Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M312973200 on March 22, 2004

J. Biol. Chem., Vol. 279, Issue 22, 23364-23375, May 28, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemantal Data
Right arrow All Versions of this Article:
279/22/23364    most recent
M312973200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, S.
Right arrow Articles by Staiger, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, S.
Right arrow Articles by Staiger, C. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

A Gelsolin-like Protein from Papaver rhoeas Pollen (PrABP80) Stimulates Calcium-regulated Severing and Depolymerization of Actin Filaments*

Shanjin Huang{ddagger}, Laurent Blanchoin§, Faisal Chaudhry{ddagger}, Vernonica E. Franklin-Tong||, and Christopher J. Staiger{ddagger}**

From the {ddagger}Department of Biological Sciences and The Purdue Motility Group, Purdue University, West Lafayette, Indiana 47907-2064, the §Laboratoire de Physiologie Cellulaire Végétale, Commissariat à l'Energie Atomique (CEA)/CNRS/Université Joseph Fourier, CEA F38054 Grenoble, France, and the ||School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

Received for publication, November 30, 2003 , and in revised form, March 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytoskeleton is a key regulator of plant morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. During the self-incompatibility response of Papaver rhoeas L. (field poppy) pollen, the actin filament network is rapidly depolymerized by a flood of cytosolic free Ca2+ that results in cessation of tip growth and prevention of fertilization. Attempts to model this dramatic cytoskeletal response with known pollen actin-binding proteins (ABPs) revealed that the major G-actin-binding protein profilin can account for only a small percentage of the measured depolymerization. We have identified an 80-kDa, Ca2+-regulated ABP from poppy pollen (PrABP80) and characterized its biochemical properties in vitro. Sequence determination by mass spectrometry revealed that PrABP80 is related to gelsolin and villin. The molecular weight, lack of filament cross-linking activity, and a potent severing activity are all consistent with PrABP80 being a plant gelsolin. Kinetic analysis of actin assembly/disassembly reactions revealed that substoichiometric amounts of PrABP80 can nucleate actin polymerization from monomers, block the assembly of profilin-actin complex onto actin filament ends, and enhance profilin-mediated actin depolymerization. Fluorescence microscopy of individual actin filaments provided compelling, direct evidence for filament severing and confirmed the actin nucleation and barbed end capping properties. This is the first direct evidence for a plant gelsolin and the first example of efficient severing by a plant ABP. We propose that PrABP80 functions at the center of the self-incompatibility response by creating new filament pointed ends for disassembly and by blocking barbed ends from profilin-actin assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The plant cytoskeleton comprises a dynamic network of actin filaments, microtubules, and accessory proteins that powers cytoplasmic streaming, prevents fungal attack, patterns the deposition of cellulosic wall polymers, and shapes cellular morphogenesis. Understanding how the cytoskeleton is organized, how it responds to environmental cues, and how its dynamics are regulated are central questions in plant biology. In addition to being essential for sexual reproduction, pollen is an ideal choice of material for studies of the cytoskeleton. Actin is one of the most abundant proteins in pollen, representing 5–20% of total cellular protein (1, 2). Cytoskeletal genes are among the most abundantly expressed classes of transcripts in Arabidopsis pollen (3), and several classes of actin-binding protein (ABP)1 have been isolated and characterized biochemically (4–6). Pollen tube growth is arguably the most dramatic example of cellular morphogenesis in plants (5, 7). A cytoplasmic extension of the vegetative cell, the pollen tube, carries the male gametes through the pistil at growth rates up to 1 cm/h. The tip growth mechanism involves carefully orchestrated delivery of cell wall materials and plasma membrane through the directed trafficking of secretory vesicles. Underlying this cellular expansion is a polar distribution of cytoplasmic organelles, oscillatory gradients of cytosolic ions, and a specific organization of the cytoskeletal machinery. Prominent actin cables support reverse fountain cytoplasmic streaming and are arranged axially throughout much of the cytoplasm. A collar-like zone of fine filament bundles, in the apical 10–15 µm, and a dynamic meshwork of fine actin filaments (F-actin) at the extreme apex are thought to organize vesicle docking and fusion. Although actin filament turnover is essential for pollen tube growth (8, 9), exactly how the cytoskeleton functions and what accessory factors modulate these events remain poorly understood.

Many plant species utilize genetic mechanisms to prevent inbreeding. The self-incompatibility (SI) response in field poppy (Papaver rhoeas) pollen involves a flood of cytosolic free calcium ([Ca2+]i) that correlates with the cessation of tip growth (10–12). Cytological and biochemical studies demonstrate that reorganization of the actin cytoskeleton is one of the earliest events triggered by SI (13, 14). Quantitative analyses of F-actin levels reveal a rapid and sustained depolymerization of actin filaments with reductions of 56 and 74% in pollen grains and pollen tubes, respectively (14). Because actin depolymerization is mimicked by treatments with calcium ionophores, attempts to model the response in vitro with known calcium-sensitive ABPs were made.

Pollen profilin binds to monomeric (G-) actin with a modest affinity and has increased G-actin sequestering activity in the presence of micromolar Ca2+ (14, 15). For native poppy profilin, however, the cellular concentration and apparent Kd at physiologically relevant [Ca2+] are inconsistent with it functioning alone to mediate the depolymerization of actin. It was proposed that profilin acts in cooperation with other, yet to be discovered, ABPs to effect the destruction of actin networks during SI (14). Profilin acts like a simple sequestering protein in the presence of capping factors that block the barbed ends of filaments (16–18). The first example of this activity, associated with a heterodimeric capping protein from Arabidopsis (AtCP), has been reported recently (19). Alternatively, proteins that sever actin filaments could enhance profilin-mediated actin depolymerization. Indirect evidence for weak severing activity by ADF/cofilin makes this a potential candidate for SI, but other examples of efficient actin filament-severing proteins from plants are lacking (6).

Gelsolin, villin, and CapG comprise a family of related calcium-regulated ABPs that are found in many vertebrate and lower eukaryotic cells (20–22). Gelsolin is composed of six conserved 125–150-amino-acid gelsolin homology domains, designated G1–G6, and has Ca2+-stimulated F-actin severing activity. Gelsolin also caps the barbed ends of actin filaments and nucleates formation of new filaments. Crystal structures for one or more of these gelsolin subdomains, coupled with spectroscopy and cryoelectron microscopy studies, have provided substantial insight about the molecular mechanisms of filament severing and capping (23). Villin has a core of six gelsolin subdomains but also contains an additional C-terminal actin-binding module called the villin headpiece (VHP) that allows it to make contact with two adjacent filaments and to cross-link filaments into bundles. In the presence of micromolar Ca2+ some, but not all, villins sever actin filaments and cap filament barbed ends (24, 25). Two actin-bundling proteins from lily pollen, 135-ABP and 115-ABP, are known (26–28), and sequence analyses reveal that both are plant villins (26, 29). Although lily villins bundle filaments in a Ca2+-/calmodulin-dependent manner, they do not have severing or capping activity (30–32). The Arabidopsis genome contains sequences for five villin-like genes (AtVLN1–5; Refs. 6 and 33) but no gelsolin-only genes. A preliminary analysis of recombinant AtVLN1 demonstrates high affinity binding to F-actin and bundle formation but no severing or capping.2

Using affinity chromatography on DNase I-Sepharose, we identified an 80-kDa ABP from poppy pollen that is an efficient nucleator of actin filaments, has potent Ca2+-stimulated severing activity, and regulates assembly by binding to filament barbed ends. De novo sequence determination by mass spectrometry indicates that ABP80 is related to gelsolin. This is the first direct evidence for a plant gelsolin and the first example of efficient actin filament-severing activity by a plant ABP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Purification—Approximately 5 g of pollen from field poppy (P. rhoeas L.) was hydrated at 37 °C for 30 min. Pollen was ground in a cooled mortar along with 1 g of fine sand (Sigma S-9887) in extraction buffer (1 M Tris, pH 7.5, 0.6 M KCl, 0.5 mM MgCl2, 0.8 mM ATP, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride (34)) supplemented with a 1:100 dilution of protease inhibitors from a stock solution (2) for 30 min. The mixture was sonicated with five 30-s bursts, and Triton X-100 was added to 4% (v/v). The sonicate was clarified with three consecutive centrifugations of 30,000, 46,000, and 130,000 x g. The supernatant was passed through two layers of Miracloth (Calbiochem) and loaded onto a DNase I-Sepharose column (34) pre-equilibrated with Buffer G (1 mM Tris, pH 8.0, 0.2 mM ATP, 0.2 mM CaCl2, 0.1 mM DTT, 0.005% NaN3). The column was washed with Buffer G, and bound proteins were eluted with Ca2+-free buffer G (1 mM Tris, pH 8.0, 0.2 mM ATP, 0.5 mM MgCl2, 5 mM EGTA, 0.1 mM DTT, 0.005% NaN3). Pooled protein fractions were dialyzed against Solution A (10 mM KCl, 1 mM DTT, 0.01% NaN3, 10 mM Tris-HCl, pH 7.0, 1 mM phenylmethylsulfonyl fluoride, 1:200 protease inhibitors). The dialyzed protein was applied to a Q-Sepharose column (Amersham Biosciences) pre-equilibrated with Solution A, and bound proteins were eluted with a linear gradient of KCl (10–500 mM). The purified PrABP80 was dialyzed against Buffer G (2 mM Tris-HCl, pH 8.0, 0.01% NaN3, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM DTT), aliquoted, frozen in liquid nitrogen, and stored at -80 °C. The protein was further clarified by centrifugation at 200,000 x g for 1 h before use. Protein concentration was determined with Bradford reagent (Bio-Rad) using bovine serum albumin as standard. PrABP80 from Q-Sepharose was probed with affinity-purified Arabidopsis VILLIN1 (AtVLN1) antibody at 1:100 dilution and developed as described previously (35). For production of recombinant AtVLN1, the first three gelsolin domains (G1–G3) of AtVLN1 (33) were amplified by PCR and cloned into pGEX-KG for the generation of a GST-VLN1(G1–G3) fusion protein. Alternatively, PrABP80 was probed with lily villin, 135-ABP serum kindly provided by T. Shimmen, Himeji Institute of Technology (28). Recombinant protein was purified from bacterial cells, and the glutathione S-transferase (GST) moiety was removed, according to the methods of Kovar et al. (36).

