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Planta (1999) 209: 435±443 Redistribution of actin, pro®lin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs Markus Braun, Frantisek BalusÏ ka, Matthias von Witsch, Diedrik Menzel Botanisches Institut, Zellbiologie der P¯anzen, UniversitaÈt Bonn, Venusbergweg 22, D-53115 Bonn, Germany Received: 11 March 1999 / Accepted: 27 May 1999 Abstract. The continuously changing polar cytoplasmic organization during initiation and tip growth of root hairs is re¯ected by a dynamic redistribution of cytoskeletal elements. The small G-actin binding protein, pro®lin, which is known to be a widely expressed, potent regulator of actin dynamics, was speci®cally localized at the tip of root hairs and co-distributed with a diusely ¯uorescing apical cap of actin, but not with subapical actin micro®lament (MF) bundles. Pro®lin and actin caps were present exclusively in the bulge of outgrowing root hairs and at the apex of elongating root hairs; both disappeared when tip growth terminated, indicating a tip-growth mechanism that involves pro®lin-actin interactions for the delivery and localized exocytosis of secretory vesicles. Phosphatidylinositol-4,5-bisphosphate (PIP2), a ligand of pro®lin, was localized almost exclusively in the bulge and, subsequently, formed a weak tip-to-base gradient in the elongating root hairs. When tip growth was eliminated by the MF-disrupting inhibitor cytochalasin D, the apical pro®lin and the actin ¯uorescence were lost. Mastoparan, which is known to aect the PIP2 cycle, probably by stimulating phospholipases, caused the formation of a meshwork of distinct actin MFs replacing the diuse apical actin cap and, concomittantly, tip growth stopped. This suggests that mastoparan interferes with the PIP2-regulated pro®linactin interactions and hence disturbs conditions indispensable for the maintenance of tip growth in root hairs. Key words: Actin ± Cytochalasin D ± Mastoparan ± Phosphatidylinositol-4,5-bisphosphate ± Pro®lin ± Tip growth Abbreviations: FITC = ¯uorescein isothiocyanate; MF = micro®lament; PIP2 = phosphatidylinositol-4,5-bisphosphate Correspondence to: M. Braun E-mail: [email protected]; Fax: 49 (228) 732677 Introduction The cytoplasmic architecture of plant cells is highly dynamic and depends on continuous remodelling of cytoskeletal elements in response to developmental and environmental signals and requirements. The arrangement and properties of the cytoskeleton are known to be regulated by associated proteins that bind either to monomeric or polymerized cytoskeletal proteins. The number of well studied and characterized cytoskeletonregulating proteins in plant cells is still very limited (for review, see Staiger et al. 1997; De Ruijter and Emons 1999). Pro®lin, a widely expressed G-actin-binding protein, which interacts with poly-L-proline and phosphoinositides (Sun et al. 1995; SchluÈter et al. 1997; Chaudhary et al. 1998; Gibbon et al. 1998) is one of the best-characterized examples. Isoforms of pro®lin have been reported to regulate cellular growth and morphogenesis by organizing actin cytoskeletal dynamics in higher plants (e.g. Staiger et al. 1994, 1997; Valster et al. 1997). The actin-binding properties of plant pro®lins have been demonstrated in vitro (Valenta et al. 1993; Giehl et al. 1994; Staiger et al. 1994; Vidali and Hepler 1997) and in situ by microinjection experiments (Cao et al. 1992; Staiger et al. 1994; Gibbon et al. 1997, 1998; Holzinger et al. 1997; Valster et al. 1997). The distribution of pro®lin in plant cells, however, has not been studied in great detail so far. Previous localization studies in pollen tubes (Mittermann et al. 1995; Vidali and Hepler 1997) and the green alga Micrasterias (Holzinger et al. 1997) have shown that pro®lin is evenly distributed throughout the cytoplasm and is not associated with any cellular structure. Nevertheless, a possible role of pro®lin as part of the signalling pathways involved in the regulation of pollen tube tip growth has been suggested (Clarke et al. 1998a). On the other hand, in animal cells such as ®broblasts, the distribution of pro®lin correlates with highly dynamic F-actin arrays but not with relatively stable actin micro®laments (MFs), suggesting that pro®lin is an essential partner involved in the regulation of actin dynamics (Buss et al. 1992). 