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Mechanics of Signal Transduction in Cell Membranes

The hardware for intracellular signal transduction consists of cascades of chemical reactions among thousands of membrane receptors and signaling molecules. At one level, these processes can be described by the ensemble of species present and the chemical transformations among them. In many cases, however, the individual properties of these molecules have proved insufficient to explain their behavior in vivo. Collective protein-protein interactions and clustering on molecular-length scales have been widely implicated in signal transduction mechanisms. More recently, coordinated rearrangement of membrane receptors into large-scale patterns is emerging as a common theme of cellular signaling as well. Hallmark examples are provided by the immunological synapses, which have now been recognized at junctions between a variety of immune cells and their respective target cells. These synapses are defined by the spatial organization of proteins that develops within the intercellular junction as populations of receptors on one cell engage their cognate ligands on the apposed cell membrane. The emergent patterns can be microns in extent, thus transcending direct protein-protein contact interactions, and exhibit strong correlations with the ensuing intracellular signaling and effector functions.

My research efforts focus on the physical mechanisms of chemical signal transduction in biology, with emphasis on the role of spatial organization. An important experimental platform for much of this work consists of synthetic cell surfaces created by self-assembling proteolipid bilayers onto inorganic materials. This configuration, known as a supported membrane, preserves key properties of the cell membrane, such as lateral fluidity. This allows membrane components to move and assemble into functional complexes, even while under control of the solid support. Using this strategy, we have developed a hybrid live cell-supported membrane interface that enables solid-state nanostructures on the substrate to guide the spatial organization of cell surface receptors and signal transduction molecules inside living cells. Thus, through physical perturbations, a rich array of essentially equivalent living cells can be generated that differ only by the spatial organization of their signal transduction molecules. The first implementation of this strategy, which we refer to as spatial mutation, targeted the T cell immunological synapse (in collaboration with Michael Dustin, New York University). The resulting discoveries proved pivotal in our elucidation of the role of spatial organization as a regulator of T cell receptor (TCR) signaling. Additionally, the hybrid live cell-supported membrane junction is forming the foundation for a substantial effort in our studies of cancer metastasis. The spatial mutation is one tool in the collection of physical strategies my research group is developing to manipulate and probe the chemistry of cells.


T Cell Receptor Signaling

Activation of T cells is an essential component of the adaptive immune response. Its aberrant regulation can result in autoimmune diseases such as rheumatoid arthritis and psoriasis. T cell activation primarily occurs through interaction of TCRs on the T cell with major histocompatibility complex proteins displaying peptide antigens (pMHC) on the antigen-presenting cell (APC). Recognition of peptide antigen by T cells involves molecular reorganization on multiple-length scales within the T cell–APC junction, which is known as the immunological synapse. On molecular-length scales, TCR engagement with antigenic pMHC nucleates small clusters of TCR-pMHC complexes. These recruit an entourage of costimulatory and signaling molecules to become functional signaling microclusters. Over larger length scales, TCR-pMHC microclusters can be actively transported long distances on the cell surface to form specific geometric patterns, such as the central supramolecular activation cluster (cSMAC). This consists of a single central region enriched in TCR-pMHC complexes that is surrounded by successive rings enriched in different proteins.

The multiple layers of molecular assembly and spatial reorganization within the immunological synapse are driven by the T cell. Remarkably, these can be recapitulated in hybrid synapses between living T cells and supported membranes, which have been functionalized with membrane-linked forms of pMHC and intercellular adhesion molecule 1 (ICAM1) (as a minimal set). The spatial mutation experiment works in the following way: Defined patterns of solid-state structures on the substrate serve as barriers to lateral diffusion and transport of lipids and proteins in the supported membrane. These effects are strictly local; the membrane remains in a fluid state, except that molecules cannot cross the barriers. Different barrier configurations, fabricated onto the substrate by electron-beam lithography, can successfully guide immunological synapse assembly into a variety of different spatially organized states.

Other than the underlying freedom-of-motion constraint, the initial distributions of proteins in the supported membrane are uniform and freely diffusing. As receptors on the living T cell surface engage their cognate ligands in the supported membrane, they too become subject to the geometrical configuration of mobility restrictions imposed by the substrate. Substrate patterns influence the transport of proteins and signaling machinery within the living cell only through their interactions with cell surface receptors; this provides the specificity. As immunological synapse assembly proceeds, the cSMAC and its surrounding ring structures are skewed into various nonnative configurations by the different geometric mobility constraints. Classical cell biological techniques can then be used to characterize the signaling activity and other cellular behaviors in these spatial mutants.

We are utilizing this strategy to probe several aspects of TCR signaling. First, it is clear that the reorganization of the T cell surface into synaptic patterns is driven largely by the actin cytoskeleton. I am particularly interested in the mechanical linkage between the cytoskeleton and signaling molecules; this is the juncture where chemical signal transduction meets mechanical control. Are the mechanisms at work in T cells unique to T cells, or could T cells be providing the first glimpse of more general chemomechanical regulatory processes? Another class of questions under current investigation concerns the precise role of clustering. How does receptor clustering modulate the overall signal input-response function? By pushing our inorganic materials fabrication capabilities to nanometer-scale features, it becomes possible to control receptor clustering in living cells almost molecule by molecule. This enables some unprecedented types of experiments, which we hope will reveal new insights into biological signaling mechanisms.


EphA2 Receptor Signaling and Cancer

Metastasis is one of the most deadly processes of cancer, and each of its phases is regulated by cell-cell contact interactions and the associated signaling systems. For example, recent studies have found the EphA2 receptor tyrosine kinase (RTK) to be frequently overexpressed and functionally altered in aggressive tumor cells (40 percent of breast cancers), and that these changes promote metastatic character. EphA2 is one of the Eph receptors, which constitute the largest family of RTKs and, together with their membrane-bound ephrin ligands, regulate a broad range of signaling processes at intercellular junctions. Since both the Eph receptors and their ephrin ligands are associated with the cell membrane, this family of cell surface signaling molecules is ideally suited to reconstitution into the hybrid live cell-supported membrane configuration. We are developing this system.


Quantitative Imaging and Spectroscopy

Quantitative physical techniques form the foundation of my research program. A new addition, which will contribute to both cell systems discussed, is fluorescence cross-correlation spectroscopy (FCCS) in live cell membranes. We have now implemented this in primary T cells, using retroviral transfection of fluorescent fusion proteins (in collaboration with Mark Davis, HHMI, Stanford University). Strong cross-correlation indicates their interaction. FCCS provides a critical probe of molecular interactions on the 10- to 200-nm-length scales not accessible by imaging or Förstner resonance energy transfer (FRET). Another emerging technique we are beginning to use to resolve molecular organization on these length scales is photoactivation localization microscopy (PALM). Collectively, these optical techniques are opening a window to high spatial and temporal resolution of molecular organization in living cells during signaling processes.


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University of California, Department of Chemistry, 424 Stanley Hall, MC3220, Berkeley, CA 94720 / Tel. (510) 666-3604 / Fax (510) 666-3603