Cellular Membrane Biology Lab

Group Leader: Associate Professor Katharina Gaus

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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-resolution 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 encompassing cell biology, biophotonics and surface chemistry.

Lipid rafts: a new research field

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. The existence of specialised domains in cell membranes could finally explain the control of signal transduction processes because domains, that dynamically regulate the association and disassociation of signalling proteins, control signalling efficiency. The Lipid Raft Hypothesis created excitement because it described lipid-based lateral segregation and elevated lipids from simple building blocks to regulatory elements. However, it remains 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.

Novel approaches to study membrane organisation

Laurdan microscopy: We have made major contributions to redefining lateral membrane organisation by designing and implementing Laurdan microscopy, a novel tool that enabled, for the first time, the visualisation and quantification of membrane structure in live cells [PNAS 2003]. The original motivation for this advance was to overcome the difficulties of raft isolation, but this technique has wider applications in discerning membrane heterogeneity in all cells. The approach is based on the membrane dye Laurdan in conjunction with two-photon laser scanning microscopy. It was the first technique to describe heterogeneity of membrane fluidity in macrophages, providing significant support for the existence of ordered domains – the biophysical hallmark of lipid rafts – in live cells. Combining Laurdan microsopy and single-molecule imaging: Demonstrating and describing a link between the assembly of protein clusters and the formation of membrane domains requires the integration of Laudan microscopy with the imaging of single molecules in live cells. To achieve this, we are designing and building the world’s first time-correlated TIRF microscope. This will 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.

Lipid domains and T-cell activation

We achieved a significant breakthrough by quantifying lipid order at T-lymphocyte activation sites [J Cell Biol 2005]. In contrast to pre-existing lipid rafts, 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, whereas the prevailing view of lipid rafts was that such domains are held together by lipid–lipid interactions. The driving force of protein complexes for raft formation is a significant change to concept of lipid rafts. Our demonstration that raft formation is protein- rather than lipid-induced has transformed the underlying principles that govern membrane organisation. It added a crucial piece to the puzzle: although the assembly of activation clusters may be independent of pre-existing raft domains, the TCR protein complexes exert an ordering effect on the lipid bilayer. This work raises the question of whether coalescent rafts 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. Thus, we demonstrated that lipid order is functionally important [PLos ONE 2008]. As part of a funded NHMRC project, we will employ this novel approach to investigate how ordered membrane domains regulate T-cell activation, kinase and phosphatase activities, receptor internalisation and actin restructuring. We will determine how dietary lipids affect 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.

Focal adhesions: the role of spatial cues

Focal adhesions are the interaction sites of cells with their surrounding, the extracellular matrix (ECM). The spatial cues derived from the ECM are transmitted through the membrane 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 showed that focal adhesions are highly ordered – in fact, more ordered than lipid rafts or caveolae, a specialised form of lipid rafts containing the structural protein caveolin [J Cell Biol 2006]. Cell detachment, and thus integrin disengagement, leads to a loss of membrane order that precedes caveolin-mediated endocytosis. This indicates that it is the protein complexes of focal adhesions that are responsible for orchestrating lipid organisation at these sites, and supports the notion of protein-induced raft formation. The wider implications are that membrane order may be a universal mechanism to regulate cell activation, similar to phosphorylation and other post-translational modifications. In endothelial cells, the structure of focal adhesions is likely to regulate proliferation and migration, and thus angiogenic transformation. Our aim is to understand the role of focal adhesion organisation in signalling. Our unpublished work shows that 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. In new research into spatial cues on membrane structure and signal transduction processes, we collaborate with Prof JJ Gooding to develop novel surface chemistries. These activating surfaces allow molecular control over receptor triggering, underpinning our investigation of the effects on focal-adhesion signalling of ligand density (uniform distribution), clustering (non-uniform distribution), and the temporal and laterally mobile presentation of ligands.

Group Members

Key Publications

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. 19, 470-475.

Gaus K, Le Lay S, Balasubramanian N, Schwartz MA. (2006) Integrin-mediated adhesion regulated membrane order. J Cell Biol. 174, 725-734.

Gaus K, Zech T, Harder T. (2006) Visualizing membrane microdomains by Laurdan 2-photon microscopy. Mol Membrane Biol. 23, 41-48.

Gaus K, Chklovskaia E, Fazekas B, Jessup W, Harder T. (2005) Formation of condensed membrane domains at T cell activation sites. J Cell Biol. 171. 121-131.

Gaus K, Rodriguez M, Ruberu KR, Gelissen I, Kritharides L, Jessup W (2005). Domain-specific lipid distribution in macrophage plasma membranes. J. Lipid. Res. 46 1526-1538.

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. 18, 575-6.

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)

Funding Sources