Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles - Archive ouverte HAL
Article Dans Une Revue Journal of visualized experiments : JoVE Année : 2022

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

Hugo Wioland
Foad Ghasemi
  • Fonction : Auteur
Jahnavi Chikireddy
  • Fonction : Auteur
Guillaume Romet-Lemonne
  • Fonction : Auteur
Antoine Jégou

Résumé

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today's availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.
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Dates et versions

hal-03799792 , version 1 (07-10-2022)

Identifiants

Citer

Hugo Wioland, Foad Ghasemi, Jahnavi Chikireddy, Guillaume Romet-Lemonne, Antoine Jégou. Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles. Journal of visualized experiments : JoVE, 2022, 183, ⟨10.3791/63891⟩. ⟨hal-03799792⟩
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