The role of the actin cytoskeleton in plant cell signaling.

The plant actin cytoskeleton provides a dynamic cellular component which is involved in the maintenance of cell shape and structure. It has been demonstrated recently that the actin cytoskeleton and its associated elements provide a key target in many signaling events. In addition to acting as a target, the actin cytoskeleton can also act as a transducer of signal information. In this review we describe some newly discovered aspects of the roles of the actin cytoskeleton in plant cell signaling. In addition to a summary of the roles played by actin-binding proteins, we also briefly review the progress made in understanding how the actin cytoskeleton participates in the self-incompatibility response in pollen tubes. Finally, the emerging importance of the actin cytoskeleton in the perception and responses to stimuli such as gravity, touch and cold stress exposure are discussed. Contents I. Introduction - the actin cytoskeleton 13 II. Actin-binding proteins 14 III. The actin cytoskeleton as a target and mediator of plant cell signaling 20 IV. Summary and conclusion 25 References 25 Acknowledgements 25.

The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas.

The integration of signals received by a cell, and their transduction to targets, is essential for all cellular responses. The cytoskeleton has been identified as a major target of signalling cascades in both animal and plant cells. Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in the inhibition of incompatible pollen. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. It has been demonstrated recently that SI induces dramatic alterations in the organization of the pollen actin cytoskeleton. This implicates the actin cytoskeleton as a key target for the SI-stimulated signals. The cytological alterations to the actin cytoskeleton that are triggered in response to SI are described here and there seem to be several stages that are distinguishable temporally. Evidence was obtained that F-actin depolymerization is also stimulated. The current understanding that the actin cytoskeleton is a target for the signals triggered by the SI response is discussed. It is suggested that these F-actin alterations may be Ca(2+)-mediated and that this could be a mechanism whereby SI-induced tip growth inhibition is achieved. The potential for actin-binding proteins to act as key mediators of this response is discussed and the mechanisms that may be responsible for effecting these changes are described. In particular, the parallels between sustained actin rearrangements during SI and in apoptosis of animal cells are considered.

Actin and actin-binding proteins in higher plants.

The actin cytoskeleton is a complex and dynamic structure that participates in diverse cellular events which contribute to plant morphogenesis and development. Plant actins and associated actin-binding proteins are encoded by large, differentially expressed gene families. The complexity of these gene families is thought to have been conserved to maintain a pool of protein isovariants with unique properties, thus providing a mechanistic basis for the observed diversity of plant actin functions. Plants contain actin-binding proteins which regulate the supramolecular organization and function of the actin cytoskeleton, including monomer-binding proteins (profilin), severing and dynamizing proteins (ADF/cofilin), and side-binding proteins (fimbrin, 135-ABP/villin, 115-ABP). Although significant progress in documenting the biochemical activities of many of these classes of proteins has been made, the precise roles of actin-binding proteins in vivo awaits clarification by detailed mutational analyses.

Chlamydomonas reinhardtii produces a profilin with unusual biochemical properties.

We report the characterization of a profilin orthologue from Chlamydomonas reinhardtii. CrPRF, probably the only profilin isoform, is present in both the cell body and flagella. Examination of vegetative and gametic cells by immunofluorescence microscopy using multiple fixation procedures also revealed enrichment of CrPRF at the anterior of the cell near the base of flagella and near the base of the fertilization tubule in mating type plus gametes. Purified, recombinant CrPRF binds to actin with a Kd value approximately 10(-7) and displaces nuclei in a live cell 'nuclear displacement' assay, consistent with profilin's ability to bind G-actin in vivo. However, when compared with other profilin isoforms, CrPRF has a relatively low affinity for poly-L-proline and for phosphatidylinositol (4,5) bisphosphate micelles. Furthermore, and surprisingly, CrPRF inhibits exchange of adenine nucleotide on G-actin in a manner similar to human ADF or DNase I. Thus, we postulate that a primary role for CrPRF is to sequester actin in Chlamydomonas. The unusual biochemical properties of CrPRF offer a new opportunity to distinguish specific functions for profilin isoforms.

SEPH, a Cdc7p orthologue from Aspergillus nidulans, functions upstream of actin ring formation during cytokinesis.