Actin was isolated from rabbit skeletal muscle acetone powder and purified by Sephacryl S-300 chromatography (37). Actin was labeled on Cys-374 with pyrene iodoacetamide (37). Mouse capping protein (MmCP; {alpha}1{beta}2 heterodimer) was kindly provided by D. R. Kovar (Yale University). Zea mays profilin (ZmPRO5) and AtCP were purified as described previously (15, 19). Human plasma gelsolin was expressed from a plasmid kindly provided by T. Pollard (Yale University) and purified from bacterial cells roughly according to Pope et al. (38) with DEAE-Sepharose and Cibacron Blue 3G-A chromatography.

Mass Spectrometry—For identification of PrABP80 by mass spectrometry, appropriate bands were excised from Coomassie-stained gels. The gel pieces were destained with 50% NH4HCO3, 50% CH3CN. Supernatant was removed, and the gel pieces were dried under vacuum for 30 min. Gel pieces were reduced with 10 mM DTT in 100 mM NH4HCO3 at 56 °C for 45 min. Supernatant was removed and replaced with 55 mM C2H4INO for 30 min in the dark. Supernatant was removed again and replaced with 100 mM NH4HCO3 for 5 min and then diluted to 50% (v/v) CH3CN for 15 min. Gel pieces were dried under vacuum for 30 min and digested with 0.01 mg/ml bovine pancreas trypsin (Sigma T-8658) for 45 min on ice. After removing the solution, gel pieces were incubated in digestion buffer (25 mM NH4HCO3;5mM CaCl2) for 16 h. The digestion buffer was removed and saved, whereas the gel pieces were extracted with 25 mM NH4HCO3 for 15 min and then diluted to 50% (v/v) CH3CN for an additional 15 min. This was followed by extraction with 5% CH2O2 for 15 min and then diluted to 50% (v/v) CH3CN for an additional 15 min. This extraction step was repeated once. The washes were pooled with the digestion buffer. DTT was added to a final concentration of 1 mM, and the peptides were dried under vacuum. The peptides were resuspended in 20% C2HF3O2, frozen in liquid N2, and stored at -80 °C.

Poros Cleanup—An aliquot of the tryptic peptides was desalted in a glass purification capillary (Protana Engineering) using a 20-µm Poros R2 (polystyrene divinylbenzene; Applied Biosystems, Inc. (ABI), Foster City, CA). Briefly, the peptide mixture was loaded onto the R2 resin, washed three times with ~7 µl of 95:5, H2O:ACN, 0.5% formic acid, and eluted with ~1.5 µl of 30:70, H2O:ACN, 0.5% formic acid into a coated nanoelectrospray capillary (Protana).

Data Acquisition Parameters—Electrospray ionization (ESI) mass spectra were acquired using a QSTAR Pulsar i (ABI) quadrupole-TOF (time-of-flight) mass spectrometer equipped with a nano-ESI source (Protana). The ESI voltage was 1000 V, the TOF region acceleration voltage was 4 kV, and the injection pulse repetition rate was 6.0 kHz. External calibration was performed using porcine renin substrate decapeptide (Sigma), which yields doubly and triply charged monoisotopic signals (879.9705 and 586.9830, respectively). Mass spectra were acquired in an automated data-dependent mode (information-dependent acquisition) in positive mode over 25 min. De novo sequencing was performed with BioAnalyst software (ABI), and MS BLAST searches were performed at www.bork.embl-heidelberg.de.

High Speed and Low Speed Co-sedimentation Assays—High and low speed co-sedimentation assays were used to examine the actin-binding and actin-cross-linking properties of PrABP80, respectively (36). In a 100-µl reaction volume, 2.0 µM G-actin alone, 200 nM PrABP80 alone, or G-actin with PrABP80 were incubated in 1x F-buffer (10x stock: 50 mM Tris-Cl, pH 7.5, 5 mM DTT, 5 mM ATP, 1 M KCl, 50 mM MgCl2). These reactions were performed in the presence of 200 µM Ca2+ or 5 mM EGTA to give free Ca2+ of 180 µM and 15 nM, respectively. Free Ca2+ in the presence of EGTA was calculated with "EGTA" software by Petesmif (P. M. Smith, University of Liverpool, Liverpool UK), available at www.liv.ac.uk/~petesmif/software/egta.htm. After a 90-min incubation at 22 °C, samples were centrifuged at either 200,000 x g for 1 h at 4 °Cor 13,500 x g for 30 min at 4 °C. Equal amounts of pellet and supernatant samples were separated by 12.5% SDS-PAGE.

Actin Nucleation Assay—Actin nucleation was carried out essentially as described by Schafer et al. (39). Monomeric actin at 2 µM (5% pyrene-labeled) was incubated with PrABP80 or AtCP for 5 min in Buffer G. Fluorescence of pyrene-actin was monitored with a PTI Alphascan spectrofluorimeter (Photon Technology International, South Brunswick, NJ) after the addition of 1/10 volume of 10x KMEI (1x contains 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole-HCl, pH 7.0). The number of ends (n) generated during the nucleation reaction was calculated from the slope of the polymerization curves at half-polymerization according to Equation 1,

(Eq. 1)
where k+ is the association rate constant at the pointed ends (1.3 µM-1 s-1) (40), k- is the dissociation rate constant (0.8 s-1), and (A) is the concentration of actin monomers. These experiments were performed at 87 nM free Ca2+, buffered with 1 mM EGTA, to minimize the effect of filament severing on the initial growth rates.

Elongation Assay to Determine the Affinity of PrABP80 for Actin Filaments—Various concentrations of PrABP80 or AtCP were incubated with 0.4 µM preformed actin filaments in KMI (50 mM KCl, 1 mM MgCl2, 0.2 mM ATP, 0.2 mM CaCl2, 0.5 mM DTT, 3 mM NaN3 and 10 mM imidazole, pH 7.0) for 5 min at room temperature. The reaction mixture was supplemented with 1 µM G-actin (5% pyrene-labeled) saturated by 4 µM human profilin 1 to initiate actin elongation at barbed ends, and the final free [Ca2+] was 160 µM. The affinity of PrABP80 or AtCP for the barbed ends of actin filaments was determined by the variation of the initial rate of elongation as a function of the concentration of PrABP80 or AtCP using Equation 2

(Eq. 2)
where Vi is the observed rate of elongation, Vif is the rate of elongation when all barbed ends are free, Vib is the rate of elongation when all barbed ends are capped, [ends] is the concentration of barbed ends, and [CP] is the concentration of capping protein or PrABP80. To test the effects of [Ca2+] on the affinity of PrABP80 for filament barbed ends, similar reactions were performed in the presence of 5 mM EGTA to give free [Ca2+] of 10 nM, 100 nM, 1 µM, and 10 µM. The data were modeled with Kaleidagraph v3.6 software (Synergy Software, Reading, PA).

Dynamics of Actin Filament Depolymerization—F-actin at 2.5 µM (40–50% pyrene-labeled) was depolymerized by the addition of an equimolar amount of ZmPRO5, in the presence or absence of PrABP80 or MmCP. The decrease in pyrene fluorescence accompanying actin depolymerization was monitored for 30 min after the addition of profilin. The final free [Ca2+] was 1 µM for most reactions. To test the requirement of Ca2+ for PrABP80-mediated depolymerization, free Ca2+ was reduced to 15 nM with 5 mM EGTA.

Fluorescence Microscopy of Actin Filaments—Individual actin filaments labeled with fluorescent phalloidin were imaged by fluorescence microscopy according to Blanchoin et al. (41, 42). To visualize actin filaments generated during nucleation, actin at 4 µM alone or together with PrABP80 were polymerized in 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.2 mM CaCl2, 0.5 mM DTT, 3 mM NaN3, and 10 mM imidazole, pH 7, at 25 °C for 30 min and labeled with an equimolar amount of rhodamine-phalloidin (Sigma) during polymerization. For these nucleation experiments, the free [Ca2+] was 87 nM. In other experiments, PrABP80 or AtCP were incubated, in the presence of 160 µM or 14.7 nM free Ca2+, with prepolymerized rhodamine-phalloidin-labeled F-actin. The polymerized F-actin was diluted to 10 nM in fluorescence buffer containing 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 100 mM DTT, 100 µg/ml glucose oxidase, 15 mg/ml glucose, 20 µg/ml catalase, 0.5% methylcellulose. A dilute sample of 3 µl was applied to a 22 x 22 mm coverslip coated with poly-L-lysine (0.01%). Actin filaments were observed by epi-fluorescence illumination under a Nikon Microphot SA microscope equipped with a 60x, 1.4 NA Planapo objective, and digital images were collected with a Hamamatsu ORCA-ER 12-bit CCD camera using Metamorph 6.0 software.

In the elongation assay, actin filaments stabilized by rhodaminephalloidin were elongated by actin monomers in presence of Alexa-488 phalloidin (Molecular Probes, Eugene OR) to stabilize the new filaments. Polymerization conditions were as given above for assembly from monomers, and the final free [Ca2+] was 76 nM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of an 80-kDa P. rhoeas Actin-binding Protein (PrABP80)—To better understand the Ca2+-mediated depolymerization of actin filaments in pollen, several independent approaches for the identification of ABPs are being pursued. During the purification of native actin from poppy pollen (14), we observed an ~80-kDa protein that bound to DNase I columns in the presence of 1 M salt. Because ABPs such as gelsolin, villin, and ADF form high affinity complexes with G-actin and are retained on DNase I-Sepharose columns (43–47), we sought to identify this putative pollen ABP. Hydrated poppy pollen was homogenized and clarified by differential centrifugation. The high speed supernatant (Fig. 1A, lane 1) was loaded onto DNase I-Sepharose, and most of the proteins passed into the flow-through (Fig. 1A, lane 2). After excessive buffer washes containing 200 µM Ca2+, actin and a major 80-kDa protein remained on the DNase I-Sepharose column (Fig. 1A, lane 3). The 80-kDa protein and some of the actin (45 kDa) were eluted with EGTA (Fig. 1A, lane 4). Following dialysis, the 80-kDa protein was purified further by chromatography on Q-Sepharose, which removed most of the actin (Fig. 1A, lane 5). Typical samples contained a small amount of a 42-/40-kDa doublet, and the average purity of PrABP80 was greater than 80%.



View larger version (77K):
[in this window]
[in a new window]
  		FIG. 1.
Purification of an 80-kDa polypeptide from poppy pollen. A, Coomassie-stained gel. Poppy pollen extracts were fractionated on DNase I-Sepharose with elution by EGTA followed by binding to Q-Sepharose and elution with a linear salt gradient. The purified protein fraction has a major polypeptide of ~80 kDa and two minor contaminants of 42 and 40 kDa. The major protein is designated PrABP80. Loadings are as follows: lane 1, total extract (25 µg); lane 2, flow-through (25 µg); lane 3, DNase I-bound proteins (3 µg); lane 4, EGTA eluate (3 µg); lane 5, Q-Sepharose eluate (3 µg). The migration of molecular mass (MM) standards is given at the left in kDa. B, protein immunoblot probed with affinity-purified, anti-AtVLN1(G1–G3) antibody. Lane 1, poppy pollen total cell extract (30 µg); lane 2, purified PrABP80 (100 ng).

 
Identification of PrABP80 as a Gelsolin-like Protein by Mass Spectrometry—To identify PrABP80, the polypeptide was isolated from Coomassie-stained polyacrylamide gels, digested with trypsin, and subjected to mass spectrometry (MS). Peptide mass fingerprinting with quadrupole-TOF yielded profiles that matched villin/gelsolin proteins in current databases. To obtain sequence information, tryptic digests of PrABP80 were performed, and seven fragments were analyzed by ESI-MS/MS (Table I). The amino acid sequences were most similar to 135-ABP from lily pollen and Arabidopsis villins (Fig. 2). PrABP80 peptides shared 36–80% amino acid sequence identity with 135-ABP or AtVLN3, verifying that it is villin- or gelsolin-like. The peptides also shared 11–38% identity with human gelsolin. At 80 kDa, the purified protein is significantly smaller than the predicted Mr for plant villins, which range from 107,000 to 135,000. Moreover, none of the sequences obtained matched the C-terminal villin-headpiece for the known plant villins. Along with the biochemical properties (see below), these data suggest that PrABP80 is pollen gelsolin and not a villin-like protein.


View this table:
[in this window]
[in a new window]
  		TABLE I
PrABP80 peptides determined by ESI-MS/MS

 



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 2.
Sequence similarity between gelsolin/villin family members and the 80-kDa ABP. Peptide sequences for PrABP80 from tryptic digests of gel-excised protein subjected to ESI-MS/MS were obtained as described under "Experimental Procedures." Alignments of the de novo sequences determined from seven fragments in comparison with other gelsolins and villins are shown. PrABP80 shares 36–80% amino acid sequence identity with corresponding regions of plant 135-ABP and 11–38% identity with human gelsolin (see Table I for additional information). Accession numbers are as follows: 135-ABP from Lilium longiflorum (AF088901 [GenBank] ), Arabidopsis thaliana VILLIN3 (AtVLN3; AF081203 [GenBank] ), human plasma gelsolin (CAA28000 [GenBank] , and mouse adseverin (AAB61682 [GenBank] . Alignments were created using the Clustal W algorithm with MacVectorTM software (v7.1.1, Accelrys, Madison WI).

 
The purified protein was recognized by an affinity-purified antibody (Fig. 1B, lane 2) raised against the G1–G3 domains of recombinant AtVLN1, suggesting that it is related to the gelsolin/villin family. It also cross-reacted with lily villin, 135-ABP antiserum (not shown). To demonstrate that PrABP80 exists within cells, a pollen extract was prepared by grinding ungerminated pollen under liquid nitrogen, and proteins were extracted with Laemmli buffer. Western blots using either antibody revealed a prominent pair of bands at ~135 kDa, as well as a distinct polypeptide of 80 kDa that comigrates with purified PrABP80 (Fig. 1B, lane 1). The higher Mr polypeptides are presumed to be similar to lily villins characterized by Shimmen and co-workers (26–32).

PrABP80 Binds to F-actin and Severs or Depolymerizes in a Ca2+-dependent Manner but Does Not Have Filament Bundling Activity—The ability of PrABP80 to bind to F-actin was assayed with high speed co-sedimentation assays (Fig. 3A). The majority of the polymerized actin (F-actin) sedimented at 200,000 x g under these conditions (Fig. 3A, lane 2); however, no detectable PrABP80 sedimented in the absence of F-actin (Fig. 3A, lane 10). In the presence of F-actin and 15 nM free Ca2+, a small amount of PrABP80 was found in the pellet (Fig. 3A, lane 8), indicating that PrABP80 can bind to the sides or ends of actin filaments. However, the amount of binding was modest when compared with side-binding proteins such as fimbrin (36) or AtVLN1 (not shown). In the presence of PrABP80 and 180 µM free Ca2+, substantially more actin stayed in the supernatant (Fig. 3A, lane 3) when compared with the controls (Fig. 3A, lanes 1 and 7). This is consistent with Ca2+-stimulated filament-severing or depolymerizing activity. Because this effect was observed at substoichiometric amounts of PrABP80 to actin, severing activity is the most plausible explanation.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 3.
PrABP80 binds but does not cross-link actin filaments. A, high speed co-sedimentation assays, in the presence or absence of calcium, were used to determine PrABP80 binding to F-actin. A mixture of F-actin (2 µM) and PrABP80 (0.2 µM) in the presence of 180 µM (high) or 15 nM (low) free Ca2+ was centrifuged at 200,000 x g for 1 h. The resulting supernatants and pellets were subjected to SDS-PAGE and Coomassie-stained. The samples include: lane 1, actin alone supernatant (high Ca2+); lane 2, actin alone pellet (high Ca2+); lane 3, actin plus PrABP80 supernatant (high Ca2+); lane 4, actin plus PrABP80 pellet (high Ca2+); lane 5, actin alone supernatant (low Ca2+); lane 6, actin alone pellet (low Ca2+); lane 7, actin plus PrABP80 supernatant (low Ca2+); lane 8, actin plus PrABP80 pellet (low Ca2+); lane 9, ABP80 alone supernatant; lane 10, PrABP80 alone pellet. B, a low speed co-sedimentation assay compared the filament bundling activity of PrABP80 with AtVLN1 (see Footnote 2). Actin alone, actin plus PrABP80, and actin plus AtVLN1 were incubated under polymerization conditions as in panel A except that the PrABP80 concentration was 0.8 µM and the reaction was centrifuged at 13,500 x g. Equivalent amounts of supernatant and pellet were separated by SDS-PAGE. Samples are: lane 1, actin alone supernatant; lane 2, actin alone pellet; lane 3, actin plus PrABP80 supernatant; lane 4, actin plus PrABP80 pellet; lane 5, actin plus AtVLN1 supernatant; lane 6, actin plus AtVLN1 pellet. The migration of molecular mass (MM) standards is given at the left.

 
Because all plant villins have the ability to bundle actin filaments, an activity that correlates with the presence of a VHP, we examined whether PrABP80 also has this property. To analyze bundling, low speed pelleting assays were performed (Fig. 3B). In the absence of PrABP80, very little F-actin sedimented at 13,500 x g (Fig. 3B, lane 2). A considerable amount of polymerized actin sedimented in the presence of AtVLN1 (Fig. 3B, lane 6); however, very little actin sedimented in the presence of PrABP80 (Fig. 3B, lane 4). The lack of PrABP80 filament bundling or cross-linking was confirmed by fluorescence microscopy, as described below. Unlike AtVLN1 or lily villins (28, 31), the 80-kDa ABP from poppy does not have bundling activity, which is consistent with it being a gelsolin-like protein.

PrABP80 Has Nucleation Activity—Gelsolin eliminates the lag period associated with initiation of actin assembly or nucleates actin filament formation (21, 22, 48). Experiments to examine the effect of PrABP80 on the dynamics of actin polymerization were performed. To minimize the potential effects of filament severing, these experiments were conducted in the presence of 1 mM EGTA to provide a free [Ca2+] of 87 nM. As shown in Fig. 4A, when pyrene fluorescence was used to monitor rabbit muscle actin polymerization kinetically, the initial lag corresponding to the nucleation step decreased with increasing PrABP80 concentration. Similar results were observed with the heterodimeric capping protein from Arabidopsis, AtCP (Fig. 4B), as shown previously (19).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4.
PrABP80 nucleates filament assembly from monomers. PrABP80 and AtCP at various concentrations were incubated for 5 min with 2 µM actin (5% pyrene-labeled) prior to polymerization. Pyrene fluorescence (arbitrary units, a.u.) was plotted versus time after the addition of polymerization salts to initiate polymerization. A, PrABP80 at various concentrations, from bottom to top: 0, 12.5, 25, 62.5, and 125 nM. B, AtCP at various concentrations, from bottom to top: 0, 20, 50, 100, 500, and 1000 nM.

 
PrABP80 Inhibits the Addition of Profilin-Actin Complexes to Filament Barbed Ends—Gelsolin caps the newly created barbed end after it severs an actin filament (49). The activity of PrABP80 was also tested with a dynamic elongation assay from F-actin nuclei. The elongation rate depends on the availability of barbed ends, under these experimental conditions: 0.4 µM preformed F-actin was incubated with varying concentrations of ABP for 5 min, and polymerization was initiated with the addition of 1 µM G-actin (5% pyrene-labeled) at a free [Ca2+] of 160 µM. The G-actin was saturated with 4 µM human profilin to prevent polymerization from pointed ends. The results showed that the initial elongation rate was decreased with substoichiometric amounts of PrABP80 (Fig. 5A), just like AtCP (19). By fitting the data to Equation 2 (see "Experimental Procedures"), an apparent Kd value for binding barbed ends of 5.5 nM was calculated (Fig. 5B). For comparison, a representative experiment with AtCP gave a Kd of 23 nM (Fig. 5B).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5.
Elongation of actin filaments by the addition of profilin-actin complex to barbed ends is inhibited by PrABP80. A, preformed F-actin (0.4 µM) was incubated with different concentrations of PrABP80. To initiate actin elongation at the barbed end, 1 µM G-actin saturated with 4 µM human profilin I was added to each reaction. The change in pyrene-actin fluorescence accompanying polymerization was plotted versus time after the addition of G-actin. From bottom to top, PrABP80 concentration is: 26.67, 13.34, 6.67, 3.33, 1.33, 0.67, and 0 nM. a.u., arbitrary units. B, initial rates of polymerization versus PrABP80 (open squares) and AtCP (closed circles) concentration were plotted for the experiment in panel A. The data were fit with Equation 2 (see "Experimental Procedures") to determine apparent binding constant values of 5.5 nM for PrABP80 and 23 nM for AtCP.

 
Similar experiments were performed in the presence of 5 mM EGTA and variable amounts of Ca2+ to examine the Ca2+ dependence of capping. Free Ca2+ between 10 nM and 10 µM had no effect on the affinity of PrABP80 for the barbed end of filaments (Table II). For PrABP80 binding to rabbit skeletal muscle actin, a Kd value of 2.74 nM at 10 µM free Ca2+ was not significantly different from the Kd of 2.02 nM at 100 nM Ca2+. Identical experiments were conducted with recombinant human plasma gelsolin. Apparent Kd values ranged from 4.4 nM at low Ca2+ to 2.0 nM at 10 µM free Ca2+ (Table II). Therefore, PrABP80 and gelsolin binding to the barbed end of actin filaments is of high affinity and independent of Ca2+.


View this table:
[in this window]
[in a new window]
  		TABLE II
PrABP80 affinity for barbed ends is not Ca2+-sensitive

 
PrABP80 Enhances Profilin-mediated Depolymerization of F-actin—The activity of PrABP80 was also measured with a dynamic depolymerization assay. The influence of PrABP80 and a capping protein (MmCP) on filament depolymerization was investigated by adding equimolar amounts of profilin (2.5 µM) to pyrene-labeled F-actin and monitoring the decrease in fluorescence. The results from a typical experiment in the presence of 1 µM free Ca2+ are shown in Fig. 6. The depolymerization rate was increased by substoichiometric amounts of PrABP80 (Fig. 6A, bottom), when compared with actin alone (Fig. 6A, middle). By comparison, the barbed end capping protein, mouse CP, inhibits profilin-mediated actin depolymerization (Fig. 6A, upper). To mimic the situation that occurs during the SI response of poppy, profilin-mediated depolymerization was performed with varying amounts of PrABP (1–10 nM) at high (1 µM) and low free Ca2+ (15 nM). The experiments in Fig. 6B show a dose-dependent, synergistic activity of PrABP and profilin that stimulates actin depolymerization. The results suggest that PrABP80 severs F-actin and produces additional ends for depolymerization. The observation that 10 nM PrABP80 in the presence of profilin caused nearly 50% depolymerization of F-actin is consistent with severing rather than monomer binding activity for the plant gelsolin. Moreover, this level of depolymerization was not observed when free calcium is reduced to 15 nM, a condition where PrABP80 should function like a barbed end cap (Fig. 6B, top curve). Indeed, at low calcium, PrABP inhibited actin depolymerization relative to the control of actin and profilin alone. The severing activity was confirmed by measuring the lengths of prepolymerized rhodamine-stabilized actin filaments incubated with PrABP80 and by a time course of individual actin filaments severed by PrABP80.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 6.
PrABP80 enhances profilin-mediated actin filament depolymerization. ZmPRO5 (2.5 µM) alone, or ZmPRO5 together with MmCP or PrABP80, was added to F-actin solutions prepared from 2.5 µM actin (40–50% pyrene-labeled). Depolymerization was recorded by reduction in pyrene fluorescence beginning from the addition of protein at time = 0. Except where noted, all reactions were performed in the presence of 1 µM free Ca2+. A, in addition to 2.5 µM F-actin, the reactions contained: 2.5 µM profilin + 50 nM CP, 2.5 µM profilin, or 10 nM PrABP80, as given on the figure. a.u., arbitrary units. B, all reactions contained 2.5 µM actin in polymer, to which 2.5 µM profilin and variable amounts of PrABP80 were added. As noted on the figure, experiments included: 10 nM PrABP80 in the presence of 15 nM free Ca2+ (+EGTA), no PrABP80, 1 nM PrABP80, 5 nM PrABP80, and 10 nM PrABP80.

 
Effects of PrABP80 on Actin Filament Length—Fluorescence microscopy was used to determine the effect of PrABP80 on the length distribution of actin filaments. When 4 µM actin was polymerized in the presence of an equimolar amount of rhodamine-phalloidin and diluted to a final concentration of 10 nM before attaching to polylysine-coated coverslips, the number of filaments per field was few, and their length distribution was approximately exponential (Fig. 7A; mean of 7.5 ± 5.1 µm). By contrast, when actin was polymerized in the presence of 9.4 (Fig. 7B), 18.8 (Fig. 7C), 47 (Fig. 7D), 94 (Fig. 7E), and 188 nM PrABP80 (Fig. 7F), the mean length of filaments was 3.2 ± 2.1, 2.9 ± 2.1, 1.4 ± 1.0, 1.0 ± 0.9, and 0.7 ± 0.6 µm (n >= 178), respectively. These results demonstrate that PrABP80 reduced actin filament length in a dose-dependent manner. This is consistent with PrABP80 blocking assembly at barbed ends, inhibiting filament annealing, as well as increasing the number of actin filaments during assembly by nucleation. Similar results are observed with AtCP and mouse CP (19). There was no evidence for the formation of bundles or cross-linked networks in the presence of PrABP80, which would be expected if it exhibited villin-like activity.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 7.
PrABP80 reduces filament length during assembly from monomeric actin. Fluorescence micrographs of individual actin filaments assembled in the presence or absence of PrABP80 are shown. The effect of PrABP80 on filament length is shown. In each case, 4 µM Mg-ATP-actin was polymerized for 30 min in the presence of 4 µM rhodamine-phalloidin. Average filament lengths were determined from measurement of at least 100 filaments. A, actin filaments formed in the absence of PrABP80 or in the presence of 9.4 nM (B), 18.8 nM (C), 47 nM (D), 94 nM (E), and 188 nM PrABP80 (F). The scale bar in panel F represents 10 µm.

