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

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 Ca 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, Ca -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 selfincompatibility 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 (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) 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 m, 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 ([Ca ]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 * 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. □S The on-line version of this article (available at http://www.jbc.org) contains four supplementary time-lapse movies of actin filament severing. ¶ Supported by an Actions Thématiques et Incitives sur Programmes et Equipes from the CNRS. ** 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: [email protected]. 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; ZmPRO5, Z. mays profilin; SI, self-incompatibility; VHP, villin headpiece; ESI, electrospray ionization; MS, mass spectrometry; TOF, time-offlight. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 22, Issue of May 28, pp. 23364–23375, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.