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.     

Arabidopsis Profilin Isoforms,PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fuorescent protein （GFP） tag and expressed in Escherichia coil and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S：：GFP-PRF1 formed a filamentous network, while 35S：：GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.     

Mouse profilin 2 regulates endocytosis and competes with SH3 ligand binding to dynamin 1

Mammalian profilins are abundantly expressed actin monomer-binding proteins, highly conserved with respect to their affinities for G-actin, poly-L-proline, and phosphoinositides. Profilins associate with a large number of proline-rich proteins; the physiological significance and regulation of which is poorly understood. Here we show that profilin 2 associates with dynamin 1 via the C-terminal proline-rich domain of dynamin and thereby competes with the binding of SH3 ligands such as endophilin, amphiphysin, and Grb2, thus interfering with the assembly of the endocytic machinery. We also present a novel role for the brain-specific mouse profilin 2 as a regulator of membrane trafficking. Overexpression of profilin 2 inhibits endocytosis, whereas lack of profilin 2 in neurons results in an increase in endocytosis and membrane recycling. Phosphatidylinositol 4,5-bisphosphate releases profilin 2 from the profilin 2-dynamin 1 complex as well as from the profilin 2-actin complex, suggesting that profilin 2 is diverging the phosphoinositide signaling pathway to actin polymerization as well as endocytosis.     

The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.     

Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-L-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-L-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-L-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.     

Distinct roles of the first introns on the expression of Arabidopsis profilin gene family members

Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (Arabidopsis thaliana), five profilins were identified. The vegetative class profilins, PRF1, PRF2, and PRF3, are expressed in vegetative organs. The reproductive class profilins, PRF4 and PRF5, are mainly expressed in pollen. In this study, we examined the role of the first intron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. In transgenic plants, we examined PRF2 and PRF5, which represent vegetative and reproductive profilins. The expression of the PRF2 promoter fused with the beta-glucuronidase (GUS) gene was observed in the vascular bundles, but transgenic plants carrying the PRF2 promoter-GUS with its first intron showed constitutive expression throughout the vegetative tissues. However, the first intron of PRF5 had little effect on the reporter gene expression pattern. Transgenic plants containing PRF5 promoter-GUS fusion with or without its first intron showed reproductive tissue-specific expression. To further investigate the different roles of the first two introns on gene expression, the first introns were exchanged between PRF2 and PRF5. The first intron of PRF5 had no apparent effect on the expression pattern of the PRF2 promoter. But, unlike the intron of PRF5, the first intron of PRF2 greatly affected the reproductive tissue-specific expression of the PRF5 promoter, confirming a different role for these introns. The results of a transient expression assay indicated that the first intron of PRF1 and PRF2 enhances gene expression, whereas PRF4 and PRF5 do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns are required for the strong and constitutive gene expression of PRF1 and PRF2 in vegetative tissues.     

X-ray structure determination of human profilin II: A comparative structural analysis of human profilins

Human profilins are multifunctional, single-domain proteins which directly link the actin microfilament system to a variety of signalling pathways via two spatially distinct binding sites. Profilin binds to monomeric actin in a 1:1 complex, catalyzes the exchange of the actin-bound nucleotide and regulates actin filament barbed end assembly. Like SH3 domains, profilin has a surface-exposed aromatic patch which binds to proline-rich peptides. Various multidomain proteins including members of the Ena/VASP and formin families localize profilin:actin complexes through profilin:poly-L-proline interactions to particular cytoskeletal locations (e.g. focal adhesions, cleavage furrows). Humans express a basic (I) and an acidic (II) isoform of profilin which exhibit different affinities for peptides and proteins rich in proline residues. Here, we report the crystallization and X-ray structure determination of human profilin II to 2.2 A. This structure reveals an aromatic extension of the previously defined poly-L-proline binding site for profilin I. In contrast to serine 29 of profilin I, tyrosine 29 in profilin II is capable of forming an additional stacking interaction and a hydrogen bond with poly-L-proline which may account for the increased affinity of the second isoform for proline-rich peptides. Differential isoform specificity for proline-rich proteins may be attributed to the differences in charged and hydrophobic residues in and proximal to the poly-L-proline binding site. The actin-binding face remains nearly identical with the exception of five amino acid differences. These observations are important for the understanding of the functional and structural differences between these two classes of profilin isoforms.     

Formin differentially utilizes profilin isoforms to rapidly assemble actin filaments

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.     

The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.

Profilins are small proteins that form complexes with G-actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly-L-proline, but the biological significance of this interaction is not yet known. The recent molecular cloning of the vasodilator-stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP- and cGMP-dependent protein kinases, revealed the presence of a proline-rich domain which prompted us to investigate a possible interaction with profilins. VASP is a microfilament and focal adhesion associated protein which is also concentrated in highly dynamic regions of the cell cortex. Here, we demonstrate that VASP is a natural proline-rich profilin ligand. Human platelet VASP bound directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly-L-proline or a peptide corresponding to a proline-rich VASP motif. Finally, the subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. Our data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation.     

Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissues

Profilin is a low-molecular weight, actin monomer-binding protein that regulates the organization of actin cytoskeleton in eukaryotes, including higher plants. Unlike the simple human or yeast systems, the model plant Arabidopsis has an ancient and highly divergent multi-gene family encoding five distinct profilin isovariants. Here we compare and characterize the regulation of these profilins in different organs and during microspore development using isovariant-specific monoclonal antibodies. We show that PRF1, PRF2, and PRF3 are constitutive, being strongly expressed in all vegetative tissues at various stages of development. These profilin isovariants are also predominant in ovules and microspores at the early stages of microsporogenesis. In contrast, PRF4 and PRF5 are late pollen-specific and are not detectable in other cell types of the plant body including microspores and root hairs. Immunocytochemical studies at the subcellular level reveal that both the constitutive and pollen-specific profilins are abundant in the cytoplasm. In vegetative cell types, such as root apical cells, profilins showed localization to nuclei in addition to the cytoplasmic staining. The functional diversity of profilin isovariants is discussed in light of their spatio-temporal regulation during vegetative development, pollen maturation, and pollen tube growth.     

Mechanism of regulation of actin polymerization by Physarum profilin

Physarum profilin reduces the rates of nucleation and elongation of F- actin and also reduces the extent of polymerization of actin at the steady state in a concentration-dependent fashion. The apparent critical concentration for polymerization of actin is increased by the addition of profilin. These results can be explained by the idea that Physarum profilin forms a 1:1 complex with G-actin and decreases the concentration of actin available for polymerization. The dissociation constant for binding of profilin to G-actin is estimated from the kinetics of polymerization of G-actin and elongation of F-actin nuclei and from the increase of apparent critical concentration in the presence of profilin. The dissociation constants for binding of Physarum profilin to Physarum and muscle actins under physiological ionic conditions are in the ranges of 1.4-3.7 microM and 11.3-28.5 microM, respectively. When profilin is added to an F-actin solution, profilin binds to G-actin which co-exists with F-actin, and then G- actin is dissociated from F-actin to compensate for the decrease of the concentration of free G-actin and to keep it constant at the critical concentration. At the steady state, free G-actin of the critical concentration is in equilibrium not only with F-actin but also with profilin-G-actin complex. The stoichiometry of 1:1 for the formation of complex between profilin and G-actin is directly shown by means of chemical cross-linking.     

纤维蛋白影响花粉肌动蛋白体外聚合分析

The effects of different ratio of native profilin on maize (Zea mays L.) pollen actin polymerization in vitro were analyzed by using ultracentrifuging sedimentation and ultraviolet absorption spectrum measurement (the molar ratio of profilin to actin was 2∶1, 1.5∶1, 1∶1, 0.5∶1, 0.1∶1 respectively). Preliminary results showed that profilin bound to G-actin and inhibited its polymerization. The inhibition of actin polymerization by profilin increased with the increasing ratio of profilin to pollen actin. The dissociation constant （Kd） value of profilin for binding to actin was (1.30±0.33) μmol/L. No stimulation effect of profilin on actin polymerization was observed, suggesting that pollen profilin may affect actin organization by sequestering the G-actin.     

Class-specific interaction of profilin and ADF isovariants with actin in the regulation of plant development

Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms. Plant genomes encode complex actin and actin binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns. In Arabidopsis thaliana, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture. Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes. We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs. To examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically coexpressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1. Our results demonstrated that the coexpression of these reproductive, but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture to the double transgenic plants. Thus, the actins and ABPs appear to have evolved class-specific, protein-protein interactions that are essential to the normal regulation of plant growth and development.     

