Analyzing Actin Dynamics at the Immunological Synapse

Methods Mol Biol. 2017;1584:7-29. doi: 10.1007/978-1-4939-6881-7_2.

Abstract

T cell signaling is inextricably linked to actin cytoskeletal dynamics at the immunological synapse (IS). This process can be imaged in living T cells expressing GFP actin or fluorescent F-actin binding proteins. Because of its planar nature, the IS provides a unique opportunity to image events as they happen, monitoring changes in actin retrograde flow in T cells interacting with different stimulatory surfaces or after pharmacological treatments. Here, we described the imaging methods and analytical procedures used to measure actin velocity across the IS in T cells spreading on planar stimulatory surfaces.

Keywords: Actin; Cytoskeleton; Immunological synapse; Integrin; Kymograph; Live cell imaging; Mobile ligands; Planar lipid bilayer; Spinning disk; T-cells.

MeSH terms

  • Actins / genetics
  • Actins / immunology*
  • Actins / metabolism
  • Animals
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / immunology
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunological Synapses / genetics
  • Immunological Synapses / immunology*
  • Immunological Synapses / metabolism
  • Jurkat Cells
  • Mice
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / metabolism

Substances

  • Actins
  • Green Fluorescent Proteins