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Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data
Published on: December 17, 2015
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Spatio-temporal image correlation spectroscopy and super-resolution microscopy to quantify molecular dynamics in T
George W Ashdown1, Dylan M Owen2
1Department of Life Sciences, Imperial College London, London, UK.
Methods (San Diego, Calif.)
|February 8, 2018
Summary
Super-resolution microscopy and advanced image analysis reveal detailed T cell actin flow dynamics. This technique visualizes filamentous (F)-actin retrograde flow, crucial for T cell immune response.
Area of Science:
- Cellular dynamics and immunology
- Molecular and cellular biology
- Biophysics and imaging
Background:
- Cellular processes rely on the spatio-temporal organization of signaling complexes, cytoskeletal components, and membranes.
- At the T cell immunological synapse, retrograde flow of cortical filamentous (F)-actin drives signaling protein microclusters towards the center.
- Conventional microscopy limits visualization of dense actin meshes due to resolution constraints.
Purpose of the Study:
- To apply super-resolution microscopy and quantitative image analysis to visualize and analyze the dynamic cortical actin meshwork in T cell synapses.
- To investigate the retrograde flow of F-actin and its role in T cell activation.
Main Methods:
- Structured Illumination Microscopy (SIM) was used for high-resolution imaging of the T cell synapse actin mesh under Total-Internal Reflection Fluorescence (TIRF) illumination.
- Spatio-temporal Image Correlation Spectroscopy (STICS) and its cross-correlation variant (STICCS) were applied to analyze molecular flow velocity and directionality.
- Small molecule inhibitors of actin polymerization were used to assess their impact on actin flow.
Main Results:
- SIM provided unprecedented detail of the dynamic cortical actin meshwork.
- STICS/STICCS generated accurate, quantitative maps of molecular flow velocity and directionality within the actin mesh.
- Actin flow was successfully disrupted using small molecule inhibitors of actin polymerization.
Conclusions:
- The combination of SIM and STICS/STICCS offers a powerful new tool for studying molecular dynamics at cellular length scales.
- Demonstrated retrograde F-actin flow is vital for clustering and dynamics of signaling proteins in T cell activation.
- Understanding these dynamics is crucial for effective T cell-mediated immune responses.
Keywords:
Cortical actinImage correlation spectroscopyStructured illumination microscopyT cell synapseMore Related Videos
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