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Automated Podosome Identification and Characterization in Fluorescence Microscopy Images

Published online by Cambridge University Press:  25 January 2013

Marjolein B.M. Meddens ,
Bernd Rieger ,
Carl G. Figdor ,
Alessandra Cambi  and
Koen van den Dries
Marjolein B.M. Meddens
Affiliation:
Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
Bernd Rieger
Affiliation:
Quantitative Imaging Group, Department of Imaging Science & Technology, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands
Carl G. Figdor
Affiliation:
Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
Alessandra Cambi*
Affiliation:
Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Nanobiophysics, MIRA Institute for Biomedical Technology and Technical Medicine and MESA+ Institute for Nanotechnology, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
Koen van den Dries
Affiliation:
Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
*
*Corresponding author. E-mail: a.cambi@ncmls.ru.nl
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Abstract

Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures contain actin, which makes podosome segmentation by automated image processing difficult. Here, we have developed a quantitative image analysis algorithm that is optimized to identify podosome cores within a typical sample stained with phalloidin. By sequential local and global thresholding, our analysis identifies up to 76% of podosome cores excluding other F-actin-based structures. Based on the overlap in podosome identifications and quantification of podosome numbers, our algorithm performs equally well compared to three experts. Using our algorithm we show effects of actin polymerization and myosin II inhibition on the actin intensity in both podosome core and associated actin network. Furthermore, by expanding the core segmentations, we reveal a previously unappreciated differential distribution of cytoskeletal adaptor proteins within the podosome ring. These applications illustrate that our algorithm is a valuable tool for rapid and accurate large-scale analysis of podosomes to increase our understanding of these characteristic adhesion structures.

Keywords

podosomesimage analysisfluorescence microscopyactinimage quantificationcytoskeletal adaptor proteins
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 180 - 189
Copyright
Copyright © Microscopy Society of America 2013

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