436 Root hair initiation is characterized by the shift of the cellular polarity from diuse expansion growth of the trichoblasts to localized and highly polarized tip growth. On the light-microscopical level, the bulging of the cell at the site of root hair emergence is the ®rst visible event. However, a number of intracellular events precede the actual outgrowth of the root hair, e.g. the sudden onset of cytoplasmic streaming, reorientation of cytoplasmic strands and endoplasmic reticulum, and a major rearrangement of cytoskeletal elements and associated proteins. During the various developmental stages of tip growth in root hairs (see Fig. 1 in Heidstra et al. 1997), the polar organization of the dierent cytoplasmic zones, including the apical vesicle-rich region, as well as the pattern of cytoplasmic streaming and the arrangement of the actin cytoskeleton change constantly (Miller et al. 1997, 1999). In this study, root hairs of three plant species were used for investigating the dynamic redistribution of actin, pro®lin and phosphatidylinositol-4,5-bisphosphate (PIP2) and the possible involvement of pro®lin in the regulation of actin-dependent processes in tipgrowing plant cells. Cytochalasin D was used to interfere with the actin cytoskeleton and mastoparan to interfer with the possible PIP2-regulated pro®lin-actin interaction. Our experiments, which include phalloidin staining and immunolabeling of actin, pro®lin and PIP2 in untreated and inhibitor-treated root hairs in dierent developmental stages of root hairs, indicate that pro®linactin interactions have an important function in tip growth of root hairs. Materials and methods Maize (Zea mays L. cv. Alarik) grains were obtained from Force Limagrain (Darmstadt, Germany), Lepidium sativum L. (cress) seeds from Chrysanth, Bonn, Germany and Arabidopsis thaliana (L.) Heynh. seeds from Lehle Seeds, Round Rock, USA. Maize grains and cress seeds were soaked for 6 h and germinated in moistened rolls of ®lter paper for 2±3 d in darkness at 20 °C. Arabidopsis seeds were germinated on agar plates containing 1% sucrose. For inhibitor treatment, germinated seedlings were adapted to aerated water for 6 h and then incubated with cytochalasin D (5± 10 lM; Sigma-Aldrich Chemie, Deisenhofen, Germany) and mastoparan (1±5 lM; Sigma) for 30 min prior to ®xation. For in-vivo observations, seedlings were mounted on a slide in a drop of water, covered with a coverslip, and inspected under a microscope connected to a video recorder. Inhibitors were applied by adding them at working concentration directly to the mounted roots. Fig. 1A±G. Detection of a pro®lin band at approx. 14 kDa by the anti-pro®lin (ZmPRO3) antibody in root extracts of Arabidopsis (A), maize (C soluble proteins, E microsomal proteins) and cress (G). Preincubation of anti-ZmPRO3 with recombinant pro®lin isoforms resulted in strongly reduced labeling of the pro®lin band of the soluble protein fraction of maize (D) and a complete suppression of the labeling in the maize microsomal fraction (F) and in the Arabidopsis (B) total protein fraction M. Braun et al.: Actin, pro®lin and PIP2 in root hairs Freeze-shattering and confocal microscopy. The freeze-shattering procedure was modi®ed after Braun and Wasteneys (1998). Either whole roots (Arabidopsis) or epidermal strips with subjacent cortical cell layers excised from roots (Lepidium, Zea), untreated and inhibitor-treated, were ®xed for 30 min with a freshly prepared ®xation solution containing 1% formaldehyde and 1% glutaraldehyde (Sigma; Grade I, stored at )20 °C), 50 mM Pipes, 5 mM EGTA and 5 mM MgSO4 (pH 7.2). After several rinses in ®xation buer without aldehydes, the buer was gradually replaced with phosphate-buered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.9 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4), incubated three times with freshly prepared 1 mg/ml NaBH4, washed again in PBS, placed into cold ()10 °C) methanol for 5 min and rinsed with PBS containing 50 mM glycine. After permeabilization with 1% Triton X-100 in PBS/glycine for 30 min, the roots were gently squashed between two polyethyleneimine-coated microscope slides and plunged into liquid nitrogen for approx. 