In the filamentous fungus, Aspergillus nidulans, multiple rounds of nuclear division occur before cytokinesis, allowing an unambiguous identification of genes required specifically for cytokinesis. As in animal cells, both an intact microtubule cytoskeleton and progression through mitosis are required for actin ring formation and contraction. The sepH gene from A. nidulans was discovered in a screen for temperature-sensitive cytokinesis mutants. Sequence analysis showed that SEPH is 42% identical to the serine-threonine kinase Cdc7p from fission yeast. Signalling through the Septation Initiation Network (SIN), which includes Cdc7p and the GTPase Spg1p, is emerging as a primary regulatory pathway used by fission yeast to control cytokinesis. A similar group of proteins comprise the Mitotic Exit Network (MEN) in budding yeast. This is the first direct evidence for the existence of a functional SIN-MEN pathway outside budding and fission yeast. In addition to SEPH, potential homologues were also identified in other fungi and plants but not in animal cells. Deletion of sepH resulted in a viable strain that failed to septate at any temperature. Interestingly, quantitative analysis of the actin cytoskeleton revealed that sepH is required for construction of the actin ring. Therefore, SEPH is distinct from its counterpart in fission yeast, in which SIN components operate downstream of actin ring formation and are necessary for ring contraction and later events of septation. We conclude that A. nidulans has components of a SIN-MEN pathway, one of which, SEPH, is required for early events during cytokinesis.

Fluorescently-labeled fimbrin decorates a dynamic actin filament network in live plant cells.

Recently it has been established, through a detailed biochemical analysis, that recombinant Arabidopsis thaliana fimbrin 1 (AtFim1) is a member of the fimbrin/plastin family of actin filament bundling or cross-linking proteins [D.R. Kovar et al. (2000) Plant J 24:625-636]. To determine whether AtFim1 can function as an F-actin-binding protein in the complex environment of the plant cell cytoplasm, we created a fluorescent protein analog and introduced it by microinjection into live Tradescantia virginiana L. stamen hair cells. AtFim1 derivatized with Oregon Green 488 had biochemical properties similar to unlabeled fimbrin, including the Kd value for binding to plant F-actin and the ability to cross-link filaments into higher-order structures. Fluorescent-fimbrin decorated an array of fine actin filaments in the cortical cytoplasm of stamen hair cells, which were shown with time-course studies to be highly dynamic. These data establish AtFim1 as a bona fide member of the fimbrin/plastin family, and represent the first use of a plant actin-binding protein as a powerful cytological tool for tracking the spatial and temporal redistribution of actin filaments in individual cells.

The characterization of ligand-specific maize (Zea mays) profilin mutants.

Profilins are low-molecular-mass (12-15 kDa) cytosolic proteins that are major regulators of actin assembly in all eukaryotic cells. In general, profilins from evolutionarily diverse organisms share the ability to bind to G-actin, poly-(L-proline) (PLP) and proline-rich proteins, and polyphosphoinositides. However, the functional importance of each of these interactions remains unclear and might differ between organisms. We investigated the importance of profilin's interaction with its various ligands in plant cells by characterizing four maize (Zea mays) profilin 5 (ZmPRO5) mutants that had single amino acid substitutions in the presumed sites of ligand interaction. Comparisons in vitro with wild-type ZmPRO5 showed that these mutations altered ligand association specifically. ZmPRO5-Y6F had a 3-fold increased affinity for PLP, ZmPRO5-Y6Q had a 5-fold decreased affinity for PLP, ZmPRO5-D8A had a 2-fold increased affinity for PtdIns(4,5)P(2) and ZmPRO5-K86A had a 35-fold decreased affinity for G-actin. When the profilins were microinjected into Tradescantia stamen hair cells, ZmPRO5-Y6F increased the rate of nuclear displacement in stamen hairs, whereas ZmPRO5-K86A decreased the rate. Mutants with a decreased affinity for PLP (ZmPRO5-Y6Q) or an enhanced affinity for PtdIns(4,5)P(2) (ZmPRO5-D8A) were not significantly different from wild-type ZmPRO5 in affecting nuclear position. These results indicate that plant profilin's association with G-actin is extremely important and further substantiate the simple model that profilin acts primarily as a G-actin-sequestering protein in plant cells. Furthermore, interaction with proline-rich binding partners might also contribute to regulating profilin's effect on actin assembly in plant cells.

AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana.

