Live Cell Studies of Adhesion Receptors by Two-photon Image Correlation Spectroscopy and Image Cross-Correlation Spectroscopy

摘要

Our ability to study the complex interactions between macromolecules within living cells has been greatly enhanced by the development of biophysical techniques such as fluorescence correlation Spectroscopy (FCS) and multiphoton microscopy. One area of great interest to cell biologists is the molecular mechanism that governs cellular adhesion. Direct physical and chemical measurements on intact living cells will be important for obtaining a better understanding of how cells control their adhesive properties at the molecular level in order to control tissue development, maintain tissue integrity, and regulate cellular migration. Cells dynamically regulate the formation and disassembly of macromolecules in focal adhesions within the basal membrane so it would be advantageous to be able to measure such phenomena in situ. By combining two-photon microscopy imaging of living cells expressing fusion proteins of adhesion molecules and mutants of the green fluorescent protein, and image correlation Spectroscopy (ICS) and image cross-correlation Spectroscopy (ICCS) analysis, we have been able to perform direct studies of the molecular transport and clustering. We report on the characterization of flow, diffusion, aggregation, and co-localization of adhesion macromolecules/fluorescent protein constructs in living cells by two-photon ICS and ICCS experiments at 37℃.