 
We confirmed that the barbed ends of actin filaments are blocked by PrABP80 using a fluorescence microscope assay for filament elongation. Actin filaments labeled with rhodaminephalloidin (Fig. 8, red) were used as prelabeled templates for subsequent elongation. The elongation was performed in the presence of Alexa-488 phalloidin so that newly elongated actin filaments were labeled green. As a control, when both ends are free, green actin elongated from both ends of red actin filaments (Fig. 8A). New growth under these conditions was asymmetric; the presumed barbed end typically had a long stretch of green filament, whereas the pointed end had a comparatively short length of green filament. In the presence of saturating amounts of PrABP80 (Fig. 8C) or AtCP (Fig. 8B), green actin filaments grew almost exclusively from one end of the red actin filaments. Moreover, the extent of green actin filament growth was short, suggesting that PrABP80 blocked all available barbed ends and green actin filaments extended only from pointed ends.



View larger version (8K):
[in this window]
[in a new window]
  		FIG. 8.
Elongation of actin filaments from the barbed end is blocked by PrABP80. Two-color overlays of actin filament assembly from rhodamine-phalloidin-labeled actin template (red). The new growth is marked by Alexa-488 phalloidin (green). A, 2 µM F-actin stabilized with rhodamine-phalloidin was incubated with 1 µM Mg-ATP-actin for 15 min in the presence of 1 µM Alexa-488 phalloidin. Because the filament density was somewhat lower, due to the increased filament length at equivalent actin concentrations to those shown in panels B and C, this image is a montage created from three different fields. B, AtCP-capped actin filaments (0.5 µM) labeled with rhodamine-phalloidin were used for new polymerization as described for panel A. C, PrABP80-capped actin filaments (100 nM) were used for polymerization. The scale bar in panel C represents 10 µm.

 
PrABP80 Severing Activity Can Be Observed with Fluorescence Microscopy—Gelsolin is able to sever phalloidin-stabilized F-actin (50). Initially, we tested whether PrABP80 can sever populations of prepolymerized F-actin stabilized with rhodamine-phalloidin. As shown in Fig. 9, PrABP80 significantly reduced the length of actin filaments (mean = 0.4 ± 0.4 µm) after incubation in the presence of 160 µM free Ca2+ for 30 min (Fig. 9D) when compared with controls (Fig. 9A, mean = 5.1 ± 3.7 µm). After incubation with 500 nM AtCP (Fig. 9B), the mean length of actin filaments, 4.9 ± 3.4 µm, was not significantly different from the control. The reduction in filament length by PrABP80 was dependent on Ca2+; reactions performed in the presence of 5 mM EGTA and 16 nM free Ca2+ had a mean filament length of 4.7 ± 3.2 µm (Fig. 9C). These results indicated that PrABP80 not only caps ends but also severs actin filaments.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 9.
The length of preformed filaments is reduced in the presence of PrABP80. Actin filaments (4 µM) labeled with rhodaminephalloidin were incubated with AtCP or PrABP80, and images were collected after a 30-min incubation. PrABP80 reduces the mean length of actin filaments in a Ca2+-dependent manner. A, actin alone; B, actin + 500 nM AtCP; C, actin + 166 nM PrABP80 in the presence of 14.7 nM free Ca2+; and D, actin + 166 nM ABP80 in the presence of 160 µM Ca2+. Bar = 10 µm.

 
The severing activity of PrABP80 was also demonstrated by monitoring the time course of actin filament length reduction directly. As shown in Fig. 10, the number of breaks (arrows)in a field of actin filaments increased over time (see also Supplemental Data; Movies 1–4). In the absence of PrABP80, a minimal amount of photobleaching was observed, but filaments were not severed or reduced in length (not shown). Many of the breaks caused by PrABP80 were associated with the presence of a kink or bend in the filament backbone, as shown previously for gelsolin by electron microscopy and light microscopy (50, 51). This provided convincing and direct evidence that PrABP80 can sever F-actin.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 10.
Severing activity of PrABP80 is observed directly by fluorescence microscopy. Prepolymerized, rhodamine-phalloidin-labeled actin filaments were incubated with PrABP80, and images were collected every 500 ms. Individual filaments showed an increasing number of breaks (arrows) as time elapsed, consistent with severing activity. Many of the breaks occurred at bends in the filament. See Supplemental Data, Movie 1, for a QuickTime movie of the entire time series.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we report the first molecular, biochemical, and cytological evidence for the existence of a gelsolin-like protein in plants. An 80-kDa polypeptide from P. rhoeas pollen was purified to near homogeneity, and its sequence was determined by mass spectrometry. A combination of recognition by an affinity-purified AtVLN antibody and de novo sequence determination identified PrABP80 as gelsolin or villin-like. Gelsolin (80–90 kDa) is a calcium- and phosphoinositide-regulated actin cytoskeleton remodeling protein identified originally from alveolar macrophages (52). Gelsolin has three main activities (21, 48, 53): it binds along the backbone of actin filaments and severs the contacts between adjacent subunits; it caps the barbed end of F-actin with high affinity, thereby preventing subunit addition and loss from the capped end; and it associates with two or three actin subunits to nucleate assembly of new filaments. We have demonstrated that, like vertebrate gelsolin, PrABP80 has a high affinity for filament barbed ends, nucleates filament formation during assembly, severs existing filaments, and stimulates actin depolymerization. However, unlike plant or non-plant villins, it does not bundle F-actin. The molecular weight, sequence, and biochemical activities, as well as direct demonstration of severing activity by fluorescence microscopy, provide overwhelming evidence that PrABP80 is a plant gelsolin.

Kinetic analyses of PrABP80 interaction with pyrene-labeled actin demonstrated that substoichiometric amounts of PrABP80 eliminate the initial lag period required for actin assembly from monomers and prevent the addition of profilin-actin complexes onto actin filament ends. These properties are consistent with high affinity binding and capping of filament barbed ends. Indeed, when a titration with ABP was used to calculate an apparent Kd for barbed ends, PrABP80 has a value of 2–6nM. The binding constant of gelsolin family members for capping barbed ends may be as low as 10 pM for platelet gelsolin in the presence of 150 µM Ca2+ (54), but more typical values range from 1 to 40 nM (55–58). The ability of PrABP80 to block subunit addition at the barbed end of filaments was confirmed by fluorescence microscopy. In a two-color filament elongation assay (41), limited green filament growth was observed from only one end of pre-existing red filaments in the presence of PrABP80, whereas controls showed growth from both ends, and one new section of filament was typically much longer than that extending from the other end.

The actin-binding activity of gelsolin is typically held to be Ca2+-regulated, but the details of this regulation are complex and somewhat controversial. Part of the complexity is due to the fact that an individual gelsolin polypeptide can bind multiple Ca2+ molecules and additional sites are coordinated with bound actin. Type-2 sites reside wholly within the gelsolin polypeptide, whereas type-1 sites require residues on both gelsolin and a bound actin. X-ray crystallographic studies indicate that gelsolin has up to six type-2 sites, one for each gelsolin-homology domain, and two type-1 sites (59–62). In the absence of Ca2+, the six homologous domains are folded into a compact, globular structure with the actin-binding sites buried (63). An overwhelming amount of biochemical and structural data lend support to a "tail latch" mechanism, whereby calcium binding induces a conformational change that releases the C-terminal helix of G6 from its contact with G2 and exposes the actinbinding site(s) (60, 62–66). However, there is some dispute about the level of Ca2+ required to undo the structural restraint. Using dynamic light scattering, Pope et al. (38) provide evidence for conformational changes at nanomolar Ca2+ levels, whereas Lin et al. (66) use endogenous tryptophan fluorescence to show that unlatching requires micromolar Ca2+. Several studies report that the capping activity of gelsolin and villin is activated half-maximally at 10–30 nM Ca2+, whereas severing activity requires micromolar Ca2+ for full activation (67, 68). Here, we find that PrABP80 has substantial capping and nucleating activity at 10–100 nM free Ca2+, consistent with unlatching and/or activation at these physiological levels of Ca2+. Moreover, the apparent Kd value for binding filament barbed ends does not change significantly over the range of 100 nM to 10 µM Ca2+, levels that are found in actively growing and stimulated pollen tubes (69, 70). This suggests that within pollen, much of the PrABP80 will be bound to the ends of actin filaments. An alternative model for the lack of Ca2+ regulation would be that PrABP80 is missing the latch helix or G6 domain. Creation of a mutant human plasma gelsolin that lacks just 3% of the C-terminal amino acid sequence alleviates Ca2+ regulation (65). This theory is consistent with the molecular weight of PrABP80 and the presence of splice variants in other plants that comprise just G1–G5. Further understanding of this phenomenon will require additional biochemical studies and isolation of a full-length cDNA for PrABP80.

Several lines of investigation provide evidence for actin filament severing by PrABP80. The rate of depolymerization of pre-existing filaments by profilin is rather limited on its own and is inhibited by barbed end capping factors. However, by creating new ends, severing proteins enhance profilin-mediated actin filament disassembly. Substoichiometric amounts of PrABP80 increased the depolymerization rate of pyrene-labeled actin filaments in a dose- and Ca2+-dependent manner. Fluorescence microscopy also showed that PrABP80 can reduce the mean length of pre-assembled actin filaments in a Ca2+-dependent manner. The most compelling and direct evidence for severing comes from time-lapse movies of individual filaments in the presence of PrABP80 (see Supplemental Data; Movies 1–4). Rhodamine-phalloidin-stabilized actin filaments developed numerous breaks along their backbone after just seconds of incubation with PrABP80.

