Generation of a complete model of how cells interpret and respond to their extracellular environment is a grand challenge of contemporary cellular and molecular biology. Failure of a cell to properly detect and process these stimuli often results in a variety of diseases and developmental problems. Traditional molecular biology approaches have provided detailed insight into individual signaling pathways, but it is becoming increasingly clear that these processes intersect and overlap in complex motifs that regulate overall cell response. This thesis focuses on the development and use of new surface micro- and nano-fabrication techniques towards a new understanding of cell signaling, specifically in the context of epithelial and immune cell function. Integrin-cadherin crosstalk is an important aspect of cell function. We first explored this signaling crosstalk using substrates micropatterned with islands of fibronectin surrounded by E-cadherin, capturing the segregation of these signals in normal tissue. While MDCK cells were able to concurrently form adhesive structures with these two proteins, engagement of fibronectin by MCF-7 cells, an adenocarcinoma cell line, inhibited response of these cells to E-cadherin. We further demonstrated that this inhibition is rigidity dependent; on soft elastomer substrates with Young's modulus in the range of tens of kiloPascals, MCF-7 cells were able to engage both integrin and cadherin ligands. Micropatterned costimulation of T cells may provide enhanced control over T cell activation. We previously introduced a platform for investigating spatially-dependent signaling, in the context of the immune synapse. Multiple rounds of microcontact printing are combined to produce glass surfaces with independently defined, micro-scale patterns of antibodies to CD3 and CD28, surrounded by ICAM-1. We demonstrated that IL-2 secretion by naive mouse CD4+ T-cells is sensitive to the position of CD28 signaling within the region of cell-substrate contact and is less sensitive to the organization of CD3. In sharp contrast, we show next that IL-2 secretion by human CD4+ T-cells requires colocalization of CD3 and CD28 signaling. Immunohistochemical staining revealed that colocalized patterns are more effective than segregated counterparts in maintaining Lck phosphorylation at Y394. No differences in Zap70, PI3K, or PKC-theta were observed. Together, these results identify a dramatic difference between mouse and human T cell physiology, and suggest that Lck may be responsible for spatial integration of CD3 and CD28 signaling. Up until now, the cell signaling studies presented have involved presenting immobilized proteins in some form of geometric configuration to cells. However, molecules associated with the outer surface of living cells exhibit complex, non-Brownian patterns of diffusion. In the last section, supported lipid bilayers were patterned with nanoscale barriers to capture key aspects of this anomalous diffusion in a controllable format. First, long-range diffusion coefficients of membrane-associated molecules were significantly reduced by the presence of the barriers, while short-range diffusion was unaffected. Second, this modulation was more pronounced for large molecular complexes than for individual lipids. Surprisingly, the quantitative effect of these barriers on long-range lipid diffusion could be accurately simulated using a simple, continuum-based model of diffusion on a nanostructured surface; we thus describe a metamaterial that captures the properties of the outer membrane of living cells.
Probing Cell Signaling CrossTalk Through Micro- And Nano- Surface Engineering.
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