The hardware for intracellular signal transduction consists of cascades of chemical reactions. At one level, these processes are described by the ensemble of species present and the chemical transformations among them. In many cases, however, such an inventory alone has proven insufficient to explain the system's behavior in living cells. 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 larger scale signaling clusters and even spatial patterns spanning the entire cell surface are emerging as common themes of cellular signaling.
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 appear to exert regulatory control over the ensuing intracellular signaling and effector functions. More recently, similarly large-scale spatial motifs have been discovered in receptor tyrosine kinase (RTK) signaling pathways.
My research efforts focus on the physical mechanisms of chemical signal transduction in biology, with an 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.
This technique, which we refer to as a spatial mutation, underlies two parallel lines of work focusing on T cell receptor (TCR) signaling and Eph receptor signaling. Though disparate with respect to their biological functions, we are discovering that the shared juxtacrine geometry of these signaling systems leads to surprising commonality in terms of the physical mechanisms by which they function. 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 clusters 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 (e.g., by electron-beam lithography or colloidal self-assembly), can successfully guide immunological synapse formation 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 TCR microclusters and their larger scale distribution are skewed into various nonnative configurations by the different geometric mobility constraints.
When the supported membrane is patterned into a grid array of isolated corrals, an additional layer of control is achieved. Since barriers block lateral transport, the molecular content of any given corral is fixed. Thus, when a cell assembles a signaling cluster, the ligand content of that cluster is now limited by the corral contents. The cell is no longer able to scavenge ligands from a wide area. This relatively simple constraint provides a powerful analytical tool with which we can probe the precise role of cluster assembly on signaling functionality.
How does receptor clustering modulate the overall signal input-response function? We have used the spatial mutation technique to perform an extensive series of experiments in which agonist density and TCR cluster size are independently and controllably varied in primary T cells. A key observation from these experiments is that the threshold for triggering calcium flux is determined by the number of activating ligands within individual TCR clusters; it is not determined by the total number encountered by the cell. This level of quantitative information on the function of signaling systems in living cells is unprecedented and raises intriguing questions concerning spatial localization of decision-making process in receptor signaling systems.
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 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 have applied the spatial mutation to EphA2-expressing human breast cancer cells using supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction are all observed. Quantitative analysis of receptor-ligand transport across a library of mammary epithelial cell lines reveals signature differences that strongly correlate with disease characteristics. Restriction of EphA2 transport by physical barriers nanofabricated onto the underlying substrate alters receptor spatial organization. This physical reorganization of EphA2 also alters the cellular response to ephrin-A1 as observed by changes in cytoskeleton morphology and recruitment of the protease ADAM10. These observations reveal a mechanism for mechanical regulation of EphA2 signaling pathways. Most importantly, it is known that mechanical differences exist between cancerous and noncancerous tissues and our recent discoveries suggest a specific molecular mechanism for how such properties may contribute to the onset and progression of the disease.
Quantitative Imaging and Spectroscopy
Quantitative physical techniques form the foundation of my research program. In recent years, we have been developing the technique of pulsed-interleaved fluorescence cross-correlation spectroscopy for studies of live cell membrane structures. A major study on the role of lipid anchors in controlling protein sorting in cell membranes challenges classical views of the membrane "raft" hypothesis. We have also used the technique collaboratively to investigate galectin-mediated cell surface reorganization (with HHMI Investigator Carolyn Bertozzi, University of California, Berkeley) and the clustering state of EGFR (with HHMI Investigator John Kuriyan, University of California, Berkeley). A fundamental take-home message from these and other ongoing experiments is that cell membrane organization is highly complex, still poorly understood, and deeply important to the natural function of living molecular systems. The good news is that emerging new tools, particularly in fluorescence imaging and spectroscopy, are poised to provide real insight into the fascinating role of the membrane in bringing molecules to life.
This work was supported in part by the National Institutes of Health and the Department of Energy, Basic Energy Sciences.
As of October 03, 2012