Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions

Christopher Jedeszko1, Mansoureh Sameni1, Mary B. Olive1, Kamiar Moin1, Bonnie F. Sloane1

1 Department of Pharmacology and Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.20
DOI:  10.1002/0471143030.cb0420s39
Online Posting Date:  June, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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. Curr. Protoc. Cell Biol. 39:4.20.1‐4.20.15. © 2008 by John Wiley & Sons, Inc.

Keywords: 3‐D culture; recombinant basement membrane; DQ‐substrates; proteases; live cell imaging; ECM

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Quantitative Measurement of Proteolytic Degradation of DQ‐Substrates in Live‐Cell Cultures Using a Laser Scanning Confocal Microscope
  • Basic Protocol 2: Quantitative Analysis of Confocal Image Z‐Stacks
  • Support Protocol 1: Prepare Cell Cultures on ECM Containing DQ‐Substrates
  • Support Protocol 2: Harvesting 3‐D Spheroids of Cells Grown in rBM: An Alternative to Directly Growing on ECM Containing DQ‐Substrates
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Quantitative Measurement of Proteolytic Degradation of DQ‐Substrates in Live‐Cell Cultures Using a Laser Scanning Confocal Microscope

  Materials
  • Cell‐permeable DNA‐binding dye [Hoechst 33342 (Invitrogen) or DRAQ5 (Biostatus Ltd)]
  • Cell culture medium
  • Prelabeled cell cultures established in ECM containing DQ‐substrates ( protocol 3), grown on coverslips in 35‐mm cell culture petri dishes
  • Upright laser scanning confocal microscope equipped with a water‐immersion lens and appropriate filter sets and lasers
  • Microscope stage incubator with humidity, temperature, and CO 2 control

Basic Protocol 2: Quantitative Analysis of Confocal Image Z‐Stacks

  Materials
  • Z‐stacks ( protocol 1)
  • Computer system capable of processing large image files: 350 mHz or faster processor, large storage capacity, and at least 256 MB video RAM and minimum 1 GB system RAM
  • Advanced image processing/analysis software capable of applying thresholds and measuring pixel intensities [e.g., ImageJ (Open Source, Public Domain), MetaMorph (Molecular Devices), and Volocity (Improvision)]

Support Protocol 1: Prepare Cell Cultures on ECM Containing DQ‐Substrates

  Materials
  • DQ‐substrates: DQ‐collagen IV, DQ‐collagen I, DQ‐gelatin (Invitrogen)
  • Cell line of interest
  • CellTracker Orange CMTMR Dye (Invitrogen)
  • Powdered bovine skin gelatin (Sigma)
  • Sucrose (Sigma)
  • 1× and 10× phosphate‐buffered saline, sterile (PBS; appendix 2A)
  • Collagen I (Cohesion Laboratories)
  • NaOH
  • Recombinant basement membrane (rBM): Matrigel (BD) or Cultrex (Trevigen)
  • Cell culture medium, phenol red–free
  • 56°C water bath
  • 0.22‐µm filter
  • 12‐mm no.1 round glass coverslips, acid‐washed and sterilized by baking
  • 35‐mm cell culture petri dishes
  • 100‐µl pipettor
  • 37°C humidified incubator
  • Additional reagents and equipment for trypsinizing and counting cells (unit 1.1)

Support Protocol 2: Harvesting 3‐D Spheroids of Cells Grown in rBM: An Alternative to Directly Growing on ECM Containing DQ‐Substrates

  Materials
  • Cell culture grown on rBM within a 60‐mm cell culture dish
  • 1× phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 6 U/mg lyophilized dispase (Roche)
  • Cell culture medium, phenol red–free
  • 37°C incubator
  • Microscope
  • 5‐ml pipet
  • 15‐ml conical tube
  • Centrifuge
  • Additional reagents and equipment for preparing cultures on freshly coated coverslips ( protocol 3)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Cavallo‐Medved, D., Mai, J., Dosescu, J., Sameni, M., and Sloane, B.F. 2005. Caveolin‐1 mediates the expression and localization of cathepsin B, pro‐urokinase plasminogen activator and their cell‐surface receptors in human colorectal carcinoma cells. J. Cell Sci. 118:1493‐1503.
   Debnath, J. and Brugge, J.S. 2005. Modelling glandular epithelial cancers in three‐dimensional cultures. Nat. Rev. Cancer 5:675‐688.
   Friedl, P. and Wolf, K. 2003. Proteolytic and non‐proteolytic migration of tumour cells and leucocytes. Biochem. Soc. Symp. 70:277‐285.
   Menges, D.A., Ternullo, D.L., Tan‐Wilson, A.L., and Gal, S. 1997. Continuous assay of proteases using a microtiter plate fluorescence reader. Anal. Biochem. 254:144‐147.
   Podgorski, I., Linebaugh, B.E., Sameni, M., Jedeszko, C., Bhagat, S., Cher, M.L., and Sloane, B.F. 2005. Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer. Neoplasia 7:207‐223.
   Sameni, M., Moin, K., and Sloane, B.F. 2000. Imaging proteolysis by living human breast cancer cells. Neoplasia 2:496‐504.
   Sameni, M., Dosescu, J. Moin, K., and Sloane, B.F. 2003. Functional imaging of proteolysis: Stromal and inflammatory cells increase tumor proteolysis. Mol. Imaging 2:159‐175.
   Schmeichel, K.L. and Bissell, M.J. 2003. Modeling tissue‐specific signaling and organ function in three dimensions. J. Cell Sci. 116:2377‐2388.
   Sloane, B.F. 1996. Suicidal tumor proteases. Nat. Biotechnol. 14:826‐827.
   Sloane, B.F., Sameni, M., Podgorski, I., Cavallo‐Medved, D., and Moin, K. 2006. Functional imaging of tumor proteolysis. Annu. Rev. Pharmacol. Toxicol. 46:301‐315.
   Tsai, K.K., Chuang, E.Y., Little, J.B., and Yuan, Z.M. 2005. Cellular mechanisms for low‐dose ionizing radiation‐induced perturbation of the breast tissue microenvironment. Cancer Res. 65:6734‐6744.
   Urbich, C., Heeschen, C., Aicher, A., Sasaki, K., Bruhl, T., Farhadi, M.R., Vajkoczy, P., Hofmann, W.K., Peters, C., Pennacchio, L.A., Amolmaali, N.D., Chavakis, E., Reinheckel, T., Zeiher, A.M., and Dimmler, S. 2005. Cathepsin L is required for endothelial progenitor cell‐induced neovascularization. Nat. Med. 11:206‐213.
   Yamada, K.M. and Cukierman, E. 2007. Modeling tissue morphogenesis and cancer in 3D. Cell 130:601‐610.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library