Biological Signaling Platforms
Campagnola, Mayer

Experimental manipulation of the location, size, and composition of signaling complexes is essential for building and validating quantitative models of spatially organized signaling networks and pathways. Researchers at CCAM are developing standardized methods to experimentally manipulate local concentrations and stoichiometries of membrane-bound and cytosolic signaling complexes (signaling platforms) in living cells. Experimentally manipulating the subcellular localization and/or local concentration of a factor in living cells provides insight to its function and resulting effects on biological activity. CCAM faculty use biological aggregrates, nano-fabrication and optical force to manipulate and study components of signaling networks. An example of a biological singaling platform is the creation of fusion transmembrane proteins that can be aggregated at the plasma membrane by monoclonal antibodies.

Holographic Optical Tweezers
Dufresne

Collaborators at Yale University have developed integrated platforms for the direct manipulation of live cells, their components, and functionalized microparticles using optical forces. These manipulations exploit recent advances in imaging, computer-generated holography, spatial-light modulation, and control systems to provide real-time independent control of up to one thousand targets in 4-D (x,y,z,t). With this technology collaborations between CCAM researchers and the Dufresne Lab, have been capable of exploring cooperative phenomena in molecular and mechanical signaling for the investigation of cell fusion, fission, trafficking, motility, polarization and chemotaxis.

In Vivo Nanofabrication
Campagnola, Mayer

Biophotonics, specifically the use of multiphoton excited (MPE) photochemistry, to crosslink endogenous proteins to create walls, barriers and channels inside the cytoplasm is being developed as a means to isolate compartments that can affect propagation of signaling molecules or assembly of cytoskeletal structures. We are determining the range of structures that can be fabricated in terms of sizes and shapes via imaging, and characterizing diffusion through the barriers and within compartments using fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS).