Research > Cellular Membrane Biology (Katharina Gaus)
Cellular Membrane Biology Lab
Group Leader: Professor Katharina Gaus
Overview of Research
Our research aims to identify the principles that govern the organisation of lipids and proteins within the plasma membrane and thus define the mechanism of signal transduction processes. The overriding quest is to determine how specialised membrane domains organise signalling pathways. Because different signalling pathways share the same signalling machinery, it is the organisation of signalling cascades in time and space that establish hierarchies and, ultimately, control signalling outcomes that determine cell function in health and disease. The way forward for breakthrough science in membrane biology is to use high- and super-resolution fluorescence microscopy to measure membrane signalling events in live cells, while controlling signal triggering on a molecular level. We aim to link membrane organisation to cell signalling by implementing single-molecule imaging techniques and using novel cell-activating surfaces. This multidisciplinary approach encompasses cell biology, biophotonics and surface chemistry.
A new area of research initiated by Dr Till Böcking focuses on elucidating the molecular mechanisms of cellular assembly and disassembly processes using a combination of biochemical and biophysical approaches. In particular we develop fluorescence imaging techniques to observe reaction pathways at the single molecule level. Using single molecule approaches we can resolve details of molecular interactions that have not been accessible with traditional techniques.
More information - Molecular Machines Unit
Lipid domains and T-cell activation
By quantifying lipid order at T-lymphocyte activation sites [J Cell Biol 2005], we revealed that activation of the T-cell receptor (TCR) leads to a condensation of the plasma membrane at these sites. We found that ordered domains at T-cell activation sites are stabilised by the actin cytoskeleton. This work raises the question whether lipid domains at activation sites are functionally important or simply ‘byproducts’ of the formation of multimolecular signalling assemblies. We use the oxysterol 7-ketocholesterol (7KC) that acts as a spacer to prevent the formation of ordered membrane domains. 7KC-enriched T cells failed to activate when the TCR was triggered. Thus, we demonstrated that lipid order is functionally important [PLoS ONE 2008]. The wider implications are that membrane order may be a universal mechanism to regulate cell activation, similar to phosphorylation and other post-translational modifications. Current and future projects are:
- To better understand the molecular mechanisms of the assembly of TCR activation clusters, we established Photo-Activation Localization Microscopy (PALM) [J Biophotonics]. This super-resolution microscopy approach images individual molecules with nanometre precision. From PALM image, we can determine the degree of protein clustering and hence measure signalling efficiencies.
- We aim to determine how dietary lipids affect signalling efficiencies and the balance of fluid/ordered membranes in T cells. This is a candidate mechanism of the underlying immune dysfunction in metabolic diseases such as obesity and diabetes. Ultimately, our goal is to image T cell migration and activation in vivo to understand how lipid disorders affect T cell function.
- Although it is known that actin restructuring is required for TCR-induced signalling and migration, it is not well understood how the organization of the actin cytoskeleton contributes to domains formation and clustering of signalling proteins.
Adhesion and migration: the role of spatial cues
To understand how receptor triggering re-organizes proteins and lipids in the plasma membrane requires molecular level control over the spatial organization of the activating signal. In collaboration with the Gooding research group (http://www.chem.unsw.edu.au/staffprofiles/gooding.html
), we have developed novel surface chemistries to preciously determine the density, clustering and patterning of receptor ligands on a surface. These surfaces will allow us to determine how cells integrate spatial cues and how external signals affect membrane organization.
Focal adhesions are the interaction sites of cells with their surrounding mediated by transmembrane integrin receptors. Signalling at focal adhesions controls numerous cell activation responses, such as cell polarisation and migration, membrane trafficking, cell-cycle progression, gene expression, and oncogenic transformation. We have previously shown that focal adhesions are highly ordered [J Cell Biol 2006]. Cell detachment, and thus integrin disengagement, leads to a loss of membrane order. Our unpublished work shows that in endothelial cells, the degree of localised integrin engagement – that is, the degree of integrin cross-linking – defines the membrane structure at these sites. Membrane structure at focal adhesions affects the activation and coordination of signalling, and hence downstream outcomes such as cell migration, proliferation and survival.
- Our aim is to understand the role of focal adhesion organisation in signalling. By using activating surfaces we aim to investigate how ligand density (uniform distribution), clustering (non-uniform distribution), and the temporal and laterally mobile presentation of ligands imposes a membrane organization onto the plasma membrane and how this organization influences signalling activities and efficiencies.
- Using super-resolution PALM microscopy, we aim to link the molecular distribution of ligands to integrin receptors in adherent and migrating cells
- We aim to investigate how sterol manipulations such a 7KC enrichment affects cell migration over 2D surfaces and through 3D scaffolds. This project involves a range of imaging techniques including video time-lapse imaging to monitor cell motility as well as molecular imaging to determine the targeting of proteins within a motile cell.
Novel approaches to study membrane organisation
Over the past ten years, the Lipid Raft Hypothesis has changed the way cell biologists view lipids and membrane organisation. It defines lipid rafts as cholesterol- and sphingolipid-rich domains within the plasma membrane, which localise and concentrate raft-associated proteins to specific sites, in particular signalling proteins. Due to their distinctive lipid composition, these domains are more ordered than their fluid surroundings. Hence, these membrane domains constitute biophysically and biochemically discrete platforms. However, lipid domains remain controversial, mainly because raft isolation does not provide clear results (see for example [J Lipid Res 2005]). New approaches to describing membrane organisation in intact, live cells have enormous potential to advance our knowledge of the lateral distribution of lipids and proteins in membranes.
