Analysis of the signalling function of Arabidopsis cyclase associated protein (CAP1) and its interaction with a novel transmembrane protein (AtCIP).

Each cell has an internal skeleton, the cytoskeleton. This is composed of three self-assembling protein filament systems: actin, microtubules and intermediate filaments. Plant cells appear only to have actin filaments and microtubules in their cytoskeleton. The cytoskeleton is responsible for many cellular processes including maintenance of cell structure, cell shape, mitosis (karyokinesis, the division of the chromosomes and cytokinesis, the division of the cell), the movement of organelles around the cell and cell expansion. The term 'Cytoskeleton' does not only include the filamentous elements but also a plethora of proteins that anchor, crossbridge or otherwise regulate the network within the cell. The cytoskeleton has to be responsive to cell signals that are generated internally or externally so that it can respond to the cells changing needs in different environments. For example, when a cell enters mitosis the cytoskeleton has to rearrange to divide the nuclei and the cell. The single celled amoeba has to move in the direction of food and the cytoskeleton governs this motility. Plants have to bend towards the light and to do this there has to be localised cell expansion. Actin and microtubules are structurally the same in animals, plants and fungae. Many of the proteins that bind and alter the dynamics of these filaments are also shared, but their sequence conservation is limited. The pathways used to regulate these proteins in plants are starting to be elucidated and significant differences are appearing. This is perhaps not surprising since the signals for cell growth and the environmental signals to which plants have to respond differ to those of animals. The focus of my laboratory is to determine the signalling pathways that control actin reorganisation in plants. The actin-binding Cyclase Associated Protein (CAP) has two roles; a signalling role and a role in controlling actin dynamics. The PJH laboratory has identified a mutation in the Arabidopsis CAP protein, and the appearance of the mutant is not what we would expect from just a defect in actin organisation. The question we are addressing here is 'What is the role of this protein in signalling in plants?'. Towards an answer to this question we have identified a plant specific protein that interacts with plant CAP and this protein localises to the plasma membrane offering the exciting possibility that the CAP1:AtCIP couple is part of a signal transduction machinery. We will be asking the question 'what is the function of the CAP1:AtCIP interaction?'

We have identified insertion mutants for the Arabidopsis Cyclase Associated Protein CAP1. The phenotype is pleiotropic, characteristic of signalling mutants. The cap1 mutants are dwarf showing a loss of apical dominance. There is a notable curling of roots and shoots which have only been observed in mutants defective in signalling e.g. gravitropic mutants and mutants in phospholipid signalling. Root hairs are bulbous and branched and the actin cytoskeleton is deformed indicating that the root hair architecture could be wholly or partly due to the abnormalities in the actin cytoskeleton. As CAP is a bifunctional protein we will establish which aspects of this phenotype are due to signalling defects and which to actin abnormalities. It is possible that both the signalling and the actin defects are integrated through the one protein which would provide a direct link between signalling and the actin cytoskeleton in plants. To analyse the signalling function we identified a protein that interacts with the N-terminal domain of Arabidopsis CAP analogous to the N-terminal signalling domain of yeast and Dictyostelium CAP. This interacting protein is a transmembrane protein with the cytoplasmic C-terminus interacting with the N-terminus of Arabidopsis CAP. This provides the exciting possibility that the AtCAP:AtCIP acts as a couple in a signal transduction machinery. We will examine the function of this plant specific CAP interacting protein in plants.