7 M). Under these conditions CD inhibits the associa- saline (PBS) containing 10 mM CaCl2 and 10 mM MgCl2, and tion of actin monomers to the fast-growing end of microfil- processed for immunofluorescence labeling as described above. In aments by 90% and thereby mimicks the activity of capping recovery experiments, cells were washed three times with prewarmed complete culture medium after CD treatment and incu- proteins (Brown and Spudich, 1979; Brenner and Korn, bated in the absence of CD for 1-4 h. 1979; Flanagan and Lin, 1980; Bonder and Mooseker, 1986; Sampath and Pollard, 1991). Previous studies carried out by others used this behaviour of CD to investigate the turnover Villin antibodies of microfilaments in living cells. The results obtained during The production and properties of the monoclonal (ID2C3) and the those studies suggested that actin monomer addition at the polyclonal antibodies against villin, as well as affinity purification uncapped barbed end of actin oligomers is necessary for the of the polyclonal antibodies have been described (Dudouet et al., extension of lamellar membrane protrusions in motile cells 1987; Coudrier et al., 1981). (Yahara et al., 1982; Forscher and Smith, 1988). Here we show that in cells producing large amounts of Fluorescence labeling of the cells villin CD impairs the formation of the spike-like F-actin In most of the experiments, transfected cells were fixed with 3% bundles that constitute the cytoskeleton of surface of paraformaldehyde, detergent permeabilized with Triton X-100 microvilli and provokes the appearance of thick cytoplas- (0.4%) and labeled as described (Reggio et al., 1983). For mic actin bundles that do not interact with the plasma mem- immunofluorescence staining of villin, cells were incubated with brane. The formation of these F-actin structures requires the purified monoclonal anti-villin antibodies (10 mg/ml) and then carboxy-terminal actin-binding site of villin (Friederich et with mouse IgG-specific antibodies conjugated to fluorescein (Amersham). In order to visualize F-actin, rhodamine-conjugated al., 1992), which is also necessary for microfilament phalloidin (0.3 mg/ml; Sigma Chem. Co.) was added to the anti- bundling in vitro (Glenney and Weber, 1981). Our results villin antibodies. In order to visualize the cell surface, living cells are in favor of the existence of uncapped actin oligomers on coverslips were washed with PBS at 20°C and then labeled for in close contact with the dorsal plasma membrane, which 5 min at 0°C with wheat germ agglutinin conjugated to rhodamine may serve as nuclei for the assembly of microfilaments sup- (10 mg/ml, Sigma Chem. Co.). After three washes with cold PBS, porting the long, rigid microvilli seen in cells producing 3% of paraformaldehyde was added and cells were allowed to large amounts of villin. warm up to 20°C. After fixation, cells were processed for immunofluorescence labeling as described above.

MATERIALS AND METHODS Fig. 1. CD inhibits the villin-induced reorganization of the actin cytoskeleton in CV-1 in a dose-dependent manner. Cells were Plasmid DNA transfected with a DNA construct encoding human villin. CD was Standard procedures as described by Sambrook and collaborators added after 24 h. Cells were incubated for further 24 h in the (1989) were used for large-scale preparation of the constructs presence of CD and then processed for double-fluo r e s c e n c e pSV51-villin and pSV51-villin D7 (Friederich et al., 1989, 1992) labeling. Untransfected cells were treated in parallel. as previously described. Pa r a f o r m a l d e h y d e - fi xed and detergent-permeabilized cells were double-labeled for villin with a villin-specific monoclonal antibody, Cell culture followed by incubation with a fluorescein-coupled second antibody, Unless otherwise indicated, CV-1 cells were grown in Dulbecco’s and for F-actin using rhodamine-coupled phalloidin. Untransfected minimum essential medium (DMEM) supplemented with 10% cells were stained for F-actin with rhodamine-coupled phalloidin. Left and middle panels are micrographs of the double-stained fetal calf serum (complete medium), at 37°C, under a 10% CO 2 transfected cells. Left panels: villin immunofluorescence stainings. atmosphere. Middle panels: micrographs of the corresponding phalloidin Transient cDNA expression in CV-1 cells labelings. Right panels: phalloidin labelings of the untransfected cells. (A, B, C) controls (no CD); (A, B) note the presence of many CV-1 cells were transfected using the DEAE-dextran-mediated long actin-containing spikes present on the dorsal face of the villin- DNA transfer method as previously described (Friederich et al., positive cell; (C) lamellar F-actin containing membrane protrusions 1989) with the following modifications. In order to limit cell (arrowheads) and stress fibers are present at the ventral face of damage, the DNA-DEAE-dextran incubation step was reduced to untransfected cells. (D, E, F) 5 nM CD; (D, E) the number of villin- 30 min. After removal of the DNA-DEAE-dextran-containing induced F-actin structures is strongly reduced. Moreover, this cell is medium, cells were treated for 2-3 h with culture medium (see devoid of stress fibers; (F) untransfected cells present an almost above) containing 50 mM chloroquine, which increased transfec- un m o d i fi ed actin cytoskeleton (lamellipodia are indicated by tion efficiency. Cells were analyzed after addition of DNA at the arrowheads). (G, H, I) 50 nM CD; (G, H) note the presence of a few time points indicated in the figure legends. thick actin rodlets and the lack of the fine, regular-shaped actin spikes; (I) these prominent F-actin structures are not detectable in Cytochalasin D treatment villin-negative cells; note the absence of lamellipodia at the cell The cytochalasin D (Sigma Chem. Co., St. Louis, MO) stock solu- periphery (arrowheads). (J, K, L) 50 nM CD followed by wash-out tion (2 mM in ethanol) was kept at - 20°C and diluted in com- of CD for 3 h; (J, K) spike-like F-actin structures are present on the plete culture medium just before use. After addition of the DNA villin-positive cells; (L) untransfected cells have lamellipodia the cells were incubated in the absence of CD for various time (arrowhead) and stress fibres. Bars, 10 mm. Cytochalasin D inhibits microvilli growth 767

Fig. 1 768 E. Friederich, T. E. Kreis and D. Louvard

Proteins cence illumination to detect the fluorescein signals of the cells Rabbit skeletal muscle actin was prepared according to the method microinjected with the DNA-FITC-dextran solution. These cells of Spudich and Watt (1971). Actin was labeled with rhodamine were then microinjected with rhodamine-actin and incubated for following to the procedure described by Kreis and colleagues 30 min at 37°C after the second injection. Microinjection of one (1982). Frozen samples were stored at - 70°C. coverslip (~30 cells) was achieved within 15 min. Time-lapse video-intensified fluorescence Sequential microinjection of pSV51-villin and microscopy rhodamine-actin into CV-1 cells In vivo observation of microinjected cells was performed as CV-1 cells were plated on 22 mm glass coverslips. Microinjec- described by Ho et al. (1990). The coverslip with the injected cells tion was performed 24 h after plating using an automated microin- was kept on the microscope stage for about 3 h mounted in a ther- jection system (Pepperkok et al., 1988) and microinjection capil- mostatically controlled culture chamber at 37°C, which contained laries (GC-100 F/10). DNA (0.2 mg/ml) mixed with 1% DMEM medium buffered with 20 mM HEPES, pH 7.4, and lacked FITC-coupled dextran (150000; Sigma Chem. Co.) was injected phenol red. Cells were discontinuously observed using an inverted in sterilized water. Before microinjection rhodamine-actin was microscope (Leitz Diavert) equipped for transmitted and fluores- diluted in G-buffer (2 mM Tris-HCl, 0.2 mM CaCl2 and 0.2 mM cence (rhodamine and fluorescein) light microscopy. ATP, pH 7.6) lacking b-mercaptoethanol to a final concentration of 0.5 mg/ml and cleared by centrifugation at 75,000 g for 30 min. The supernatant was kept on ice and immediatly used for microin- RESULTS jection. Cells were microinjected in HEPES-buffered DMEM medium lacking NaHCO3. First, the pSV51-villin DNA was microinjected into the nucleus of the cells. After microinjection We assessed the effect of low concentrations of cytocha- of DNA, cells were incubated for 6 h at 37°C. Cells were then lasin D on the villin-induced morphogenesis of microvilli. exposed to 50 nM CD for a further 12 h, by which time villin- CV-1 cells were transiently transfected with a DNA con- induced reorganization of the actin cytoskeleton had occurred. The struct encoding human villin and CD was added at various microscope used for microinjection was equipped with fluores- times after transfection. The effects of low doses of CD on

Fig. 2. The formation of rodlet-like F-actin structures in the presence of CD depends on large amounts of villin. Cells were transfected with a DNA construct encoding human villin. CD was added 8 h later and was present until 24 h after transfection. Cells were processed for fluorescence double-labeling of villin and F-actin as described in Fig. 1. Left panels are micrographs of the villin immunofluorescence stainings. Right panels are micrographs of the corresponding phalloidin labelings. (A,B) no CD, note the absence of F-actin spikes. Villin staining accumulates at the cell periphery and colocalizes with cortical microfilaments. (C, D) 100 nM CD. The villin-label colocalizes with the CD-resistant cortical actin filments. No rodlet-like actin structures are detectable. Bar, 10 mm. Cytochalasin D inhibits microvilli growth 769 the reorganization of the microfilament network into spike- untreated cells (Fig. 2A,B; and Friederich et al., 1989). In like F-actin structures and on the formation of microvilli parallel, villin-positive cells exposed to CD (100 nM) did on the dorsal cell face were analyzed by double-labeling not exhibit thick cytoplasmic F-actin rodlets (Fig. 2C,D). fluorescence microscopy. This finding indicates that the formation of these F-actin structures was dependent on large amounts of villin. Inter- Exposure of CV-1 cells to CD impairs the estingly, villin codistributed with CD-resistant microfila- reorganization of the actin cytoskeleton induced ments that were frequently found at the periphery of non- by large amounts of villin confluent cells (Fig. 2C,D). Analysis of transfected cultures of cells showed that the reorganization of the actin cytoskeleton in villin-producing Parallel to the inhibition of the formation of F-actin CV-1 cells occurred within 24 to 48 h after addition of spikes, low CD concentrations inhibit the growth DNA (Figs 1A,B, 2A,B). Thus, to investigate the effect of of long microvilli CD on this process, cells were exposed to the drug during The organization of the dorsal cell surface of transfected this period. The effect of a range of various concentrations cells was investigated by fluorescence double-labeling for of CD (2-100 nM) were determined. After 48 h cells were villin and for the outer plasma membrane using flu o r e s- double-stained for F-actin using rhodamine-coupled phal- cent wheat germ agglutinin as a cell surface probe (Fig. loidin and for villin by indirect immunostaining. As eval- 3). Cells that were not treated with CD displayed numer- uated from the intensity of the immunofluorescence signal, ous, elongated membrane projections on their dorsal face cells examined under these conditions contained large (Fig. 3A,B), previously identified by transmission and amounts of villin. In the absence of CD, cells exhibited a scanning electron microscopy as long microvilli reorganized actin cytoskeleton. Fluorescent-phalloidin (Friederich et al., 1989). Exposure of cells to increasing labeling of microfilaments of a villin-positive cell showed concentrations of CD resulted in a dose-dependent reduc- the redistribution of F-actin into long spike-like structures tion of the density of these long microvilli (Fig. 3C,D,E,F). located at the dorsal face of the cell (Fig. 1A,B). Villin- Villin-positive cells treated with CD concentrations equal label was localized in these structures. Villin-negative cells to or higher than 20 nM contained F-actin rodlets, as indi- had normal, well-developed stress fibers and numerous rectly visualized here by the associated villin label (Fig. membrane protrusions under these conditions (Fig. 1C). CD 3E,F). The fact that these cells were free of long surface concentrations equal to or higher than 5 nM affected F- projections strongly suggests that the F-actin rodlets are actin spike formation (Fig. 1). Villin-positive cells treated not extending the dorsal plasma membrane. However, it is with 5 nM CD exhibited a reduced number of spike-like important to stress that even in the presence of 50 to 100 actin structures and lacked stress fibres (Fig. 1D,E). In con- nM CD rudimentary microvilli were still found on most trast, villin-negative cells had almost unaffected stress fibres cells (Fig. 3G,H). The morphology of these microvilli was at their ventral faces (Fig. 1F). Exposure to CD concentra- not affected by the presence or absence of villin. Thus, tions higher than 20 nM completely impaired the appear- only the formation of villin-induced long microvilli ance of fine needle-like F-actin structures in villin-positive appeared to be inhibited by CD. cells. Cells incubated with 50 nM CD are shown in Fig. The effect of CD on the formation of long microvilli was 1G,H,I. Surprisingly, villin-containing cells exhibited actin also reversible. Villin-positive cells that, after long-term rodlets to which the villin-label was associated (Fig. 1G,H). exposure to 50 nM CD, were incubated for 3 h in the These F-actin structures were never observed in CD-treated absence of the drug displayed long membrane protrusions villin-negative cells (Fig. 1I). However, under these con- on their dorsal face (Fig. 3I,J). These microvilli could be ditions not only villin-producing cells but also those lack- cleary distinguished by their length and shape from those ing villin had a reduced number of the stress fibres (Fig. found on villin-negative cells. 1I). Moreover, CD impaired the formation of lamellar F- actin-containing membrane extensions (Fig. 1I, arrow- Removal of CD results, after a lag phase, in the heads). rapid disassembly of the cytoplasmic F-actin We also investigated the reversibility of the CD effect. rodlets After exposure to 50 nM CD the drug was washed out In order to gain an insight into the contribution of CD to for 3 h (Fig. 1J,K). Villin-producing cells exhibited spike- the stabilization of the F-actin rodlets we investigated their like F-actin structures, very similar to those observed in disassembly dynamics after removal of CD. Living villin- the untreated villin-positive cell (Fig. 1A,B). However, positive CV1 cells that had been treated with 50 nM CD while in villin-negative cells (Fig. 1L) F-actin-containing were microinjected with rhodamine-actin as a probe for the membrane protrusions (Fig. 1L, arrowheads) had reap- dynamic distribution of actin, and then observed by time- peared already 30 min after CD removal, the disassem- lapse video-intensified fluorescence microscopy (Fig. 4). bly of the thick actin bundles and the subsequent forma- Most of the microinjected rhodamine-actin was incorpo- tion of fine F-actin spikes required longer incubation rated into the thick F-actin rodlets, as visualized in Fig. periods (2 h). 4A, immediately before wash-out of CD (Fig. 4). In par- Cells were also exposed to CD between 6 and 24 h after allel, microinjected cells that had been fixed and flu o r e s- transfection of villin cDNA, a time period when levels of cently immunostained for villin displayed a clear colocal- synthesized villin are low, as evaluated from the weak ization of villin and rhodamine-actin label (data not immunofluorescence signal (Fig. 2). Under these con- shown). The micrograph in Fig. 4B represents an enlarged ditions, villin did not reorganize the actin cytoskeleton in area of the same cell, recorded 50 min after CD removal 770 E. Friederich, T. E. Kreis and D. Louvard

Fig. 3 Cytochalasin D inhibits microvilli growth 771 and shows that F-actin rodlets were still present in the cytoplasm. After this lag phase, the actin rodlet rapidly disassembled; the most prominent rodlet shortened within 9 min from 13 to 4 mm (Fig. 4C). It is interesting to note that the actin rodlet appeared to disassemble exclusively at one extremity (Fig. 4B,C; see arrowheads). Assuming that the rodlet is formed exclusively by long protoﬁlaments of 13 mm, the disassembly rate of such a ﬁlament, which theo- retically has 4,745 subunits (Egelman et al., 1982), would be 6 subunits s- 1. Exposure to CD causes rapid shortening of spike- like F-actin structures and slow subsequent formation of cytoplasmic F-actin bundles The data reported above indicate that villin-induced growth of microvilli is extremely sensitive to CD. We have also explored the sensitivity of ready-formed F-actin spikes to CD. It is important to stress that assembled F-actin spikes were only affected when cells were exposed to ³ 20 nM CD. An untreated villin-positive cell and a cell exposed for 15 min to 100 nM CD are shown in Fig. 5. While the untreated cell exhibited long needle-like structures (Fig. 5A,B), the cell incubated in the presence of CD contained numerous short F-actin structures (Fig. 5C,D). F-actin rodlets were exclusively observed in cells exposed for 2 h or longer to CD (data not shown).

The carboxy-terminal actin-binding site of villin is required for the formation of cytoplasmic actin bundles We put forward the hypothesis that villin may participate in the formation of the prominent F-actin rodlets by its bundling activity. To test this hypothesis we used a villin variant lacking seven amino acid residues from the carboxy terminus of the headpiece domain. In vitro as well as in vivo studies allowed us to demonstrate that this truncation inactivates an actin-binding site (Friederich et al., 1992), which has been shown by others to be required for microfilament bundling (Glenney et al., 1981b). Untreated cells that produced this truncate (Fig. 6A,B) contained an Fig. 4. Disassembly dynamics of the prominent F-actin rodlets Fig. 3. CD inhibits the villin-induced growth of surface microvilli after removal of CD. Cells were sequentially microinjected. in a dose-dependent manner. Cells were transfected with a DNA First, the villin-encoding DNA construct was microinjected construct encoding human villin. CD was added 24 h after into the nucleus. Since the reorganization of the actin transfection. Cultures of cells were incubated in the presence of cytoskeleton occurred 8 h after microinjection of villin cDNA CD for further 24 h and then analyzed. Living cells were labeled (data not shown), 50 nM CD was added 6 h after the fir s t in the cold (4°C) with rhodamine-conjugated wheat germ injection. After 18 h of incubation, rhodamine-coupled G-actin agglutinin. After paraformaldehyde fixation and detergent (0.5 mg ml- 1) was microinjected into the cytoplasm of the permeabilization, cells were stained for villin as described in the same cells. Microinjected living cells were observed by video- legend to Fig. 1. Left panels are micrographs of the villin i n t e n s i fied fluorescence microscopy. A representative immunofluorescence stainings. Right panels are micrographs of experiment is shown. Time-lapse images of the same cell are wheat germ agglutinin stainings. (A,B) no CD, note the presence represented. (A) + 50 nM CD. The image has been recorded of many long surface projections (B) present on the dorsal face of just before CD wash-out. Most of the rhodamine-actin has been the villin-positive cell (A). (C,D) 5 nM CD; the number of villin- incorporated into the F-actin rodlets. (B,C) Wash-out of CD. induced long surface projections is reduced. (E,F) and (G,H) 50 Video-images represent a 2.5-fold magnification of the area in nM CD. (E) Note the presence of thick actin rodlets containing the square (A). (B) 50 min after removal of CD. Actin rodlets villin. (F) This cell lacks long surface projections. However, under are still present. (C) 59 min after removal of CD. The most these conditions a large fraction of transfected cells exbibits prominent actin rodlet has shortened. The disassembly of the dotted surface structures corresponding to rudimentary microvilli actin rodlet apparently occurred exclusively at one end. Bars, (G,H). These structures are also observed in untransfected cells. 5 mm . (I,J) 50 nM CD followed by wash-out of CD for 3 h. Long surface structures are present on the villin-positive cell. Bar, 10 mm. 772 E. Friederich, T. E. Kreis and D. Louvard

Fig. 5. Villin-induced F-actin spikes shorten after addition of CD. Cultures of cells were transfected with a DNA construct encoding human villin. After 48 h cells were treated with 100 nM CD for 15 min. Cells were processed for double-labeling of villin and F-actin as described for Fig. 1. Left panels are micrographs of the immunofluorescence stainings. Right panels are micrographs of the corresponding phalloidin labelings. (A,B) no CD, note the presence of F-actin spikes. (C,D) 100 nM CD. The villin-positive cell (dorsal face) exhibits short F-actin structures. Bar, 10 mm. unmodified stress fiber system at their ventral face and cytoskeleton in transfected CV-1 cells, accompanied by the lacked spike-like F-actin structures. The villin variant accu- growth of microvilli. Although we cannot exclude the pos- mulated at the cell periphery, near the plasma membrane sibility that CD may have another target besides actin, (Fig. 6A). When exposed to 100 nM CD, cells producing recent work on the effects of cytochalasin on cells con- this villin truncate exhibited a highly reduced number of taining a cytochalasin-resistant b-actin variant has provided stress fibres as did the surrounding untransfected cells (Fig. direct evidence that cytochalasin interacts exclusively with 6C,D) but never contained actin rodlets as observed with actin in living cells (Ohmori et al., 1992). Long-term expo- wild-type villin. sure to low concentrations of CD did not irreversibly damage the cells, since the effects of CD on the cytoskeleton were always fully reversible in our study. Our results demonstrate that the villin-induced microvilli DISCUSSION and the formation of spike-like F-actin structures are affected by low concentrations of CD at which untrans- CD inhibits the villin-induced morphogenesis of fected control cells exhibited an almost unmodified actin long surface microvilli cytoskeleton. Since stress fibers of villin-positive cells were In the present study we further characterized the mecha- disrupted, even when cells were almost free of spike-like nism of assembly of surface microvilli. We took advantage F-actin structures, it is likely that the disassembly of stress of the fact that villin induces the reorganization of the actin fibers is not a direct consequence of the recruitment of actin Cytochalasin D inhibits microvilli growth 773

Fig. 6. The formation of rodlet-like actin structures in the presence of CD is dependent on an actin-binding site located at the carboxy terminus of the villin headpiece domain. Cultures of cells were transfected with a DNA construct encoding the D7 villin truncate (Friederich et al., 1992). CD was added after 24 h. Cells were incubated in the presence of CD for further 24 h and then analyzed. Cells were processed for double-labeling of villin and F-actin as described for Fig. 1, with the difference that polyclonal villin-specific antibodies were used as first antibodies and fluorescein-coupled rabbit IgG-specific antibodies were used as second antibodies. Left panels are micrographs of the immunofluorescence stainings. Right panels are micrographs of the corresponding phalloidin labelings. (A,B) no CD; note the absence of F-actin spikes. (C,D) 100 nM CD; note the absence of F-actin rodlets. Villin label colocalizes with CD-resistant cortical F-actin. Bar, 10 mm. monomers into F-actin-containing surface structures. CD we cannot exclude such an interaction, the presence of short was used at concentrations that have been shown to inhibit actin-containing surface protrusions in CD-treated cells the association of actin monomers to the plus-end of micro- suggests that actin filaments still persist near the microvil- filaments in vitro. Therefore, we consider that the most lar plasma membrane. likely mechanism for the inhibition of microvilli growth by CD is the inhibition of the elongation of microfilaments at The formation of actin rodlets is a cooperative their fast-growing end. It is unlikely, however, that CD effect of villin and CD might act by competing with villin, since in vitro experi- At CD concentrations that completely impaired the growth ments demonstrated that villin and the actin-binding drug of long surface microvilli, villin induced the formation of cytochalasin B, which is closely related to CD, bind to two randomly distributed cytoplasmic actin rodlets. The forma- different sites at the barbed end of microfilaments (Cribbs tion of these F-actin structures was dependent on large et al., 1982). amounts of villin and was never observed in untransfected We also considered the possibility that CD may prevent cells. This surprising finding suggests that blocking nucle- attachment of microfilaments to the plasma membrane by ation sites near the plasma membrane results in the competing with proteins involved in this process. Although ‘random’ nucleation of microfilaments throughout the cyto- 774 E. Friederich, T. E. Kreis and D. Louvard plasm and stresses the important role of the plasma mem- filaments are unblocked. Thus, if villin nucleates actin fil- brane in the organization of the actin cytoskeleton. After aments this should occur by a mechanism that results in wash-out of CD, the actin rodlets disassembled with an esti- uncapped or in unstably capped barbed filament ends. mated rate of 6 monomers s- 1, a value that is very close to Villin may, for instance, stabilize actin filaments by lateral that of the off-rate of ADP-actin from the barbed end of interactions. This hypothesis is strengthened by the obser- actin filaments in vitro (7 monomers s- 1) (Pollard, 1986). vation that the actin-binding site of villin, which we This suggests that the fast-growing ends of these microfil- demonstrated here to be essential for the formation of the aments were blocked by CD and that the rodlets may have cytoplasmic actin rodlets, is not only involved in microfil- formed slowly by monomer addition at the pointed and/or ament bundling but also induces G-actin to adopt a con- the CD-capped barbed ends. The fast disassembly at one formation favorable for microfilament formation extremity of the rodlet suggests a polarized orientation of (Friederich et al., 1992). This property may also play a role the microfilaments. It remains to be investigated whether in the stabilization of F-actin oligomers near the plasma villin is responsible for this organization by bundling micro- membrane as well as of the actin rodlets formed in the filaments in an oriented manner. presence of CD. The finding that the villin truncate is not able to induce The effect of cytochalasin B has been previously tested the formation of the thick actin rodlets in the presence of on developing microvilli of intestinal epithelial cells, using CD: (1) strongly suggests that the carboxy-terminal actin- cultured explants of chick embryo intestines (Burgess and binding site is required for the formation of these struc- Grey, 1974). Since cytochalasin B was added at embry- tures; (2) provides indirect evidence that the bundling onic stages (day 12 to 18) where intestinal epithelial cells activity of villin is implicated in this process; (3) is further already presented microvilli with an organized actin core proof that the formation of actin rodlets is dependent on the made of actin-binding proteins such as fimbrin and villin presence of villin. Moreover, our results qualitatively (Shibayama et al., 1987; Ezzell et al., 1989), it is difficult demonstrate that in living cells CD not only causes the dis- to compare our findings on CV-1 cells with those obtained assembly of existing actin structures but may also con- during these studies. However, it is interesting to note that tribute to the formation of other new ones. The fact that the microvilli of developing intestinal epithelial cells were sen- formation of these structures is dependent on villin demon- sitive to cytochalasin, which altered their shape, length and strates that the action of CD can be modulated by actin- spatial distribution. As we observed in transfected CV-1 binding proteins. Moreover, this finding may explain why cells, cytochalasin caused the formation of new F-actin controversial results have been obtained in quantitative structures. Treatment of explants with cytochalasin B studies measuring the G/F-actin content in CD-treated within a range of 0.1-2.5 mg/ml resulted in the formation living cells (Morris and Tannenbaum, 1980; Casella et al., of thick actin bundles plunging deeply into the cytoplasm 1981; Fox and Phillips, 1981; Rao et al., 1992). (Burgess and Grey, 1974). In contrast to the actin rodlets that we observed in the cell body of transfected cells, these Implications of CD-impaired villin-induced long bundles supported, on their upper part, the plasma microvilli formation on the mechanism of the membrane, forming normal-sized microvilli. Very high assembly of microvilli concentrations of cytochalasin B induced branching or Our data suggest that CD impairs the elongation rather than elongation of part of the microvilli as well as the redistri- the formation of short microvilli containing membrane-asso- bution of their spatial arrangement, some areas of the ciated actin oligomers. Thus it is very likely that the fast- apical cell surface being devoid of microvilli (Mak et al., growing ends of the microvillar microfilaments are not 1974). In contrast to the microvilli of embryonic cells, capped, or, are only unstably capped, at least during the those of adult intestinal cells are resistant to cytochalasin elongation phase, which is critical for the extension of the B (Mak et al., 1974), indicating that the dynamics of the microvillar plasma membrane. This is in line with the find - microvillar actin cytoskeleton change during development. ings of Mooseker and colleagues (1982), who observed actin The observation that villin-induced microvilli in CV-1 cells monomer addition at the barbed end of isolated intestinal were still sensitive to CD may reflect the fact that these brush border microvilli. Although the present approach does structures are lacking factor(s) that protect the actin not allow us to determine the polarity of the microvillar cytoskeleton of intestinal microvilli from the effect of this mi c r o fi laments, we predict from our findings that the barbed drug. ends of the microvillar microfilaments are oriented towards the tip of the villin-induced microvilli. Such an orientation We thank Dr Rainer Pepperkok for advice with microinjection of membrane-associated microfilaments has been found in of cells. Thanks are also due to Dr Monique Arpin and Dr Tony other membrane protrusions such as microspikes (Small et Pugsley for carefully reading the manuscript and for their con- al., 1978). It will be important in the future to verify this structive comments, to Jean-Claude Benichou for his help with prediction by myosin S1 subfragment decoration. the photographic work and to Raphaële Carré for help in the prepa- A possible role for villin in the formation of long ration of the text. microvilli may be the nucleation or stabilization of actin This work was supported by grants from: the Association pour la Recherche sur le Cancer (ARC - no. 6379), the Fondation pour m i c r o filaments near the plasma membrane. In vitro exper- la Recherche Médicale, the Institut National de la Santé et de la iments have shown that nucleation of actin polymerization Recherche Médicale (INSERM no. 920204), the Ligue Nationale by villin is followed by capping of the barbed end (Glen- Française contre le Cancer. ney et al., 1981a). However, our results suggest that during Evelyne Friederich is also supported by a short-term EMBO the elongation phase the barbed ends of microvillar actin fellowship. Cytochalasin D inhibits microvilli growth 775

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