Identification of Actin-Binding Proteins from Maize Pollen

Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells

Arabidopsis VILLIN1 generates actin filament cables that are resistant to depolymerization

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Signalling and the cytoskeleton of pollen tubes of Papaver rhoeas

Self-incompatibility (SI) is a genetically controlled system used by many flowering plants to prevent self-pollination, often by the inhibition of pollen tube growth. The importance of cytosolic free calcium, [Ca2+]i, for the regulation of pollen tube growth is well known. We have established, using calcium imaging, that the SI response in Papaver rhoeas L. pollen involves a calcium-mediated intracellular signalling pathway. Tip growth of cells is dependent upon a typical configuration of the actin cytoskeleton, which is controlled by actin binding proteins. In animal cells, the actin-binding protein, profilin, is thought to act as a key intermediate between signalling pathways and actin rearrangements. Profilin is an abundant component of pollen. To better understand the signalling cascades that modulate pollen tip growth and actin dynamics, we are investigating a possible signalling role for profilin. We have demonstrated that profilin modulates the phosphorylation of pollen proteins in vitro. This implicates a role for profilin in altering protein kinase or phosphatase activity. Furthermore, we demonstrate for the first time that profilin from pollen can be phosphorylated in vitro. This provides compelling evidence that profilin interacts with signalling pathways in angiosperms. Finally, we demonstrate that in the SI response, the actin cytoskeleton of incompatible pollen tubes is dramatically rearranged. Our data strongly support a role for the cytoskeleton and actin-binding proteins interacting with signalling pathways involved in the regulation of pollen tube growth