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A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions.

Authors
  • Saha, Tanumoy1
  • Rathmann, Isabel1
  • Galic, Milos2
  • 1 DFG Cluster of Excellence 'Cells in Motion', (EXC 1003), Institute of Medical Physics and Biophysics, University of Muenster.
  • 2 DFG Cluster of Excellence 'Cells in Motion', (EXC 1003), Institute of Medical Physics and Biophysics, University of Muenster; [email protected]
Type
Published Article
Journal
Journal of Visualized Experiments
Publisher
MyJoVE Corporation
Publication Date
Jul 11, 2017
Issue
125
Identifiers
DOI: 10.3791/55653
PMID: 28745622
Source
Medline
Language
English
License
Unknown

Abstract

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

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