Plasma-membrane protein / actin cytoskeleton interactions
Lead Research Organisation:
Oxford Brookes University
Department Name: Faculty of Health and Life Sciences
Abstract
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.
Technical Summary
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.
Publications
Feraru E
(2011)
PIN polarity maintenance by the cell wall in Arabidopsis.
in Current biology : CB
Jaffé FW
(2012)
G protein-coupled receptor-type G proteins are required for light-dependent seedling growth and fertility in Arabidopsis.
in The Plant cell
Kleine-Vehn J
(2011)
Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane.
in Molecular systems biology
Luu DT
(2012)
Fluorescence recovery after photobleaching reveals high cycling dynamics of plasma membrane aquaporins in Arabidopsis roots under salt stress.
in The Plant journal : for cell and molecular biology
Martinière A
(2012)
Salt stress triggers enhanced cycling of Arabidopsis root plasma-membrane aquaporins.
in Plant signaling & behavior
Martinière A
(2013)
Protein diffusion in plant cell plasma membranes: the cell-wall corral.
in Frontiers in plant science
Martinière A
(2011)
Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton.
in The Plant journal : for cell and molecular biology
Martinière A
(2011)
Homeostasis of plasma membrane viscosity in fluctuating temperatures.
in The New phytologist
Martinière A
(2012)
Cell wall constrains lateral diffusion of plant plasma-membrane proteins.
in Proceedings of the National Academy of Sciences of the United States of America
Salleh FM
(2012)
A novel function for a redox-related LEA protein (SAG21/AtLEA5) in root development and biotic stress responses.
in Plant, cell & environment
Description | The most important finding from this research was that proteins of the plant plasma membrane are immobilised by interaction with the cell wall. Protein localisation in specific domains within the plasma membrane is important for cell signal reception and perception of the external environment. Cells use many mechanisms to immobilise and concentrate proteins at their site of action and many of these mechanisms were investigated during this research. Individual experiments were conducted to study the ways in which proteins attach to membranes and to study protein localisation as mediated by cytoskeleton, plasma-membrane lipid compostion, protein-protein interactions, and the cell wall. |
Exploitation Route | There is the potential to use the techniques developed in this study to investigate protein dynamics of the plant plasma membrane in various stressful situations. These situations would include biotic stresses such as pathogen attack, and abiotic stresses such as temperature extremes or under drought conditions. Manipulation of protein dynamics in such conditions will be useful in producing plants better able to withstand pathogens and envoronmental extremes. There is the potential to use the techniques developed in this study to investigate protein dynamics of the plant plasma membrane in various stressful situations. These situations would include biotic stresses such as pathogen attack, and abiotic stresses such as temperature extremes or under drought conditions. Manipulation of protein dynamics in such conditions will be useful in producing plants better able to withstand pathogens and envoronmental extremes. |
Sectors | Agriculture, Food and Drink |
Description | Findings from this research represent understanding of fundamental cell biology and, as such, have been incorporated by plant scientists working on the questions of how plants respond to their environments. Specifically, this research demonstrated techniques for monitoring individual molecules in membranes and these techniques have been taken into further research in my own and other labs. Economic impact will derive from this research as the findings have implications for crop protection and food security. |
First Year Of Impact | 2012 |
Sector | Agriculture, Food and Drink |
Impact Types | Economic |
Description | Cell-surface mechanism for stabilisation of plasma membrane protein dynamics |
Amount | £471,344 (GBP) |
Funding ID | BB/K009370/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2013 |
End | 06/2016 |
Title | Single molecule tracking of cell-surface receptors |
Description | Photoactivatable fluorescent proteins have been used for the first time in plant cells in an experiment employing TIRF microscopy to track single membrane protein dynamics. This technique has been refined in both imaging technology and analysis since first developed in 2012 |
Type Of Material | Biological samples |
Year Produced | 2012 |
Provided To Others? | Yes |
Impact | We are using this technique currently in the study of altered cell surface protein dynamics after pathogen challenge. |
Description | Imaging at Central Laser Facility - Harwell (STFC) |
Organisation | Rutherford Appleton Laboratory |
Department | Central Laser Facility |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | My research team produce plant lines that are genetically modified so that plasma membrane proteins and proteins in other organelles can be tracked and measured using the advanced imaging equipment at the CLF. |
Collaborator Contribution | CLF partners maintain and operate the advanced imaging platforms and contribute to the image-analysis aspect of our research. |
Impact | Several papers have resulted from this collaboration. All report findings that involve very high resolution imaging of membrane proteins. This research is multi-disciplinary in that the CLF collaborators are mathematicians and physicists while we are biologists. |
Start Year | 2009 |
Description | BBC Radio - weekly science discussion |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | I appear on BBC radio Oxford each week to discuss current science stories. To date, I have appeared on radio 185 time and discussed over 400 science topics. Many of these are research in my area or in BBSRC-remit areas. |
Year(s) Of Engagement Activity | 2012,2013,2014,2015,2016 |
Description | TEDx presentation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | A TEDx talk that used the background and findings from our research on plant cell communication to illustrate how we are attempting to make agriculture more sustainable. |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.ted.com/tedx/events/21790 |