1 min. The frozen slides were rapidly separated, but then joined back together again and pressed in order to fracture the frozen tissue. After thawing, the root fragments were incubated with the ®rst antibodies, anti-actin (clone C4 from ICN, Costa Mesa, USA; 1:400), anti-pro®lin (maize isoform ZmPRO3; Karakesisoglou et al. 1996; 1:200), anti-PIP2 (Perseptive Biosystems, Framingham, USA; Bubb et al. 1998; 1:200) for 3 h at 37 °C or overnight at room temperature. After three rinses in the same buer, the cells were incubated with ¯uorescein isothiocyanate (FITC)-conjugated second antibodies for 2 h (Sigma, 1:100) at 37 °C. For double-labeling of pro®lin and actin, root fragments were sequentially incubated with anti-pro®lin for 10 h, with anti-actin (C4) for 3 h, with FITC-conjugated antirabbit (Sigma) for 2 h and with Alexa 546-conjugated anti-mouse (Molecular Probes, Eugene, Ore., USA) for 2 h. Washing was performed after each incubation step. Stained samples were rinsed three times with PBS/glycine and mounted in 0.1% para-phenylene diamine and 50% glycerol to minimize fading of the ¯uorescent conjugate. Images of immuno¯uorescently labeled samples were collected using a confocal microscope (TCS4D; Leica, Heidelberg, Germany). Several controls were performed to determine the speci®city of the antibody labelings, including incubation with buer only and with primary antibodies only as well as incubation with secondary antibodies only. Preabsorption of anti-ZmPRO3 with recombinant pro®lins was carried out by incubating 1 ml of diluted antibody with 60 lg of ZmPRO3 and 20 lg of ZmPRO1, 2, and 4 overnight at 4 °C. Anti-PIP2 (Perseptive Biosystems) was preabsorbed with PIP2 from bovine brain (Sigma) overnight at 4 °C. Recombinant Pro®lin was also used as an inappropriate antigen to preabsorb the anti-PIP2-antibody and PIP2 was used as an inappropriate antigen to preabsorb anti-ZmPRO3. Oregon-green phalloidin and rhodamine phalloidin (both Molecular Probes) stock solutions (0.3 lM, in 100% methanol) were diluted 1:100 with SoÈrensen-phosphate buer (pH 7.2) and labelings were imaged using the confocal microscope. Preparation of protein extracts and immunoblotting. Roots of 3-dold maize and cress seedlings grown on wet ®lter paper and whole seedlings of Arabidopsis thaliana grown on agar for 5 d were collected and homogenized in a buer containing 50 mM Tris (pH 7.4), 300 mM sucrose, 5 mM KCl, NaCl and EDTA, 2 mM ascorbic acid, 10 mM freshly added DTT, and a cocktail of protease inhibitors (10 lg/ml each of pepstatin A, leupeptin, aprotinin, benzamidin and 1 lg/ml phenanthrolin). The Arabidopsis and cress homogenates were each centrifuged for 10 min at 20,000 g and 4 °C to remove cell debris and large organelles. The resulting supernatants, containing membrane vesicles and soluble proteins, were subjected to SDS-PAGE, using 15% mini slab gels at 15 lg of protein per lane. Gels were wet-blotted onto nitrocellulose, which was used for incubation with anti-ZmPRO3 and preimmune serum at a dilution of 1:500 in TTBS (15 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4). As a control, preabsorption of anti-ZmPRO3 with recombinant pro®lins was carried out by incubating 1 ml of diluted antibody with 60 lg of ZmPRO3 and 20 lg of ZmPRO1, 2, and 4. The maize homogenate was centrifuged at 6,000 g for 15 min M. Braun et al.: Actin, pro®lin and PIP2 in root hairs at 4 °C to remove cell debris and large organelles. The pellet was discarded and the supernatant was then centrifuged at 100,000 g for 60 min at 4 °C. This supernatant containing soluble proteins and the pellet containing microsomal proteins were subjected to SDSPAGE and immunoblotting as described above. Results Immunoblot analysis. The antibody against the pro®lin isoform ZmPRO3 (Karakesisoglou et al. 1996) recognized a polypeptide band at approx. 