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament-crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin-binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament-crosslinking protein. AtFim1 binds pollen actin filaments (F-actin) in a calcium-independent manner, with an average dissociation constant (Kd) of 0.55+/-0.21 microM and with a stoichiometry at saturation of 1:4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F-actin by a calcium-independent mechanism, in contrast to crosslinking of plant actin by human T-plastin, a known calcium-sensitive actin-crosslinking protein. When micro-injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2-4 min. Using the 'nuclear displacement assay' as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)-induced depolymerization, in a dose-dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin-mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium-independent, actin filament-crosslinking protein that interacts with the actin cytoskeleton in living plant cells.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Characterization of Ricinus communis phloem profilin, RcPRO1.

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.

Maize profilin isoforms are functionally distinct.

Profilin is an actin monomer binding protein that, depending on the conditions, causes either polymerization or depolymerization of actin filaments. In plants, profilins are encoded by multigene families. In this study, an analysis of native and recombinant proteins from maize demonstrates the existence of two classes of functionally distinct profilin isoforms. Class II profilins, including native endosperm profilin and a new recombinant protein, ZmPRO5, have biochemical properties that differ from those of class I profilins. Class II profilins had higher affinity for poly-l-proline and sequestered more monomeric actin than did class I profilins. Conversely, a class I profilin inhibited hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate by phospholipase C more strongly than did a class II profilin. These biochemical properties correlated with the ability of class II profilins to disrupt actin cytoplasmic architecture in live cells more rapidly than did class I profilins. The actin-sequestering activity of both maize profilin classes was found to be dependent on the concentration of free calcium. We propose a model in which profilin alters cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration. These results provide strong evidence that the maize profilin gene family consists of at least two classes, with distinct biochemical and live-cell properties, implying that the maize profilin isoforms perform distinct functions in the plant.

Latrunculin B has different effects on pollen germination and tube growth.

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.

Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses.

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1-1Delta mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1-1Delta mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1-1Delta mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

A potential signaling role for profilin in pollen of Papaver rhoeas.

Regulation of pollen tube growth is known to involve alterations in intracellular calcium levels and phosphoinositide signaling, although the mechanisms involved are unclear. However, it appears likely that pollination events involve a complex interplay between signaling pathways and components of the actin cytoskeleton in pollen. In many eukaryotic cells, actin binding proteins function as stimulus-response modulators, translating signals into alterations in the cytoplasmic architecture. In this study, we examined whether profilin, which is a member of this class of signaling intermediate, might play a similar role in pollen. We have analyzed the functional properties of native profilin from pollen of Papaver rhoeas and have investigated the effects of profilin on the phosphorylation of pollen proteins in vitro by adding a slight excess of profilin to cytosolic pollen extracts. We present clear evidence that profilin interacts with soluble pollen components, resulting in dramatic alterations in the phosphorylation of several proteins. We also show, albeit in vitro, the involvement of profilin in modulating the activity of a signaling component(s) affecting protein phosphorylation. Our data, which suggest that pollen profilin can regulate actin-based cytoskeletal protein assembly and protein kinase or phosphatase activity, indicate a possible role for the involvement of profilin in signaling pathways that may regulate pollen tube growth.

Pollen profilin function depends on interaction with proline-rich motifs.

The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells. Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms. Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L-proline and monomeric actin from maize pollen. In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen. The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen. In contrast, the affinity of ZmPRO4 for poly-L-proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms. When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1. A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L-proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4. In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L-proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells.

Profilin is associated with the plasma membrane in microspores and pollen.

In higher plants, a large number of isoforms for the actin monomer-binding protein profilin have been identified, whereas other organisms express only few profilins. Furthermore, plant profilin isoforms are expressed in a tissue-specific manner. These observations raise questions concerning functional and locational differences between isoforms of plant profilins. In this paper, we introduce three polyclonal antisera and one monoclonal antibody developed against purified pollen profilins from Zea mays and against recombinant maize profilin. Immunoblot analyses of native profilins and four recombinant maize pollen profilin isoforms show that three of the antibodies display a preference for certain isoforms. In situ immunofluorescence of pollen of Zea mays and two developmental stages of microspores of Betula pumila indicates that all antibodies label plasma membrane-associated domains. Thus, we show that at least some profilin isoforms are located at a distinct subcellular domain within developing microspores and, less distinctly, in mature pollen. This contrasts previously reported uniform distributions throughout the cytoplasm of mature pollen and pollen tubes. The results are discussed in light of the large number of profilins co-expressed in plants and with reference to accumulating evidence for functional differences between profilin isoforms.

