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

2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês

10.1074/jbc.m312973200

ISSN

1083-351X

Autores

Shanjin Huang, Laurent Blanchoin, Faisal Chaudhry, Vernonica E. Franklin‐Tong, Christopher J. Staiger,

Tópico(s)

Plant and animal studies

Resumo

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. 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. 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 (1Liu X. Yen L.-F. Plant Physiol. 1992; 99: 1151-1155Crossref PubMed Scopus (40) Google Scholar, 2Ren H. Gibbon B.C. Ashworth S.L. Sherman D.M. Yuan M. Staiger C.J. Plant Cell. 1997; 9: 1445-1457Crossref PubMed Google Scholar). Cytoskeletal genes are among the most abundantly expressed classes of transcripts in Arabidopsis pollen (3Honys D. Twell D. Plant Physiol. 2003; 132: 640-652Crossref PubMed Scopus (412) Google Scholar), and several classes of actin-binding protein (ABP) 1The 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.1The 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. have been isolated and characterized biochemically (4Yokota E. Shimmen T. Staiger C.J. Baluska F. Volkmann D. Barlow P. Actin: A Dynamic Framework for Multiple Plant Cell Functions. Kluwer Academic Publishers, Dordrecht, The Netherlands2000: 103-118Google Scholar, 5Vidali L. Hepler P.K. Protoplasma. 2001; 215: 64-76Crossref PubMed Scopus (121) Google Scholar, 6Staiger C.J. Hussey P.J. Hussey P.J. The Plant Cytoskeleton in Cell Differentiation and Development. Blackwell Publishers, UK2004: 32-80Google Scholar). Pollen tube growth is arguably the most dramatic example of cellular morphogenesis in plants (5Vidali L. Hepler P.K. Protoplasma. 2001; 215: 64-76Crossref PubMed Scopus (121) Google Scholar, 7Hepler P.K. Vidali L. Cheung A.Y. Annu. Rev. Cell Dev. Biol. 2001; 17: 159-187Crossref PubMed Scopus (577) Google Scholar). 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 (8Gibbon B.C. Kovar D.R. Staiger C.J. Plant Cell. 1999; 11: 2349-2363Crossref PubMed Scopus (330) Google Scholar, 9Vidali L. McKenna S.T. Hepler P.K. Mol. Biol. Cell. 2001; 12: 2534-2545Crossref PubMed Scopus (253) Google Scholar), 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 (10Franklin-Tong V.E. Ride J.P. Read N.D. Trewavas A.J. Franklin F.C.H. Plant J. 1993; 4: 163-177Crossref Scopus (156) Google Scholar, 11Franklin-Tong V.E. Hackett G. Hepler P.K. Plant J. 1997; 12: 1375-1386Crossref Scopus (107) Google Scholar, 12Franklin-Tong V.E. Plant Cell. 1999; 11: 727-738Crossref PubMed Scopus (257) Google Scholar). Cytological and biochemical studies demonstrate that reorganization of the actin cytoskeleton is one of the earliest events triggered by SI (13Geitmann A. Snowman B.N. Emons A.M.C. Franklin-Tong V.E. Plant Cell. 2000; 12: 1239-1251Crossref PubMed Scopus (135) Google Scholar, 14Snowman B.N. Kovar D.R. Shevchenko G. Franklin-Tong V.E. Staiger C.J. Plant Cell. 2002; 14: 2613-2626Crossref PubMed Scopus (147) Google Scholar). 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 (14Snowman B.N. Kovar D.R. Shevchenko G. Franklin-Tong V.E. Staiger C.J. Plant Cell. 2002; 14: 2613-2626Crossref PubMed Scopus (147) Google Scholar). 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+ (14Snowman B.N. Kovar D.R. Shevchenko G. Franklin-Tong V.E. Staiger C.J. Plant Cell. 2002; 14: 2613-2626Crossref PubMed Scopus (147) Google Scholar, 15Kovar D.R. Drøbak B.K. Staiger C.J. Plant Cell. 2000; 12: 583-598Crossref PubMed Scopus (157) Google Scholar). 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 (14Snowman B.N. Kovar D.R. Shevchenko G. Franklin-Tong V.E. Staiger C.J. Plant Cell. 2002; 14: 2613-2626Crossref PubMed Scopus (147) Google Scholar). Profilin acts like a simple sequestering protein in the presence of capping factors that block the barbed ends of filaments (16Pantaloni D. Carlier M.-F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 17Perelroizen I. Didry D. Christensen H. Chua N.