Visualizing protease activity in living cells: from two dimensions to four dimensions.
Proteolytic degradation of extracellular matrix (ECM) components by cells is an important metabolic activity as cells grow, remodel, and migrate through the ECM. The ability to analyze ECM degradation can be valuable in the study of developmental processes as well as pathologies, such as cancer. In this unit we describe an in vitro live cell-based method to image and quantitatively measure the degradation of ECM components by live cells. Cells are grown in the presence of fluorescent dye-quenched protein substrates (DQ-gelatin, DQ-collagen I, and DQ-collagen IV) that are mixed with protein matrices. Upon proteolytic cleavage, fluorescence is released that directly reflects the level of proteolysis by the cells. Using confocal microscopy and advanced imaging software, the fluorescence is detected and accurate measurements of proteolytic degradation in three and four dimensions can be assessed.
Department of Pharmacology and Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
Christopher
C
Jedeszko
Mansoureh
M
Sameni
Mary B
MB
Olive
Kamiar
K
Moin
Bonnie F
BF
Sloane
D001485:Basement Membrane / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D010450:Endopeptidases / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal; D011499:Protein Processing, Post-Translational; D018874:Spheroids, Cellular / Q000378:metabolism
D005456:Fluorescent Dyes; D010450:Endopeptidases