To date, only limited and circumstantial evidence for severing activity by plant ABPs exists in the literature. Weak severing activity is known for non-plant ADF/cofilin proteins (71). Severing has never been demonstrated directly for a plant ADF, although it is often inferred from depolymerization and polymerization assays (72, 73). The relative contribution of severing versus enhancing the off-rate at pointed ends to ADF/cofilin depolymerizing activity remains controversial (74–76). There is limited biochemical evidence for a Ca2+-dependent ABP from Mimosa pudica, the sensitive plant, that is related to gelsolin/fragmin-like proteins (77). When extracts of mimosa plants were applied to a DNase I column in the presence of Ca2+, two polypeptides, a major protein of 42 kDa and a less abundant 90-kDa protein, were eluted with EGTA. After EGTA was removed, the eluate increased the amount of actin in the supernatant by sedimentation analysis and generated short actin filaments as assessed by electron microscopy. Moreover, the two proteins facilitated actin polymerization from monomers in the presence of 200 µM Ca2+ and decreased the lag period for assembly. These properties are diagnostic for either actin capping or severing proteins. However, depolymerization assays or direct analysis of severing to distinguish between these possibilities were not performed. The 42-kDa protein has limited sequence similarity with Arabidopsis and lily villins, with one of four peptides sharing 31–46% identity. However, the three other peptide sequences failed to align with plant villins.

The biochemical evidence for a plant gelsolin is somewhat surprising, based solely on analysis of the Arabidopsis genome data base (6, 78). Although both gelsolin and villin are constructed from the same core of six tandem gelsolin subdomains, villin contains an additional actin-binding module (20, 21). This 8.5-kDa VHP confers Ca2+-independent actin bundling activity upon the villin family members, a biochemical property that is missing from gelsolins. All five gelsolin/villin-related genes in Arabidopsis (AtVLN1–5) contain a VHP, suggesting that they encode villin-like proteins (6, 33). Gelsolin could be generated as an alternative splicing product from any of the AtVLN transcripts. Indeed evidence for a CapG-like splice variant for AtVLN1 (33)2 and a G1–G5 protein from AtVLN3 (GenBankTM accession number AY093052 [GenBank] ) exists in current databases.

The role of gelsolin in plant cells could include the regulation of filament dynamics in response to fluxes in calcium or PPIs. In pollen tubes, a tip high oscillatory gradient of [Ca2+]i is essential for tip growth (69, 70, 79, 80). A remarkable correspondence between actin organization at the tips of pollen tubes, the [Ca2+]i gradients, and evidence for oscillatory actin dynamics (81), suggest a role for actin dynamics in apical tip growth (82). A Ca2+-stimulated actin filament-severing protein, such as PrABP80, could act as a sensor for the oscillating [Ca2+]i and contribute to the creation of a dynamic population of actin filaments at the apex of pollen tubes. Several groups have invoked gelsolin-like activities for Ca2+-mediated actin rearrangements in response to ionophores or extracellular signals (13, 83, 84). The identification of PrABP80 lends some credence to these hypotheses, but further experimentation is required to verify that this particular class of ABP is involved in actin reorganization during each of these responses.

Actin depolymerization is a hallmark of the SI response of field poppy and can be mimicked by drugs that raise [Ca2+]i (13, 14). The rapid and sustained 50–80% reduction in polymer levels is unrivaled by most eukaryotic cells. Reconstitution experiments with native pollen profilin, a G-actin-binding protein that has increased sequestering activity at elevated Ca2+, fail to account for the level of depolymerization observed in vivo (14). We proposed previously that profilin acts in cooperation with other ABPs to mediate this depolymerization. Two proteins, capping protein (19) and PrABP80, may be such partners of profilin. The former is predicted to assist profilin in maintaining a large pool of unpolymerized actin by blocking assembly of the profilin-actin complex onto filament barbed ends and allowing profilin to function as a simple actin sequestering protein (18). PrABP80 could also assist filament depolymerization by Ca2+-activated severing and the creation of new pointed ends for depolymerization by profilin. The feasibility of this mechanism is supported by experiments in which equimolar amounts of profilin were added to pre-existing actin filaments along with nM amounts of PrABP80. Up to 50% actin depolymerization was observed with just 10 nM PrABP80, but only in the presence of µM free Ca2+. This is remarkably similar to the situation that occurs during SI. To test this molecular model thoroughly, we will need to determine the cellular concentrations for CP and PrABP80 in pollen, measure their affinity for plant actin filament ends, and determine the efficiency of severing at various physiological [Ca2+].

In summary, we have obtained the first direct evidence for a gelsolin-like protein in plants. Our characterization of PrABP80 provides the first demonstration of efficient Ca2+-regulated severing activity for a plant ABP. PrABP80 also caps the barbed ends of actin filaments with high affinity in the presence or absence of Ca2+ and can nucleate filament assembly from monomers. Its properties suggest that PrABP80 may assist profilin with Ca2+-mediated depolymerization of actin filaments during SI in poppy.


   FOOTNOTES
 
* This work was supported by grants from the Department of Energy, Energy Biosciences Division (DE-FG02-99ER20337A01) (to C. J. S.) and from the Biotechnology and Biological Sciences Research Council, UK (to V. E. F. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains four supplementary time-lapse movies of actin filament severing. Back

Supported by an Actions Thématiques et Incitives sur Programmes et Equipes from the CNRS. Back

** To whom correspondence should be addressed: Dept. of Biological Sciences, Purdue University, 333 Hansen Life Sciences Bldg., 201 S. University St., West Lafayette, IN 47907-2064. Tel.: 765-496-1769; Fax: 765-496-1496; E-mail: cstaiger{at}bilbo.bio.purdue.edu.

1 The abbreviations used are: ABP, actin-binding protein; CP, capping protein; AtCP, Arabidopsis CP; AtVLN, Arabidopsis villin; DTT, dithiothreitol; G-actin, globular or monomeric actin; F-actin, filamentous actin; MmCP, mouse CP; PrABP80, P. rhoeas 80-kDa ABP; Zm-PRO5, Z. mays profilin; SI, self-incompatibility; VHP, villin headpiece; ESI, electrospray ionization; MS, mass spectrometry; TOF, time-offlight. Back