Cloning and functional characterization of a formin-like protein (AtFH8) from Arabidopsis

The actin cytoskeleton is required for many cellular processes in plant cells. The nucleation process is the rate-limiting step for actin assembly. Formins belong to a new class of conserved actin nucleator, which includes at least 2 formin homology domains, FH1 and FH2, which direct the assembly of unbranched actin filaments. The function of plant formins is quite poorly understood. Here, we provide the first biochemical study of the function of conserved domains of a formin-like protein (AtFH8) from Arabidopsis (Arabidopsis thaliana). The purified recombinant AtFH8(FH1FH2) domain has the ability to nucleate actin filaments in vitro at the barbed end and caps the barbed end of actin filaments, decreasing the rate of subunit addition and dissociation. In addition, purified AtFH8(FH1FH2) binds actin filaments and severs them into short fragments. The proline-rich domain (FH1) of the AtFH8 binds directly to profilin and is necessary for nucleation when actin monomers are profilin bound. However, profilin inhibits the nucleation mediated by AtFH8(FH1FH2) to some extent, but increases the rate of actin filament elongation in the presence of AtFH8(FH1FH2). Moreover, overexpression of the full-length AtFH8 in Arabidopsis causes a prominent change in root hair cell development and its actin organization, indicating the involvement of AtFH8 in polarized cell growth through the actin cytoskeleton.     

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.     

Characterization of renatured profilin purified by urea elution from poly-L-proline agarose columns

We present evidence that native profilin can be purified from cellular extracts of Acanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly-L-proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6-8 M. Acanthamoeba profilin-I and profilin-II can subsequently be separated by cation exchange chromatography. The yield of Acanthamoeba profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: 1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly-L-proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; 2) dialyzed profilins form 3-D crystals under the same conditions as native profilins; 3) dialyzed Acanthamoeba profilin-I has an NMR spectrum identical with that of native profilin-I; and 4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyostelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly-L-proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin-II or alpha-actinin in crude cell extracts can be bound selectively to the poly-L-proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin-II from both Acanthamoeba and Dictyostelium cell extracts and alpha-actinin from Acanthamoeba cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly-L-proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.     

Phosphorylation of Amoeba G-actin and its effect on actin polymerization

Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.     

Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion.The actin cytoskeleton provides tracks for the deposition of cell wall materials and plays important roles during many cellular processes, such as cell expansion and morphogenesis, vesicle trafficking, and the response to biotic and abiotic signals (Baskin, 2005; Smith and Oppenheimer, 2005; Szymanski and Cosgrove, 2009; Ehrhardt and Bezanilla, 2013; Rounds and Bezanilla, 2013). Plant cells respond to diverse internal and external stimuli by regulating the turnover and rearrangement of actin cytoskeleton networks in the cytoplasm (Staiger, 2000; Pleskot et al., 2013). How these actin rearrangements sense the cellular environment and what accessory proteins modulate specific aspects of remodeling remain an area of active investigation (Henty-Ridilla et al., 2013; Li et al., 2014a, 2015).Using high spatial and temporal resolution imaging afforded by variable-angle epifluorescence microscopy (VAEM; Konopka and Bednarek, 2008), we quantified the behavior of actin filaments in Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells (Staiger et al., 2009). There are two types of actin filament arrays in the cortical cytoplasm of epidermal cells: bundles and single filaments. Generally, actin bundles are stable with higher pixel intensity values, whereas individual actin filaments are fainter, more ephemeral, and constantly undergo rapid assembly and disassembly through a mechanism that has been defined as “stochastic dynamics” (Staiger et al., 2009; Henty et al., 2011; Li et al., 2012, 2015). Elongating actin filaments in the cortical cytoskeleton originate from three distinct locations: the ends of preexisting actin filaments, the side of filaments or bundles, and de novo in the cytoplasm. Plant actin filaments elongate at rates of 1.6 to 3.4 μm/s, which is the fastest assembly reported in eukaryotic cells. Distinct from the mechanism of treadmilling and fast depolymerization in vitro, however, the disassembly of single actin filaments occurs predominately through prolific severing activity (Staiger et al., 2009; Smertenko et al., 2010; Henty et al., 2011). A commonly held view is that the dynamic actin network in plant cells is regulated by the activities of conserved and novel actin-binding proteins (ABPs). Through reverse-genetic approaches and state-of-the-art imaging modalities, we and others have demonstrated that several key ABPs are involved in the regulation of stochastic actin dynamic properties in a wide variety of plants and cell types (Thomas, 2012; Henty-Ridilla et al., 2013; Li et al., 2014a, 2015). Through these efforts, the field has developed a working model for the molecular mechanisms that underpin actin organization and dynamics in plant cells (Li et al., 2015).Profilin is a small (12–15 kD), conserved actin-monomer binding protein present in all eukaryotic cells (dos Remedios et al., 2003). Profilin binds to actin by forming a 1:1 complex with globular (G-)actin, suppresses spontaneous actin nucleation, and inhibits monomer addition at filament pointed ends (Blanchoin et al., 2014). The consequences of profilin activity on actin filament turnover differ based on cellular conditions and the presence of other ABPs. In vitro studies show that the profilin-actin complex associates with the barbed ends of filaments and promotes actin polymerization by lowering the critical concentration and increasing nucleotide exchange on G-actin (Pollard and Cooper, 1984; Pantaloni and Carlier, 1993). When barbed ends are occupied by capping protein, profilin acts as an actin-monomer sequestering protein. These opposing effects of profilin might be a regulatory mechanism for profilin modulation of actin dynamics in cells. In addition to actin, profilin interacts with Pro-rich proteins, as well as polyphosphoinositide lipids in vitro (Machesky et al., 1994). Formin is an ABP that mediates both actin nucleation and processive elongation using the pool of profilin-actin complexes (Blanchoin et al., 2010). The primary sequence of formin includes a Pro-rich domain, named Formin Homology1 (FH1). Evidence from fission and budding yeast shows that profilin can increase filament elongation rates by binding to the FH1 domain (Kovar et al., 2003; Moseley and Goode, 2005; Kovar, 2006). The FH1 domain of Arabidopsis FORMIN1 (AtFH1) is also reported to modulate actin nucleation and polymerization in vitro (Michelot et al., 2005). Recently, two groups reported that profilin functions as a gatekeeper during the construction of different actin networks generated by formin or ARP2/3 complex in yeast and mammalian cells (Rotty et al., 2015; Suarez et al., 2015). These studies highlight the importance of profilin regulation in coordinating the different actin arrays present in the same cytoplasm of eukaryotic cells. However, direct evidence for how profilin facilitates formin-mediated actin nucleation or barbed end elongation in cells remains to be established.Genomic sequencing and isolation of PROFILIN (PRF) cDNAs from plants reveal that profilin is encoded by a multigene family. For example, moss (Physcomitrella patens) has three isovariants (Vidali et al., 2007) and maize (Zea mays) has five (Staiger et al., 1993; Kovar et al., 2001). In Arabidopsis, at least five PRF genes have been identified (Christensen et al., 1996; Huang et al., 1996; Kandasamy et al., 2002). Studies in maize show that the biochemical properties of profilin isoforms differ in vitro (Kovar et al., 2000). Moreover, the localization of profilin isoforms reveals organ-specific expression patterns. Detection of protein levels in vivo with isovariant-specific profilin antibodies demonstrate that Arabidopsis PRF1, PRF2, and PRF3 are constitutively expressed in vegetative tissues, whereas PRF4 and PRF5 are expressed mainly in flower and pollen tissues (Christensen et al., 1996; Huang et al., 1996; Ma et al., 2005).Several genetic studies on the functions of profilin in plants have been conducted. Reduction of profilin levels in P. patens results in the inhibition of tip growth, disorganization of F-actin, and formation of actin patches (Vidali et al., 2007). Moreover, it was shown that the interaction between profilin and actin or Pro-rich ligands is critical for tip growth in moss. Arabidopsis PRF1 has been demonstrated to be involved in cell elongation, cell shape maintenance, and control of flowering time through overexpression and antisense PRF1 transgenic plants, and further, the reduction of PRF1 inhibits the growth of hypocotyls (Ramachandran et al., 2000). However, investigation of a prf1-1 mutant, which contains a T-DNA insertion in the promoter region of the PRF1 gene, indicates that cell expansion of seedlings is promoted and that protein levels of PRF1 are regulated by light (McKinney et al., 2001). Recently, Müssar et al. (2015) reported a new Arabidopsis T-DNA insertion allele, prf1-4, that shows an obvious dwarf seedling phenotype. To date, however, there has not been a critical examination of the impact of the loss of profilin on the organization and dynamics of bona-fide single actin filaments in vivo.Here, we use a combination of genetics and live-cell imaging to investigate the role of PRF1 in the control of actin dynamics and its effect on axial cell expansion. We observed a significant decrease in the overall filament nucleation frequency in prf1 mutants, which is opposite to expectations if profilin suppresses spontaneous nucleation. Through a pharmacological approach, we found that nucleation frequency in wild-type cells treated with a formin inhibitor, SMIFH2, phenocopied prf1 mutants. We also analyzed the dynamic turnover of individual filaments in prf1 mutants and observed a significant decrease in the rate of actin filament elongation and maximum length of actin filaments. Specifically, we found that PRF1 favors the growth of a subpopulation of actin filaments that elongate at rates greater than 2 μm/s and similar results were obtained in cells after SMIFH2 treatment. Our results provide compelling evidence that Arabidopsis PRF1 contributes to stochastic actin dynamics by modulating formin-mediated actin nucleation and filament elongation during axial cell expansion.     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。