ACTIN BINDING PROTEIN29 fromLiliumPollen Plays an Important Role in Dynamic Actin Remodeling

Abstract

Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca 2þ -and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.

ArabidopsisFIMBRIN5, an Actin Bundling Factor, Is Required for Pollen Germination and Pollen Tube Growth

“…Pharmacological disruption of actin and genetic manipulation of the actin regulatory system have revealed that the actin cytoskeleton is a major mediator of pollen tube growth (Gibbon et al, 1999;Chen et al, 2002;Cheung and Wu, 2004;Xiang et al, 2007). Furthermore, imaging of actin filaments has shown that distinct actin structures form in pollen tubes across different species.…”

Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein, Functions in Cross-linking and Stabilizing Actin Filaments

“…High and Low Speed Co-sedimentation Assays-A high speed co-sedimentation assay was used to determine the F-actin binding activity of GST-CROLIN1 following a published method (32).…”

“…Fluorescence Microscopy and Electron Microscopy Visualization of F-actin-To visualize F-actin using fluorescence microscopy, the samples were labeled with Alexa 488-phalloidin (Molecular Probes), as described in a previously published method (32). F-actin was observed using a Leica DFC420C fluorescence microscope equipped with a 5-megapixel CCD and Leica Application Suite software.…”

“…Actin Depolymerization Assays-To test the effects of CROLIN1 on actin depolymerization, a depolymerization assay was performed, as described by a published method (32). The decrease in pyrene fluorescence intensity accompanying actin depolymerization was monitored for 500 s at room temperature using a FluoroMax-4 spectrofluorometer (HORIBA Jobin Yvon).…”

“…6A, CROLIN1 reduced the rate of depolymerization in a concentration-dependent manner. ABP29 was used as a positive control, as described previously (32).…”