-H. Carlier M.-F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 18Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The first example of this activity, associated with a heterodimeric capping protein from Arabidopsis (AtCP), has been reported recently (19Huang S. Blanchoin L. Kovar D.R. Staiger C.J. J. Biol. Chem. 2003; 278: 44832-44842Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 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 (6Staiger C.J. Hussey P.J. Hussey P.J. The Plant Cytoskeleton in Cell Differentiation and Development. Blackwell Publishers, UK2004: 32-80Google Scholar). Gelsolin, villin, and CapG comprise a family of related calcium-regulated ABPs that are found in many vertebrate and lower eukaryotic cells (20Friederich E. Louvard D. Kries T. Vale R. Guidebook to the Cytoskeletal and Motor Proteins. 2nd Ed. Oxford University Press, New York1999: 175-179Google Scholar, 21Yin H.L. Kreis T. Vale R. Guidebook to the Cytoskeletal and Motor Proteins. 2nd Ed. Oxford University Press, New York1999: 99-102Google Scholar, 22Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (325) Google Scholar). 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 (23McGough A.M. Staiger C.J. Min J.-K. Simonetti K.D. FEBS Lett. 2003; 552: 75-81Crossref PubMed Scopus (155) Google Scholar). 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 (24Glenney Jr., J.R. Bretscher A. Weber K. Proc. Natl. Acad. Sci. (U. S. A.). 1980; 77: 6458-6462Crossref PubMed Scopus (97) Google Scholar, 25Janmey P.A. Matsudaira P.T. J. Biol. Chem. 1988; 263: 16738-16743Abstract Full Text PDF PubMed Google Scholar). Two actin-bundling proteins from lily pollen, 135-ABP and 115-ABP, are known (26Yokota E. Vidali L. Tominaga M. Tahara H. Orii H. Morizane Y. Hepler P.K. Shimmen T. Plant Cell Physiol. 2003; 44: 1088-1099Crossref PubMed Scopus (65) Google Scholar, 27Yokota E. Shimmen T. Plant Cell Physiol. 1995; 36S: s132Google Scholar, 28Yokota E. Takahara K.-i. Shimmen T. Plant Physiol. 1998; 116: 1421-1429Crossref PubMed Scopus (82) Google Scholar), and sequence analyses reveal that both are plant villins (26Yokota E. Vidali L. Tominaga M. Tahara H. Orii H. Morizane Y. Hepler P.K. Shimmen T. Plant Cell Physiol. 2003; 44: 1088-1099Crossref PubMed Scopus (65) Google Scholar, 29Vidali L. Yokota E. Cheung A.Y. Shimmen T. Hepler P.K. Protoplasma. 1999; 209: 283-291Crossref Scopus (71) Google Scholar). Although lily villins bundle filaments in a Ca2+-/calmodulin-dependent manner, they do not have severing or capping activity (30Yokota E. Muto S. Shimmen T. Plant Physiol. 2000; 123: 645-654Crossref PubMed Scopus (69) Google Scholar, 31Yokota E. Shimmen T. Planta. 1999; 209: 264-266Crossref PubMed Scopus (55) Google Scholar, 32Nakayasu T. Yokota E. Shimmen T. Biochem. Biophys. Res. Comm. 1998; 249: 61-65Crossref PubMed Scopus (43) Google Scholar). The Arabidopsis genome contains sequences for five villin-like genes (AtVLN1–5; Refs. 6Staiger C.J. Hussey P.J. Hussey P.J. The Plant Cytoskeleton in Cell Differentiation and Development. Blackwell Publishers, UK2004: 32-80Google Scholar and 33Klahre U. Friederich E. Kost B. Louvard D. Chua N.-H. Plant Physiol. 2000; 122: 35-47Crossref PubMed Scopus (90) Google Scholar) 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. 2S. Huang, L. Blanchoin, T. Matsumoto, R. Robinson, and C. J. Staiger, unpublished data.2S. Huang, L. Blanchoin, T. Matsumoto, R. Robinson, and C. J. Staiger, unpublished data. 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. 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 (34Schafer D.A. Jennings P.B. Cooper J.A. Cell Motil. Cytoskeleton. 1998; 39: 166-171Crossref PubMed Scopus (31) Google Scholar)) supplemented with a 1:100 dilution of protease inhibitors from a stock solution (2Ren H. Gibbon B.C. Ashworth S.L. Sherman D.M. Yuan M. Staiger C.J. Plant Cell. 1997; 9: 1445-1457Crossref PubMed Google Scholar) 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 × g. The supernatant was passed through two layers of Miracloth (Calbiochem) and loaded onto a DNase I-Sepharose column (34Schafer D.A. Jennings P.B. Cooper J.A. Cell Motil. Cytoskeleton. 1998; 39: 166-171Crossref PubMed Scopus (31) Google Scholar) 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 × 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 (35Karakesisoglou I. Schleicher M. Gibbon B.C. Staiger C.J. Cell Motil. Cytoskeleton. 1996; 34: 36-47Crossref PubMed Scopus (54) Google Scholar). For production of recombinant AtVLN1, the first three gelsolin domains (G1–G3) of AtVLN1 (33Klahre U. Friederich E. Kost B. Louvard D. Chua N.-H. Plant Physiol. 2000; 122: 35-47Crossref PubMed Scopus (90) Google Scholar) 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 (28Yokota E. Takahara K.-i. Shimmen T. Plant Physiol. 1998; 116: 1421-1429Crossref PubMed Scopus (82) Google Scholar). Recombinant protein was purified from bacterial cells, and the glutathione S-transferase (GST) moiety was removed, according to the methods of Kovar et al. (36Kovar D.R. Staiger C.J. Weaver E.A. McCurdy D.W. Plant J. 2000; 24: 625-636Crossref PubMed Google Scholar). Actin was isolated from rabbit skeletal muscle acetone powder and purified by Sephacryl S-300 chromatography (37Pollard T.D. J. Cell Biol. 1984; 99: 769-777Crossref PubMed Scopus (187) Google Scholar). Actin was labeled on Cys-374 with pyrene iodoacetamide (37Pollard T.D. J. Cell Biol. 1984; 99: 769-777Crossref PubMed Scopus (187) Google Scholar). Mouse capping protein (MmCP; α1β2 heterodimer) was kindly provided by D. R. Kovar (Yale University). Zea mays profilin (ZmPRO5) and AtCP were purified as described previously (15Kovar D.R. Drøbak B.K. Staiger C.J. Plant Cell. 2000; 12: 583-598Crossref PubMed Scopus (157) Google Scholar, 19Huang S. Blanchoin L. Kovar D.R. Staiger C.J. J. Biol. Chem. 2003; 278: 44832-44842Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 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. (38Pope B. Gooch J.T. Weeds A.G. Biochemistry. 1997; 36: 15848-15855Crossref PubMed Scopus (54) Google Scholar) 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 (36Kovar D.R. Staiger C.J. Weaver E.A. McCurdy D.W. Plant J. 2000; 24: 625-636Crossref PubMed Google Scholar). In a 100-μl reaction volume, 2.0 μm G-actin alone, 200 nm PrABP80 alone, or G-actin with PrABP80 were incubated in 1× F-buffer (10× 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 × g for 1 h at 4 °Cor 13,500 × 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. (39Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (333) Google Scholar). 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 10× KMEI (1× 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,Slope=k+(A)(n)−k−(n)(Eq. 1) where k+ is the association rate constant at the pointed ends (1.3 μm-1 s-1) (40Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (578) Google Scholar), 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 2Vi=Vif+(Vib−Vif)((Kd+[ends]+[CP]−(Kd+[ends]+[CP])2−(4[ends][CP])2[ends])(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. (41Blanchoin L. Pollard T.D. Mullins R.D. Curr. Biol. 2000; 10: 1273-1282Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 42Blanchoin L. Amann K.J. Higgs H.N. Marchand J.-B. Kaiser D.A. Pollard T.D. Nature. 2000; 404: 1007-1011Crossref PubMed Scopus (430) Google Scholar). 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 × 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 60×, 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. 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 (14Snowman B.N. Kovar D.R. Shevchenko G. Franklin-Tong V.E. Staiger C.J. 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The high speed supernatant (Fig. 1A, lane 1) was loaded onto DNase I-Sepharose, and most of the p

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