- We are in the process of establishing Fluorescence Correlation Spectroscopy (FCS) under Total Internal Reflectance Fluorescence (TIRF) and Stimulated Emission Depletion (STED) excitation. These novel approaches will enable us to directly measure lipid-lipid, protein-lipid and protein-protein interactions.
- Our new microscopy techniques enable us to discover how membrane fluidity affects local protein concentrations, protein diffusion, collision probabilities and protein–protein interactions, and how formation of protein complexes affects membrane order. We aim to integrate these parameters into a conceptual framework using Monte Carlo simulation or other mathematical modelling approaches.
New frontiers: imaging of whole tissue and organisms
- Olfactory receptor neurons (ORNs), embedded in the olfactory epithelium in the nasal cavity, are highly polarized neurons that need to combine specializations of two different cell types within a single cell. ORNs require a membrane organization with specific lipid composition and membrane structure that allows them to extend thin cilia into the mucus for odour recognition and signal transduction. At the same time, ORNs function as neurons generating action potentials at the cell body when odorants are recognized by G-protein coupled receptors. The molecular machinery for these signal transduction processes is embedded or associated with the plasma membrane raising the question how membrane composition and structure affects odour-induced signaling and neuronal functionality. Our aim is to employ our high- and super-resolution microscopes to understand the structure-function relationship of neuronal membranes. This Human Frontier Science Program-funded project is a collaboration with Dr Johannes Reisert, Monell Chemical Senses Center, Philadelphia.
- In collaboration with Dr Arindam Majumdar, Uppsala University, we have recently succeeded in imaging membrane order in whole, living zebrafish embryos [Biophysical J]. This breakthrough opens the door to new and exciting research: How does membrane order aid the development of tissue? Do polarity proteins affect membrane order and development? What is the link between membrane organization and cell migration in vivo?
|Professor Katharina Gaus||Group Leader|
|Dr Till Böcking||ARC Future Fellow, Unit Leader (Molecular Machines)|
|Dr Dylan Owen||ARC Postdoctoral Fellow |
|Dr Ales Benda||Vice-Chancellor’s Postdoctoral Fellow|
|Dr Astrid Magenau||Postdoctoral Fellow|
|Dr Jérémie Rossy||Postdoctoral Fellow|
|Mr Ahmed Abu-Siniyeh||Postgraduate student|
|Ms Carola Benzing||Postgraduate student|
|Ms Siân Cartland||Postgraduate student (Joint supervisor: Prof Jessup)|
|Ms Rhea Cornely||Postgraduate student|
|Mr Alexandre Kross||Postgraduate student|
|Ms Siti Hawa Ngalim||Postgraduate student (Joint Supervisor: Dr Böcking)|
|Ms Abigail Pollock||Postgraduate student|
|Mr David Williamson||Postgraduate student|
|Ms Jeannette Hallab||Research Assistant|
|Ms Zhengmin Yang||Research Assistant|
Williamson DJ, Owen DM, Rossy J, Magenau A, Wehrmann M, Gooding JJ, Gaus K. (2011) Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat Immunol
12, 655–662 .
Owen DM, Magenau A, Majumdar A, Gaus K. (2010) Imaging membrane lipid order in whole, living vertebrate organisms. Biophysical J
Owen, DM, Rentero C, Rossy J, Magenau A, Williamson D, Rodriguez M, Gaus K. (2010) PALM Imaging and Cluster Analysis of Protein Heterogeneity at the Cell Surface. J Biophotonics
Ngalim SH, Magenau A, Le Saux G, Gooding JJ, Gaus K (2010) How do cells make decisions: engineering micro- and nanoenvironments for cell migration. J Oncol
Owen DM, Williamson D, Rentero C, Gaus K. (2009) Quantitative microscopy: protein dynamics and membrane organization. Traffic
Rentero C, Zech T, Quinn CM, Engelhardt K, Williamson D, Grewal T, Jessup W, Harder T, Gaus K (2008). Functional implications of plasma membrane condensation for T cell activation. PLoS ONE
. In Press.
Harder T, Rentero C, Zech T, and Gaus K. (2007) Plasma membrane segregation during T cell activation: probing the order of domains. Curr Opinion Immunology.
Gaus K, Le Lay S, Balasubramanian N, Schwartz MA. (2006) Integrin-mediated adhesion regulated membrane order. J Cell Biol.
Gaus K, Zech T, Harder T. (2006) Visualizing membrane microdomains by Laurdan 2-photon microscopy. Mol Membrane Biol.
Gaus K, Chklovskaia E, Fazekas B, Jessup W, Harder T. (2005) Formation of condensed membrane domains at T cell activation sites. J Cell Biol.
Gaus K, Rodriguez M, Ruberu KR, Gelissen I, Kritharides L, Jessup W (2005). Domain-specific lipid distribution in macrophage plasma membranes. J. Lipid. Res.
Gaus K, Kritharides L, Schmitz G, Boettcher A, Drobnik W, Langmann T, Quinn CM, Death A, Dean RT, Jessup W. (2004) Apolipoprotein A-1 interaction with plasma membrane lipid rafts control cholesterol export from macrophages. FASEB J.
Gaus K, Gratton E, Kable EPW, Jones AS, Gelissen I, Kritharides L, Jessup W. (2003) Visualizing lipid structure and raft domains in living cells with 2-photon microscopy. Proc. Natl. Acad. Sci. U.S.A.
100, 15554-9. IF=9.64 (104)