Actin Purified from Maize Pollen Functions in Living Plant Cells.

A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal [alpha]-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate [alpha]-actin and had a critical concentration for assembly of 0.6 [mu]M. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.

Characterization of maize (Zea mays) pollen profilin function in vitro and in live cells.

Profilin is a small, 12-15 kDa, actin-binding protein that interacts with at least three different ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mechanism for sequestering actin monomers in the cytoplasm. It has recently become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-rich proteins, or due to the ability to lower the critical concentration for actin assembly at the fast-growing barbed end of actin filaments. Because actin-binding proteins have been shown to behave differently with heterologous sources of actin, we characterized the interaction between maize pollen profilins and plant G-actin. The equilibrium dissociation constants measured by tryptophan fluorescence quenching were similar to those of other CaATP-G-actin-profilin complexes (Kd=1.0-1.5 microM). The ability of maize profilin isoforms to bind poly-l-proline was analysed, and the Kd values for recombinant pollen and human profilins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-l-proline was substantially lower than that of any of the recombinant proteins by one of these assays. The possibility of post-translational modification of profilin in the mature pollen grain is discussed. Finally, we quantified the effects of microinjection of each profilin isoform on the cytoarchitecture of Tradescantia stamen hair cells and show that the resultant disruption can be used to compare actin-binding proteins in living cells. The results are discussed in relation to a recent model of the interphase actin array in these plant cells.

Plant profilins rescue the aberrant phenotype of profilin-deficient Dictyostelium cells.

To characterize the function of plant profilins in vivo, we expressed two pollen specific Zea mays (maize) profilin isoforms in profilin-minus Dictyostelium discoideum mutants. In maize, profilins exist as a multigene family containing 4 or more members which are highly similar to each other but substantially less similar to profilins from animals and lower eukaryotes. Previously we have shown that D. discoideum profilin-minus cells have an aberrant phenotype due to defects in cell shape, cytokinesis, and development. These defects could be rescued by introducing the pollen-specific profilins 1 or 2 from maize using a newly constructed expression vector. Expression of the heterologous profilins in Dictyostelium clones was assayed by affinity purification of the pollen profilins with poly-proline agarose and by immunoblotting with a polyclonal antiserum raised against maize pollen profilin. In contrast to the profilin-minus mutants, Dictyostelium cells expressing plant profilins showed normal cell shape, contained less F-actin, and were able to form fruiting bodies. These data provide genetic evidence that maize pollen profilins, even though they are specific for a distinct developmental stage, share functional properties with profilin from a lower eukaryote and apparently act as G-actin-sequestering proteins in this system.

Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments.

BACKGROUND: Cytoplasmic streaming is a conspicuous feature of plant cell behaviour, in which organelles and vesicles shuttle along cytoplasmic strands that contain actin filaments. The mechanisms that regulate streaming and the formation of actin filament networks are largely unknown, but in all likelihood involve actin-binding proteins. The monomeric actin-binding protein, profilin, is a key regulator of actin-filament dynamics in animal cells and it has recently been identified in plants as a pollen allergen. We set out to determine whether plant profilin can act as a monomeric actin-binding protein and influence actin dynamics in plant cells in vivo. RESULTS: Recombinant birch-pollen profilin was purified by polyproline affinity chromatography and microinjected into Tradescantia blossfeldiana stamen hair cells. After profilin injection, a rapid and irreversible change in cellular organization and streaming was observed: within 1-3 minutes the transvacuolar cytoplasmic strands became thinner and snapped, and cytoplasmic streaming ceased. Fluorescein-labelled-phalloidin staining confirmed that this was due to depolymerization of actin filaments. To confirm that the effects observed were due to sequestration of monomeric actin, another monomeric actin-binding protein, DNase I, was injected and found to produce comparable results. CONCLUSIONS: Profilin can act as a potent regulator of actin organization in living plant cells. Its rapid effect on the integrity of cytoplasmic strands and cytoplasmic streaming supports a model in which organelle movements depend upon microfilaments that exist in dynamic equilibrium with the pool of monomeric actin.