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. 2017 Jul 11:(125):55653.
doi: 10.3791/55653.

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

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

Tanumoy Saha et al. J Vis Exp. .

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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References

    1. Dunaevsky A, Tashiro A, Majewska A, Mason C, Yuste R. Developmental regulation of spine motility in the mammalian central nervous system. Proc Natl Acad Sci U S A. 1999;96(23):13438–13443. - PMC - PubMed
    1. Matus A, Brinkhaus H, Wagner U. Actin dynamics in dendritic spines: a form of regulated plasticity at excitatory synapses. Hippocampus. 2000;10(5):555–560. - PubMed
    1. Galic M, et al. Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons. Elife. 2014;3:e03116. - PMC - PubMed
    1. Hotulainen P, et al. Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. J Cell Biol. 2009;185(2):323–339. - PMC - PubMed
    1. Leijnse N, Oddershede LB, Bendix PM. Helical buckling of actin inside filopodia generates traction. Proc Natl Acad Sci U S A. 2015;112(1):136–141. - PMC - PubMed

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