Plasma-membrane protein / actin cytoskeleton interactions

Animal and plant cells are able to respond to cues from their surroundings using their protein signalling networks. Chemicals or physical sensations from outside the cell result in changes in the biochemical machinery inside the cell. Interactions of proteins are the molecular switches used by cells to respond to environmental cues. One of the primary signalling pathways in animal cells relys on the connection between proteins in the outer cell membrane and the actin cytoskeleton. Actin forms a series of filaments inside the cell membrane that support the cell and over which other proteins and organelles can move. When a receptor protein in the membrane receives a signal from outside the cell, it signals this to the actin by changing shape and this, in turn, results in activation of any of a number of cellular processes. Plant cells, like animal cells, are capable of responding to stimuli from their environment. They also have proteins in their outer membrane and an actin cytoskeleton. We don't, however, know how these membrane proteins interact with the actin. Many of the proteins have been shown in the lab to have actin binding properties but this has not been demonstrated in living cells. How will we determine if there is a physical interaction between the proteins and the actin? I have developed a technique to monitor protein movement using a confocal microscope. This type of microscope uses lasers to make a protein in the membrane of cells fluorescent. By fusing this protein - which is called Green Fluorescent Protein (GFP) - to a membrane protein I can visualise movement of the membrane protein. GFP acts as a 'marker.' The GFP I use can be activated by a short pulse of laser light enabling me to activate a small region of membrane and follow movement of the protein for longish periods of time (up to several hours). Using mathematics, I can describe the movement of the protein, i.e. its speed and direction of movement. My hypothesis is that if the protein interacts with the actin cytoskeleton then it will move differently if I destroy the cytoskeleton. There are chemicals that cause the cytoskeleton to breakdown and these can be applied to the cells so that protein movement can be measure when the cytoskeleton is absent. In addition, there are mutant plants that have defects in their actin cytoskeletons. These plants are abnormal looking as a result of improper actin structure. I predict that membrane proteins will move differently in these plants if they associagte with the abnormal actin cytoskeleton. This work will be the first step towards understanding the connection between plasma membrane and actin that is so essential in plant cell signalling.

The principal objective of this research is to measure protein dynamics within several different membranes of the plant cell secretory pathway. To do this, we will use a photoactivation approach. Proteins-of-interest within the endoplasmic reticulum, tonoplast, and plasma membrane will be fused with photoactivatableGFP (PAGFP) and expressed in tobacco leaves. Activation of a small membrane region with the 405nm laser of a Zeiss LSM 510 confocal microscope will allow tracking of activated proteins in vivo. Analysis of the dispersion pattern of activated GFP yields diffusion coefficient and mobility fraction data for a given protein. Quantitative measurements of the radial dispersion rate are based on calculating the magnitude and direction of the centre of mass of the fluorescence intensity and its angular concentration using circular statistics. We will build up a library of protein mobility data using this technique. Proteins to be studied include: i) a set of proteins that are not predicted to have interactions with other proteins. These include the calnexin transmembrane domain in the ER and LTI6b in the plasma membrane. ii) The ER translocon subunit Sec61A which is part of a heterogeneous oligomeric protein complex. iii) AFH1 and PLD1B which are localised to the plasma membrane and which interact with the actin cytoskeleton. For ii-iii we will study the dynamics of full-length proteins as well as those that have had interaction domains deleted. Finally we will study dynamics of these proteins after depolymerisation of the actin cytoskeleton to evaluate the strength and effect of the interaction. Arabidopsis lines stably expressing these proteins under their native promoters will be generated and studied by the same technique to see if expression levels can alter the membrane dynamics of a protein.