14.2 kDa in root extracts of all three species, Arabidopsis (Fig. 1A), maize (Fig. 1C,E) and cress (Fig. 1G). For a control, antiZmPRO3 antibody solution was preincubated with the recombinant maize pro®lin isoforms ZmPRO1, 2, 3 and 4. This resulted in a strongly reduced pro®lin labeling in the fraction of soluble maize proteins (Fig. 1D) and even a complete inhibition of labeling in the fraction of microsomal maize proteins (Fig. 1F) and Arabidopsis proteins (Fig. 1B). Pro®lin labeling in the soluble protein fraction (Fig. 1C) and the microsomal fraction (Fig. 1E) indicates that pro®lins are not only associated with the plasma membrane but that they are also present in the cytoplasm. 437 Root hair initiation. The results obtained by immuno¯uorescence labeling were mostly identical in the three species, cress, maize, and Arabidopsis and are, therefore, not discussed in detail for each of the species, unless necessary. A dramatic reorientation of the actin cytoskeleton was one of the ®rst characteristic features of the initiation of root hairs following the appearance of a bulge in root epidermal cells. After the appearance of the bulge, the mainly longitudinally arranged actin MF bundles of the epidermal cell surrounding the large vacuole and the nucleus (Fig. 2) begin to focus towards the bulge of the outgrowing root hair (Figs. 3A, 4). The apex of the immuno¯uorescently labeled outgrowing root hair itself did not contain distinct actin MFs, but a brightly ¯uorescing cap of diusely labeled actin (Fig. 3A). Double-labeling with anti-actin and antipro®lin (ZmPRO3) antibodies revealed a cap-like profilin pattern (Fig. 3B) which spatially co-localized with the diusely ¯uorescing actin cap (Fig. 3A), but not with the actin MF bundles. Neither of the cap-like ¯uorescence patterns coincided completely with the apical cytoplasmic portion; they constituted only a small, distal part of the apical cytoplasm. An actin depletion of the vesicle-rich region at the outermost tip as was reported by Miller et al. (1999) but was not observed here. Fig. 2. Immuno¯uorescence image of the actin distribution in cress epidermal root cells. The mainly longitudinally oriented actin MFs reverse direction at the cross-walls Fig. 3A±C. Double-labeling of actin (A) and pro®lin (B) in cress root trichoblasts during root hair initiation. The corresponding bright®eld image is shown in C. Actin MFs run into the bulge of the outgrowing root hair and merge in a diusely ¯uorescing cap of actin at the tip (A) which co-localizes with the cap-like pro®lin immuno¯uorescence (B) Fig. 4. Immuno¯uorescence image showing the actin redistribution in maize epidermal cells during the outgrowth of root hairs from bulges Fig. 5. Immuno¯uorescence labeling of PIP2 in a maize trichoblast. The nucleus (N ), the outgrowing root hair and the cytoplasmic portion beneath show bright ¯uorescence signals Figs. 2±5. Bars = 10 lm 438 Phosphatidylinositol-4,5-bisphosphate (PIP2) epitopes were immuno¯uorescently localized exclusively in the outgrowing bulge, in the cytoplasmic portion beneath the bulge and in the nucleus (Fig. 5). Optical sectioning by confocal microscopy revealed that PIP2 was not exclusively associated with the plasma membrane but is also present in the cytoplasm. Control experiments in which samples were incubated with buer only, with primary antibodies only, and with secondary antibodies only, as well as incubating samples with the preabsorbed antibodies followed by the secondary antibody, resulted in no or little background ¯uorescence (not shown). Using inappropriate antigens to preabsorb the anti-PIP2 and anti-ZmPRO3 antibody did not aect the speci®c staining (not shown). These control experiments demonstrate the speci®city of the antibodies used in this study. In addition, the anti-PIP2 antibody has previously been shown to bind exclusively to PIP2 (Bubb et al. 1998). Elongating root hairs. In elongating cress root hairs, the immuno¯uorescently labeled diuse actin cap is more prominent and appears to be separated from the axially oriented bundles of actin MFs by a gap zone with strongly reduced or even absent actin immuno¯uorescence (Fig. 6A). Although such a gap was found in fastgrowing root hairs of all three species, it was less clearly recognizable in root hairs of maize and Arabidopsis. Labeling of the actin cytoskeleton by Oregon-green phalloidin and rhodamine phalloidin (Fig. 7) was similar to the immuno¯uorescence labeling (Fig. 6A), but the apical actin cap had a more ®lamentous appearance and no gap beneath the actin cap was visualized. Median optical sections demonstrate that actin is spread throughout the whole distal portion of the apical cytoplasm (Fig. 6A), whereas pro®lin ¯uorescence is most prominent near the apical membrane (Figs. 6B, 8B). The pro®lin caps were more pronounced in cress (Fig. 6B) and Arabidopsis (Fig. 8A) root hair tips than in those of maize (Fig. 8B), where pro®lin immuno¯uorescence was restricted to the outermost apical plasma membrane. In elongating root hairs, anti-PIP2 immuno¯uorescence was found distributed uniformly throughout the cytoplasm of short root hairs (Fig. 9A, arrow). In most longer root hairs (Fig. 9A, arrowhead), the immuno¯uorescence was brightest at the apex. In fully grown, mature root hairs, PIP2 was no longer observed (Fig. 9B). Tip-growth-terminating root hairs. Following the rapidelongation growth phase, root hairs showed reduced growth rates and eventually tip growth stopped. In root hairs that were terminating tip growth, the actin-depleted subapical zone disappeared and the longitudinal actin MFs made contact with the actin cap which became smaller and ®nally disappeared (Fig. 10A). Simultaneously, the cap-shaped anti-pro®lin ¯uorescence was strongly reduced (Fig. 10B) and disappeared after tipgrowth had stopped. The nucleus, which had become spindle-shaped after entering the root hair, still showed M. Braun et al.: Actin, pro®lin and PIP2 in root hairs c Fig. 6A,B. Double-labeling of actin (A) and pro®lin (B) in short, rapidly elongating cress root hairs. A Actin immunolabeling after freeze-shattering reveals a clear zonation of the cytoarchitecture. Actin MF bundles are present in the trichoblast and in the base of the root hair. An actin-depleted area in the subapical region separates the actin MF bundles from the bright, diusely ¯uorescing cap of actin in the tip. Projection of 12 serial sections (1.0 lm each). B Pro®lin immuno¯uorescence is highest near the apical plasma membrane, but is also present in the apical cytoplasm. Projection of 3 median serial sections (0.8 lm each) Fig. 7. The Oregon-green phalloidin labeling pattern of short maize root hairs resembles that of the immunolabeling except for the absence of a subapical actin-depleted zone Fig. 8A,B. Immunolocalization of pro®lin in root hairs of Arabidopsis (A) and maize (B) after freeze-shattering. A The cap-like pro®lin immuno¯uorescence is most extensive in the apex of rapidly elongating root hairs (arrows); ¯uorescence is reduced in long, growth-terminating root hairs (arrowheads) and hardly visible in root hairs which have terminated tip growth and have lost their polar cytoplasmic zonation (asterisks). Projection of 10 serial sections (1.2 lm each). B Pro®lin immuno¯uorescence in maize root hairs is limited to a narrow region at the apical cytoplasmic membrane. Projection of 4 median serial images (0.8 lm each) Fig. 9A,B. Immuno¯uorescence image showing the distribution of PIP2 in growing (A) and in mature, non-growing (B) maize root hairs. A In short root hairs, PIP2 is evenly distributed but forms a weak tipto-base gradient in longer maize root hairs. Projection of 6 serial images collected at 0.8-lm intervals. B Mature root hairs do not show a speci®c ¯uorescence pattern. Projection of 6 serial images collected at 0.8-lm intervals Fig. 10A,B. Immuno¯uorescence labeling of actin (A) and pro®lin (B) in a growth-terminating Arabidopsis root hair. A Actin MF bundles have invaded the apical dome. B Pro®lin labeling is limited to the spindle-shaped nucleus and a strongly reduced, weakly ¯uorescing apical cap. Both images are projections of 10 serial sections (0.8 lm each) Fig. 11. Thick bundles of actin MFs reverse at the tip of a full-grown cress root hair and cytoplasmic streaming occurs up to the outermost tip. Projection of 8 serial sections (0.8 lm each) Fig. 12. Pro®lin immuno¯uorescence is no longer visible in fullgrown cress root hairs. Projection of 8 serial sections (0.8 lm each) Figs. 6±12. Bars = 10 lm anti-pro®lin ¯uorescence (Fig. 10B). In mature, nongrowing root hairs, thick longitudinally oriented bundles of actin MFs continued into the apex, performed U-turns and returned back to the base of the cell (Fig. 11). Pro®lin (Fig. 12) and PIP2 immunolabeling (Fig. 9B) could no longer be detected in the tips of mature root hairs. Inhibitor studies. Application of cytochalasin D (5± 10 lM) stopped tip growth of root hairs and resulted in a complete depolymerization of the actin MFs in root epidermal cells (Fig. 13A,B) as well as in root hairs (Fig. 14A). The cells were depleted of actin immuno¯uorescence except for thick spike-like structures of actin within the nucleus of root epidermal cells (Fig. 13A,B). Cytochalasin D also aected the distribution of pro®lin. The cap-shaped pattern was gradually replaced by a punctate pattern in the subapical region (Fig. 14B) before it ®nally disappeared completely (not shown). Treatment with mastoparan, which was applied for 30 min prior to ®xation, resulted in fast termination of M. Braun et al.: Actin, pro®lin and PIP2 in root hairs tip growth accompanied by a drastic rearrangement of the actin cytoskeleton in the apex. Instead of the diuse actin cap, a distinct actin MF network was visualized after mastoparan treatment in the bulge of outgrowing (Fig. 15, arrows) and the tip of longer root hairs (Figs. 16A, 17). The pro®lin cap dispersed and ®nally disappeared completely (Fig. 16B). The cytoplasmic streaming, however, was not visibly aected by mastoparan treatment. Discussion The polarity and cytoplasmic architecture of diusely expanding root epidermal cells changes dramatically 439 with the appearance of a bulge at the site of root hair outgrowth. The emergence of the tube-like root hair requires assembly of a tip-growth machinery for localized exocytosis of cell wall and membrane material. Actin MFs have been localized in root hairs and appear to be essentially involved in the cytoplasmic organization and the process of tip growth, despite the fact that actin MFs have never been shown to extend up to the extreme tips of elongating root hairs (Seagull and Heath 1979; Emons 1987; Ridge 1988), whereas this feature is typical of mature root hairs (CaÂrdenas et al. 1998). It has been shown that tip growth of root hairs, but not the formation of bulges from which the root hairs originate require a tip-focused gradient of calcium (Wymer et al. 1997; De Ruijter et al. 1998) and an apical array of ®ner 440 Fig. 13A,B. Cytochalasin D treatment causes complete disruption of actin MFs in maize epidermal cells (outlined). Instead, spike-like actin structures are formed in mitotic (A) and interphase nuclei (B). Projection of 8 serial images (1.0 lm each) Fig. 14A,B. Double-labeling showing the distribution of actin (A) and pro®lin (B) in cytochalasin D-treated cress root hairs. A Actin MFs and the apical actin cap of cress root hairs disappeared completely after treatment with cytochalasin D. B The apical cap-like pro®lin immuno¯uorescence is reduced and a punctate staining pattern appeared in the subapical zone. Projection of 10 serial images (0.8 lm each) Fig. 15. Actin-immunolabeling of maize epidermal cells with bulges of outgrowing root hairs (arrows) after treatment with mastoparan. The apices contain a meshwork of actin MF bundles instead of the diuse cap-like actin ¯uorescence in untreated root hairs (Figs. 3A, 4). Projection of 6 serial images (1.2 lm each) Fig. 16A,B. Double-labeling of actin (A) and pro®lin (B) in mastoparan-treated cress root hairs. A The apical actin cap has disappeared, but the actin MF bundles are still present. B A speci®c pro®lin distribution is no longer visualized in the mastoparan-treated root hair. Projections of 10 serial images (1.2 lm each) Fig. 17. The maize root hair had stopped tip growth after mastoparan treatment for 30 min and was subsequently immuno¯uorescently labeled for actin. Thick actin MF bundles have split into a dense apical meshwork of ®ner actin MFs which have replaced the former diuse actin cap in the apex. Projection of 10 serial images (0.8 lm each) Figs. 13±17. Bars = 10 lm M. Braun et al.: Actin, pro®lin and PIP2 in root hairs bundles, including an actin-depleted area at the outermost tip (Miller et al. 1999). In this study, we have demonstrated a drastic rearrangement of the actin cytoskeleton following bulge formation, and the presence of diusely ¯uorescing actin caps in the bulges of outgrowing root hairs. Actin MFs become focused towards the site of outgrowth which is preceded and accompanied by the onset of vigorous cytoplasmic streaming in the trichoblasts. Diuse apical actin ¯uorescence was detected not only during the early developmental stages, but also in elongating and maturing root hairs. Intriguingly, in tip-growth-terminating and non-growing root hairs, devoid of the apical cytoplasmic and vesicle-rich region, actin caps were not observed. Instead, actin MF bundles formed in the apical dome, as was also reported by Miller et al. (1999). In elongating root hairs the patterns of apical actin visualized by immuno¯uorescence and phalloidin labeling suggest that the actin cap is composed of both monomeric actin and delicate actin MFs. An actindepleted zone spatially coinciding with the vesicle-rich region was not observed with our freeze-shattering method which avoided cell wall digestion with enzymes, but included chemical ®xation. Immuno¯uorescence double-labeling demonstrates that pro®lin co-localizes exactly with the diusely M. Braun et al.: Actin, pro®lin and PIP2 in root hairs ¯uorescing actin cap in the tip of outgrowing and elongating root hairs. This pattern was most easily observed in cress root hairs, whereas it was less prominent in maize and Arabidopsis. This speci®c apical co-localization of actin and pro®lin is strictly correlated with the process of tip growth. With the outgrowth of root hairs both the actin and the pro®lin caps appeared after bulge formation and became progressively reduced as root hairs approached termination of tip growth. It has been argued that pro®lin can have at least dual eects: actin sequestration and actin polymerization. On the one hand, pro®lins can bind G-actin, forming 1:1 complexes, thereby initiating disassembly or preventing actin MF assembly (Cao et al. 1992; Fechheimer and Zigmond 1993; Valenta et al. 1993). On the other hand, however, pro®lin can stimulate and promote actin polymerization at the barbed end (Tilney et al. 1983; Pollard and Cooper 1984; Pring et al. 1992; Perelroizen et al. 1996) depending on the cellular location and the presence of other actin-binding proteins (reviewed by Staiger et al. 1997). Thus, in root hairs, the high concentration of pro®lin-actin complexes in the apex could contribute to actin MF elongation towards the subapical part of root hairs. Pro®lin could also mediate cytoskeleton-membrane interactions when associated with the plasma membrane by binding actin and phosphoinositides simultaneously (Hartwig et al. 1989; Machesky and Pollard 1993), thereby coordinating fast delivery and apical exocytosis of secretory vesicles. Such a scenario corresponds well with the observation that pro®lin and actin caps are most prominent in fastgrowing root hairs, whereas both become smaller and disappear when tip growth slows down and ®nally stops. In these cells, actin MFs form thick undulating bundles in the apex which U-turn in the outermost tip; the rotational cytoplasmic streaming runs through the outermost tip and polar cytoplasmic organization is lost (see Fig. 1 in Heidstra et al. 1997). Phosphatidylinositol-4,5-bisphosphate, which is discussed as a regulator involved in pro®lin-actin interactions (De Corte et al. 1987; Hansson et al. 1988; Drobak and Watkins 1994; Clarke et al. 1998b), was immuno¯uorescently localized in the bulge of the outgrowing root hair and mainly accumulated in the apex, but also throughout the cytoplasm of elongating maize root hairs. In contrast, PIP2 immuno¯uorescence in Acanthamoeba, for instance, has been found to be mainly limited to the plasma membrane (Bubb et al. 1998). There is increasing evidence, however, that phosphatidylinositol 4-kinase is associated with cytoskeletal elements in the cytoplasm and, furthermore, that the lipid components of the phosphoinositide cycle may directly be involved in regulation of the cytoskeletal structure (Xu et al. 1992; Drobak et al. 1996; Clarke et al. 1998b). This may also explain why PIP2 immuno¯uorescence was not detected in mature root hairs, where the actin MF bundles are relatively stable. However, the possibility of artifacts due to the ®xation method can not be ruled out. In a recent paper, PIP2 was reported to accumulate in the apical plasma membrane of pollen tubes, where it may reorganize the MF system 441 (Kost et al. 1999). It was suggested that PIP2 may also directly control exocytosis by recruiting required proteins or altering membrane lipid composition. Mastoparan was used to interfere with pro®lin and cytochalasin D was used to interfere with the actin cytoskeleton. Treating elongating root hairs with cytochalasin D disrupted the subapical actin MF bundles and disturbed the maintenance of the actin and pro®lin caps. Apparently, this eliminated tip growth, indicating a prominent role and intimate interaction of actin and pro®lin in the apical dome. Mastoparan was reported for instance to activate or stimulate phospholipases A2, C, and D, thereby aecting PIP2 turnover and protein phosphorylation in animal and in plant cells (e.g. Lassing and Lindberg 1985; Drobak and Watkins 1994; Clarke et al. 1998a; Munnik et al. 1998; Pingret et al. 1998; Senda et al. 1998). Application of mastoparan resulted in a strong reduction or even a loss of pro®lin ¯uorescence in the tip. In particular, it initiated the transformation of the diuse actin cap into a ®ne, netlike con®guration of actin MFs that merge into the thick actin MF bundles of the root hair shaft. Despite the fact that the eects of mastoparan are diverse and not fully understood, it seems likely that it causes alterations in PIP2 concentration. This might destabilize the apical pro®lin-actin complexes to the extent that polymerization of actin MFs is promoted, which in turn would lead to the termination of tip growth. In contrast to root hairs, tip-growing cell types that actively adjust their direction of growth according to light or gravity stimuli with internal perception mechanisms such as gravity-sensing rhizoids and protonemata of the green alga Chara, seem to require a highly organized actin MF system in the apical dome (Braun and Wasteneys 1998). Both cell types exhibit an unique SpitzenkoÈrper composed of an aggregation of endoplasmic reticulum membranes in its centre and an accumulation of secretory vesicles organized by the extensive apical actin MF system. Tip-growth of root hairs, however, is not directed by internal orienting mechanisms, but can be redirected by obstacles or by arti®cially generating an asymmetrical calcium in¯ux across the root hair tip (Bibikova et al. 1997). Having overcome the obstacles, root hairs tend to return to their original growth direction. A vesicle-rich apical dome containing a pool of pro®lactin and a delicate network of short, probably oligomeric actin MFs might be sucient or even advantageous for coordinating this mode of tip growth. Our ®nding that pro®lin is speci®cally located at the tip of growing root hairs is in contradiction to ®ndings in other cell types where pro®lin was evenly distributed throughout the cytoplasm (Mittermann et al. 1995; Holzinger et al. 1997). Even in another tip-growing cell type, the pollen tube, pro®lin was reported to be evenly spread throughout the cytoplasm in chemically ®xed, freeze-substituted and microinjected samples (Vidali and Hepler 1997). The basis for this dierence is unknown; however, it is noteworthy that characean rhizoids and protonemata which reorient their growth direction according to external physical stimuli do not exhibit apical pro®lin caps (data not shown). The growth 442 direction of root hairs is only transiently reoriented by arti®cially setting a new lateral calcium gradient (Bibikova et al. 1997), and is continually being reset to the original direction. Therefore, it seems likely that a tipgrowth mechanism coordinated by pro®lin-actin interactions is limited to the speci®c mode of root hair tip growth. It may deserve attention that pro®lin and PIP2 are not only found in the apices of root hairs, but also within the nuclei of trichoblasts and other root epidermal cells. This observation is in agreement with other reports presenting speci®c localization of pro®lin (Holzinger et al. 1997), PIP2 (Mazotti et al. 1995), the actin depolymerizing factor ZmADF3 (Jiang et al. 1997) and actin (Schindler and Jiang 1989) within the nucleus. 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