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Visualization of Actin Assembly and Filament Turnover by In Vitro Multicolor TIRF Microscopy

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Chemotaxis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1407))

Abstract

In response to chemotactic signals, motile cells develop a single protruding front to persistently migrate in direction of the chemotactic gradient. The highly dynamic reorganization of the actin cytoskeleton is an essential part during this process and requires the precise interplay of various actin filament assembly factors and actin-binding proteins (ABPs). Although many ABPs have been implicated in cell migration, as yet only a few of them have been well characterized concerning their specific functions during actin network assembly and disassembly. In this chapter, we describe a versatile method that allows the direct visualization of the assembly of single actin filaments and higher structures in real time by in vitro total internal reflection fluorescence microscopy (TIRF-M) using purified and fluorescently labeled actin and ABPs.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) by grants to J.F (FA-330/9-1 and FA-330/10-1). We additionally thank Laurent Blanchoin for fruitful discussions.

Author information

Authors and Affiliations

  1. Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Moritz Winterhoff, Stefan Brühmann, Christof Franke & Jan Faix

  2. NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany

    Dennis Breitsprecher

Authors
  1. Moritz Winterhoff
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  2. Stefan Brühmann
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  3. Christof Franke
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  4. Dennis Breitsprecher
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  5. Jan Faix
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Corresponding author

Correspondence to Jan Faix .

Editor information

Editors and Affiliations

  1. Nat Inst of Allergy& Infectious Dis, NIH, Laboratory of Immunogenetics, Rockville, Maryland, USA

    Tian Jin

  2. National Institutes of Health, Nat Inst Alcohol Abuse & Alcoholism, Rockville, Maryland, USA

    Dale Hereld

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Winterhoff, M., Brühmann, S., Franke, C., Breitsprecher, D., Faix, J. (2016). Visualization of Actin Assembly and Filament Turnover by In Vitro Multicolor TIRF Microscopy. In: Jin, T., Hereld, D. (eds) Chemotaxis. Methods in Molecular Biology, vol 1407. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3480-5_21

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  • DOI: https://doi.org/10.1007/978-1-4939-3480-5_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3478-2

  • Online ISBN: 978-1-4939-3480-5

  • eBook Packages: Springer Protocols

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