Novel Single Cell Fluorescence Approaches in the Investigation of Signaling at the Cellular Level

摘要: Treatment of human diseases relies on a clear understanding the molecularmechanisms and cellular events behind pathophysiological processes. In mostcancers, one or more steps in transduction signals from extracellularenvironment to nucleus are subverted, leading directly formation andevolution tumor. Consequently, drugs targeting transmembrane signalingproteins can be used as an effective selective treatment approach (Sausville etal. 2003). Classical molecular biological methods (blotting,immunoprecipitation,chemical cross-linking) have given important insight into details trans-membrane signaling Most these approaches include disruptionof integrity cell (e.g. by detergent treatment), thereby precluding thestudy intricate signal its native, unperturbedenvironment.Additionally, inherently unable reveal cell-to-cell differences caused heterogeneous, endogenous ectopic expression ofproteins. Biochemical often lack quantitative information needed todescribe physico-chemical processes accurately. situ labeling biomole-cules fluorescence offers valuable alternative biochemical methods.Detec-tion fluorescently labeled molecules is (1) sensitive: under favorable conditionseven single detected; (2) not harmful envi-ronment because use ionizing radiation avoided; (3) flexible andspecific: antibodies genes engineered code for proteinswith fluorescent tag accurately report subcellular distribution pro-teins.In this chapter we give brief overview application inquantitative studies,with special emphasis Forster-type fluores-cence resonance energy transfer (FRET) intracellular trafficking,and addressthe advantages potential pitfalls different detection strategiesfrom standpoint biologist.