2 S. Huang, L. Blanchoin, T. Matsumoto, R. Robinson, and C. J. Staiger, unpublished data. Back


   ACKNOWLEDGMENTS
 
Microscopy facilities were established with partial support from the National Science Foundation (0217552-DBI). We recognize the Mass Spectrometry Consortium for the Life Sciences at the University of Minnesota and various supporting agencies, including the National Science Foundation for Grant 9871237-MRI used to purchase the MS instruments. We gratefully acknowledge the help and advice from members of the Purdue Motility Group and thank D. R. Kovar (Yale University) and A. McGough (Purdue) for critical review of the manuscript. We also thank T. Pollard (Yale University) for providing the human plasma gelsolin clone and T. Shimmen (Himeji Institute, Japan) for the antibody against 135-ABP.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Liu, X., and Yen, L.-F. (1992) Plant Physiol. 99, 1151-1155[Abstract/Free Full Text]
  2. Ren, H., Gibbon, B. C., Ashworth, S. L., Sherman, D. M., Yuan, M., and Staiger, C. J. (1997) Plant Cell 9, 1445-1457[Abstract]
  3. Honys, D., and Twell, D. (2003) Plant Physiol. 132, 640-652[Abstract/Free Full Text]
  4. Yokota, E., and Shimmen, T. (2000) in Actin: A Dynamic Framework for Multiple Plant Cell Functions (Staiger, C. J., Baluska, F., Volkmann, D., and Barlow, P., eds) pp. 103-118, Kluwer Academic Publishers, Dordrecht, The Netherlands
  5. Vidali, L., and Hepler, P. K. (2001) Protoplasma 215, 64-76[CrossRef][Medline] [Order article via Infotrieve]
  6. Staiger, C. J., and Hussey, P. J. (2004) in The Plant Cytoskeleton in Cell Differentiation and Development (Hussey, P. J., ed) pp. 32-80, Blackwell Publishers, UK
  7. Hepler, P. K., Vidali, L., and Cheung, A. Y. (2001) Annu. Rev. Cell Dev. Biol. 17, 159-187[CrossRef][Medline] [Order article via Infotrieve]
  8. Gibbon, B. C., Kovar, D. R., and Staiger, C. J. (1999) Plant Cell 11, 2349-2363[Abstract/Free Full Text]
  9. Vidali, L., McKenna, S. T., and Hepler, P. K. (2001) Mol. Biol. Cell 12, 2534-2545[Abstract/Free Full Text]
  10. Franklin-Tong, V. E., Ride, J. P., Read, N. D., Trewavas, A. J., and Franklin, F. C. H. (1993) Plant J. 4, 163-177
  11. Franklin-Tong, V. E., Hackett, G., and Hepler, P. K. (1997) Plant J. 12, 1375-1386[CrossRef]
  12. Franklin-Tong, V. E. (1999) Plant Cell 11, 727-738[Free Full Text]
  13. Geitmann, A., Snowman, B. N., Emons, A. M. C., and Franklin-Tong, V. E. (2000) Plant Cell 12, 1239-1251[Abstract/Free Full Text]
  14. Snowman, B. N., Kovar, D. R., Shevchenko, G., Franklin-Tong, V. E., and Staiger, C. J. (2002) Plant Cell 14, 2613-2626[Abstract/Free Full Text]
  15. Kovar, D. R., Drøbak, B. K., and Staiger, C. J. (2000) Plant Cell 12, 583-598[Abstract/Free Full Text]
  16. Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014[CrossRef][Medline] [Order article via Infotrieve]
  17. Perelroizen, I., Didry, D., Christensen, H., Chua, N.-H., and Carlier, M.-F. (1996) J. Biol. Chem. 271, 12302-12309[Abstract/Free Full Text]
  18. Kang, F., Purich, D. L., and Southwick, F. S. (1999) J. Biol. Chem. 274, 36963-36972[Abstract/Free Full Text]
  19. Huang, S., Blanchoin, L., Kovar, D. R., and Staiger, C. J. (2003) J. Biol. Chem. 278, 44832-44842[Abstract/Free Full Text]
  20. Friederich, E., and Louvard, D. (1999) in Guidebook to the Cytoskeletal and Motor Proteins (Kries, T., and Vale, R., eds) 2nd Ed., pp. 175-179, Oxford University Press, New York
  21. Yin, H. L. (1999) in Guidebook to the Cytoskeletal and Motor Proteins (Kreis, T., and Vale, R., eds) Vol. 2nd Ed., pp. 99-102, Oxford University Press, New York
  22. Kwiatkowski, D. J. (1999) Curr. Opin. Cell Biol. 11, 103-108[CrossRef][Medline] [Order article via Infotrieve]
  23. McGough, A. M., Staiger, C. J., Min, J.-K., and Simonetti, K. D. (2003) FEBS Lett. 552, 75-81[CrossRef][Medline] [Order article via Infotrieve]
  24. Glenney, J. R., Jr., Bretscher, A., and Weber, K. (1980) Proc. Natl. Acad. Sci. (U. S. A.) 77, 6458-6462[Abstract/Free Full Text]
  25. Janmey, P. A., and Matsudaira, P. T. (1988) J. Biol. Chem. 263, 16738-16743[Abstract/Free Full Text]
  26. Yokota, E., Vidali, L., Tominaga, M., Tahara, H., Orii, H., Morizane, Y., Hepler, P. K., and Shimmen, T. (2003) Plant Cell Physiol. 44, 1088-1099[Abstract/Free Full Text]
  27. Yokota, E., and Shimmen, T. (1995) Plant Cell Physiol. 36S, s132
  28. Yokota, E., Takahara, K.-i., and Shimmen, T. (1998) Plant Physiol. 116, 1421-1429[Abstract/Free Full Text]
  29. Vidali, L., Yokota, E., Cheung, A. Y., Shimmen, T., and Hepler, P. K. (1999) Protoplasma 209, 283-291[CrossRef]
  30. Yokota, E., Muto, S., and Shimmen, T. (2000) Plant Physiol. 123, 645-654[Abstract/Free Full Text]
  31. Yokota, E., and Shimmen, T. (1999) Planta 209, 264-266[CrossRef][Medline] [Order article via Infotrieve]
  32. Nakayasu, T., Yokota, E., and Shimmen, T. (1998) Biochem. Biophys. Res. Comm. 249, 61-65[CrossRef][Medline] [Order article via Infotrieve]
  33. Klahre, U., Friederich, E., Kost, B., Louvard, D., and Chua, N.-H. (2000) Plant Physiol. 122, 35-47[Abstract/Free Full Text]
  34. Schafer, D. A., Jennings, P. B., and Cooper, J. A. (1998) Cell Motil. Cytoskeleton 39, 166-171[CrossRef][Medline] [Order article via Infotrieve]
  35. Karakesisoglou, I., Schleicher, M., Gibbon, B. C., and Staiger, C. J. (1996) Cell Motil. Cytoskeleton 34, 36-47[CrossRef][Medline] [Order article via Infotrieve]
  36. Kovar, D. R., Staiger, C. J., Weaver, E. A., and McCurdy, D. W. (2000) Plant J. 24, 625-636[CrossRef][Medline] [Order article via Infotrieve]
  37. Pollard, T. D. (1984) J. Cell Biol. 99, 769-777[Abstract/Free Full Text]
  38. Pope, B., Gooch, J. T., and Weeds, A. G. (1997) Biochemistry 36, 15848-15855[CrossRef][Medline] [Order article via Infotrieve]
  39. Schafer, D. A., Jennings, P. B., and Cooper, J. A. (1996) J. Cell Biol. 135, 169-179[Abstract/Free Full Text]
  40. Pollard, T. D. (1986) J. Cell Biol. 103, 2747-2754[Abstract/Free Full Text]
  41. Blanchoin, L., Pollard, T. D., and Mullins, R. D. (2000) Curr. Biol. 10, 1273-1282[CrossRef][Medline] [Order article via Infotrieve]
  42. Blanchoin, L., Amann, K. J., Higgs, H. N., Marchand, J.-B., Kaiser, D. A., and Pollard, T. D. (2000) Nature 404, 1007-1011[CrossRef][Medline] [Order article via Infotrieve]
  43. Bretscher, A., and Weber, K. (1980) Cell 20, 839-847[CrossRef][Medline] [Order article via Infotrieve]
  44. Harris, H. E., and Gooch, J. (1981) FEBS Lett. 123, 49-53[CrossRef][Medline] [Order article via Infotrieve]
  45. Maekawa, S., Nishida, E., Ohta, Y., and Sakai, H. (1984) J. Biochem. 95, 377-385[Abstract/Free Full Text]
  46. Ashino, N., Sobue, K., Seino, Y., and Yabuuchi, H. (1987) J. Biochem. 101, 609-617[Abstract/Free Full Text]
  47. Kurth, M. C., Wang, L.-L., Dingus, J., and Bryan, J. (1983) J. Biol. Chem. 258, 10895-10903[Abstract/Free Full Text]
  48. Sun, H. Q., Yamamoto, M., Mejillano, M., and Yin, H. L. (1999) J. Biol. Chem. 274, 33179-33182[Free Full Text]
  49. Harris, H. E., and Weeds, A. G. (1984) FEBS Lett. 177, 184-188[CrossRef][Medline] [Order article via Infotrieve]
  50. Bearer, E. L. (1991) J. Cell Biol. 115, 1629-1638[Abstract/Free Full Text]
  51. McGough, A., Chiu, W., and Way, M. (1998) Biophys. J. 74, 764-772[Medline] [Order article via Infotrieve]
  52. Yin, H. L., and Stossel, T. P. (1979) Nature 281, 583-586[CrossRef][Medline] [Order article via Infotrieve]
  53. Janmey, P. A., Stossel, T. P., and Allen, P. G. (1998) Chem. Biol. 5, R81-R85[CrossRef][Medline] [Order article via Infotrieve]
  54. Selve, N., and Wegner, A. (1986) Eur. J. Biochem. 160, 379-387[Medline] [Order article via Infotrieve]
  55. Way, M., Pope, B., and Weeds, A. G. (1992) J. Cell Biol. 116, 1135-1143[Abstract/Free Full Text]
  56. Coué, M., and Korn, E. D. (1985) J. Biol. Chem. 260, 15033-15041[Abstract/Free Full Text]
  57. Weber, A., Pring, M., Lin, S.-L., and Bryan, J. (1991) Biochemistry 30, 9327-9334[CrossRef][Medline] [Order article via Infotrieve]
  58. Janmey, P. A., Chapponier, C., Lind, S. E., Zaner, K. S., Stossel, T. P., and Yin, H. L. (1985) Biochemistry 24, 3714-3723[CrossRef][Medline] [Order article via Infotrieve]
  59. Narayan, K., Chumnarnsilpa, S., Choe, H., Irobi, E., Urosev, D., Lindberg, U., Schutt, C. E., Burtnick, L. D., and Robinson, R. C. (2003) FEBS Lett. 552, 82-85[CrossRef][Medline] [Order article via Infotrieve]
  60. Choe, H., Burtnick, L. D., Mejillano, M., Yin, H. L., Robinson, R. C., and Choe, S. (2002) J. Mol. Biol. 324, 691-702[CrossRef][Medline] [Order article via Infotrieve]
  61. Robinson, R. C., Mejillano, M., Le, V. P., Burtnick, L. D., Yin, H. L., and Choe, S. (1999) Science 286, 1939-1942[Abstract/Free Full Text]
  62. McLaughlin, P. J., Gooch, J. T., Mannherz, H.-G., and Weeds, A. G. (1993) Nature 364, 685-692[CrossRef][Medline] [Order article via Infotrieve]
  63. Burtnick, L. D., Koepf, E. K., Grimes, J., Jones, E. Y., Stuart, D. I., McLaughlin, P. J., and Robinson, R. C. (1997) Cell 90, 661-670[CrossRef][Medline] [Order article via Infotrieve]
  64. Way, M., Gooch, J., Pope, B., and Weeds, A. G. (1989) J. Cell Biol. 109, 593-605[Abstract/Free Full Text]
  65. Kwiatkowski, D. J., Janmey, P. A., and Yin, H. L. (1989) J. Cell Biol. 108, 1717-1726[Abstract/Free Full Text]
  66. Lin, K.-M., Mejillano, M., and Yin, H. L. (2000) J. Biol. Chem. 275, 27746-27752[Abstract/Free Full Text]
  67. Kinosian, H. J., Newman, J., Lincoln, B., Selden, L. A., Gershman, L. C., and Estes, J. E. (1998) Biophys. J. 75, 3101-3109[Medline] [Order article via Infotrieve]
  68. Northrop, J., Weber, A., Mooseker, M. S., Franzini-Armstrong, C., Bishop, M. F., Dubyak, G. R., Tucker, M., and Walsh, T. P. (1986) J. Biol. Chem. 261, 9274-9281[Abstract/Free Full Text]
  69. Messerli, M., and Robinson, K. R. (1997) J. Cell Sci. 110, 1269-1278[Abstract]
  70. Pierson, E. S., Miller, D. D., Callaham, D. A., van Aken, J., Hackett, G., and Hepler, P. K. (1996) Dev. Biol. 174, 160-173[CrossRef][Medline] [Order article via Infotrieve]
  71. McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771-781[Abstract/Free Full Text]
  72. Gungabissoon, R. A., Jiang, C.-J., Drøbak, B. K., Maciver, S. K., and Hussey, P. J. (1998) Plant J. 16, 689-696[CrossRef]
  73. Gungabissoon, R. A., Khan, S., Hussey, P. J., and Maciver, S. K. (2001) Cell Motil. Cytoskeleton 49, 104-111[CrossRef][Medline] [Order article via Infotrieve]
  74. Carlier, M.-F., Laurent, V., Santolini, J., Melki, R., Didry, D., Xia, G.-X., Hong, Y., Chua, N.-H., and Pantaloni, D. (1997) J. Cell Biol. 136, 1307-1322[Abstract/Free Full Text]
  75. Ressad, F., Didry, D., Xia, G.-X., Hong, Y., Chua, N.-H., Pantaloni, D., and Carlier, M.-F. (1998) J. Biol. Chem. 273, 20894-20902[Abstract/Free Full Text]
  76. Ressad, F., Didry, D., Egile, C., Pantaloni, D., and Carlier, M.-F. (1999) J. Biol. Chem. 274, 20970-20976[Abstract/Free Full Text]
  77. Yamashiro, S., Kameyama, K., Kanzawa, N., Tamiya, T., Mabuchi, I., and Tsuchiya, T. (2001) J. Biochem. 130, 243-249[Abstract/Free Full Text]
  78. Hussey, P. J., Allwood, E. G., and Smertenko, A. P. (2002) Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 791-798[Abstract/Free Full Text]
  79. Miller, D. D., Callaham, D. A., Gross, D. J., and Hepler, P. K. (1992) J. Cell Sci. 101, 7-12[Abstract/Free Full Text]
  80. Rathore, K. S., Cork, R. J., and Robinson, K. R. (1991) Dev. Biol. 148, 612-619[CrossRef][Medline] [Order article via Infotrieve]
  81. Fu, Y., Wu, G., and Yang, Z. (2001) J. Cell Biol. 152, 1019-1032[Abstract/Free Full Text]
  82. Holdaway-Clarke, T. L., and Hepler, P. K. (2003) New Phytol. 159, 539-563[CrossRef]
  83. Kohno, T., and Shimmen, T. (1987) Protoplasma 141, 177-179[CrossRef]
  84. Cárdenas, L., Vidali, L., Domínguez, J., Pérez, H., Sánchez, F., Hepler, P. K., and Quinto, C. (1998) Plant Physiol. 116, 871-877[Abstract/Free Full Text]
  85. Papayannopoulos, I. A. (1995) Mass Spectrom. Rev. 14, 49-71[CrossRef]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J Exp BotHome page
G. Cai and M. Cresti
Organelle motility in the pollen tube: a tale of 20 years
J. Exp. Bot., February 1, 2009; 60(2): 495 - 508.
[Abstract] [Full Text] [PDF]

Home page
 Mol PlantHome page
T. Wang, Y. Xiang, J. Hou, and H.-Y. Ren
ABP41 is Involved in the Pollen Tube Development via Fragmenting Actin Filaments
Mol Plant, November 20, 2008; (2008) ssn073v1.
[Abstract] [Full Text] [PDF]

Home page
 Mol PlantHome page
M. Bosch, N. S. Poulter, S. Vatovec, and V. E. Franklin-Tong
Initiation of Programmed Cell Death in Self-Incompatibility: Role for Cytoskeleton Modifications and Several Caspase-Like Activities
Mol Plant, November 1, 2008; 1(6): 879 - 887.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
H.-J. Wang, A.-R. Wan, and G.-Y. Jauh
An Actin-Binding Protein, LlLIM1, Mediates Calcium and Hydrogen Regulation of Actin Dynamics in Pollen Tubes
Plant Physiology, August 1, 2008; 147(4): 1619 - 1636.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
L. Cardenas, A. Lovy-Wheeler, J. G. Kunkel, and P. K. Hepler
Pollen Tube Growth Oscillations and Intracellular Calcium Levels Are Reversibly Modulated by Actin Polymerization
Plant Physiology, April 1, 2008; 146(4): 1611 - 1621.
[Abstract] [Full Text] [PDF]

Home page
 J Exp BotHome page
M. Bosch and V. E. Franklin-Tong
Self-incompatibility in Papaver: signalling to trigger PCD in incompatible pollen
J. Exp. Bot., February 1, 2008; 59(3): 481 - 490.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
F. Chaudhry, C. Guerin, M. von Witsch, L. Blanchoin, and C. J. Staiger
Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin
Mol. Biol. Cell, August 1, 2007; 18(8): 3002 - 3014.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
Y. Xiang, X. Huang, T. Wang, Y. Zhang, Q. Liu, P. J. Hussey, and H. Ren
ACTIN BINDING PROTEIN29 from Lilium Pollen Plays an Important Role in Dynamic Actin Remodeling
PLANT CELL, June 1, 2007; 19(6): 1930 - 1946.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
B. McClure
Pollen-Pistil Signaling in Self-Incompatible Poppy: Does It Allow More Efficient Resource Allocation in the Pistil?
Sci. Signal., April 24, 2007; 2007(383): pe17 - pe17.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
A. Lovy-Wheeler, J. G. Kunkel, E. G. Allwood, P. J. Hussey, and P. K. Hepler
Oscillatory Increases in Alkalinity Anticipate Growth and May Regulate Actin Dynamics in Pollen Tubes of Lily
PLANT CELL, September 1, 2006; 18(9): 2182 - 2193.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
S. G. Thomas, S. Huang, S. Li, C. J. Staiger, and V. E. Franklin-Tong
Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen
J. Cell Biol., July 17, 2006; 174(2): 221 - 229.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
G. M. Yoon, P. E. Dowd, S. Gilroy, and A. G. McCubbin
Calcium-Dependent Protein Kinase Isoforms in Petunia Have Distinct Functions in Pollen Tube Growth, Including Regulating Polarity
PLANT CELL, April 1, 2006; 18(4): 867 - 878.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
E. Yokota, M. Tominaga, I. Mabuchi, Y. Tsuji, C. J. Staiger, K. Oiwa, and T. Shimmen
Plant Villin, Lily P-135-ABP, Possesses G-Actin Binding Activity and Accelerates the Polymerization and Depolymerization of Actin in a Ca2+-Sensitive Manner
Plant Cell Physiol., October 1, 2005; 46(10): 1690 - 1703.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
A. Michelot, C. Guerin, S. Huang, M. Ingouff, S. Richard, N. Rodiuc, C. J. Staiger, and L. Blanchoin
The Formin Homology 1 Domain Modulates the Actin Nucleation and Bundling Activity of Arabidopsis FORMIN1
PLANT CELL, August 1, 2005; 17(8): 2296 - 2313.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
R. Zhong, D. H. Burk, C. J. Nairn, A. Wood-Jones, W. H. Morrison III, and Z.-H. Ye
Mutation of SAC1, an Arabidopsis SAC Domain Phosphoinositide Phosphatase, Causes Alterations in Cell Morphogenesis, Cell Wall Synthesis, and Actin Organization
PLANT CELL, May 1, 2005; 17(5): 1449 - 1466.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
Y. Gu, Y. Fu, P. Dowd, S. Li, V. Vernoud, S. Gilroy, and Z. Yang
A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes
J. Cell Biol., April 11, 2005; 169(1): 127 - 138.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
S. Huang, R. C. Robinson, L. Y. Gao, T. Matsumoto, A. Brunet, L. Blanchoin, and C. J. Staiger
Arabidopsis VILLIN1 Generates Actin Filament Cables That Are Resistant to Depolymerization
PLANT CELL, February 1, 2005; 17(2): 486 - 501.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
D. Takemoto and A. R. Hardham
The Cytoskeleton as a Regulator and Target of Biotic Interactions in Plants
Plant Physiology, December 1, 2004; 136(4): 3864 - 3876.
[Full Text] [PDF]

Home page
 Plant Physiol.Home page
G. O. Wasteneys and Z. Yang
New Views on the Plant Cytoskeleton
Plant Physiology, December 1, 2004; 136(4): 3884 - 3891.
[Full Text] [PDF]

Home page
 Plant Physiol.Home page
X. Fan, J. Hou, X. Chen, F. Chaudhry, C. J. Staiger, and H. Ren
Identification and Characterization of a Ca2+-Dependent Actin Filament-Severing Protein from Lily Pollen
Plant Physiology, December 1, 2004; 136(4): 3979 - 3989.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemantal Data
Right arrow All Versions of this Article:
279/22/23364    most recent
M312973200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, S.
Right arrow Articles by Staiger, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, S.
Right arrow Articles by Staiger, C. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement