Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Summary

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Abstract

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

Introduction

The goal of the experiment is to image and characterize the molecular flow of filamentous- (F-) actin in living T cells, beyond the diffraction limit in order to establish flow direction and magnitude during immunological synapse (IS) formation. This technique will be carried out using a structured illumination microscope (SIM) in total internal reflection fluorescence (TIRF) mode, imaging Jurkat T-cells forming an artificial synapse on activating coverslips coated with anti-CD3- and anti-CD28 antibodies. The IS forms between a T cell and its target; an antigen presenting cell (APC) upon T cell receptor (TCR) activation^1,2. As this is the first step in dete....

Protocol

Note: Stages 1 and 2 take place before the day of imaging; ensure all media and supplements to be used before or on the day of imaging are equilibrated. Equilibration is achieved through warming of media and supplements to 37 °C in an incubator set to 5% CO₂. All cell culture work and coverslip plating steps take place in sterile conditions under a laminar flow hood.

1. Cell Transfection

1. Transfect the T cells to be imaged 24 hr before the experiment with a plasmid encoding a fluorescent fusion construct for F-actin GFP labelling and imaging. Note: Cells should be split 24 hr prior to transfection (48 hr prior to ....

Results

Microscope settings

After successfully achieving all steps the researcher will have accomplished structured illumination (SIM) imaging of F-actin flow in a T cell synapse (Video 1), and quantified this molecular flow by spatio-temporal image correlation spectroscopy (Figure 4¹¹). Before recording images, ensure the sample illumination is uniform across the nine raw images and that structured illuminat.......

Discussion

This technique will allow researchers to image and quantify previously unresolvable details in cells expressing fluorescence. In our results we show that F-actin flows in a symmetrical fashion from the cell periphery towards the cell center in the T cell IS. These findings are consistent with previous results from 1D line profile kymograph data¹³ and extend the capacity of analysis to 2D and super-resolution data.

The presented technique is a progression from kymograph imaging which.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Consumables:
Jurkat E6.1 cells	APCC	ATTC TIB-152
RPMI	Life Technologies	21875-091
FBS	Sigma	F7524-500ML
Penicillin-Strepromycin	Life Technologies	15140-122
HBSS	Life Technologies	14025-050
HEPES	Life Technologies	15630-056
#1.5 8-well Labtek coverslips	NUNC, Labtek	155409
LifeAct GFP construct	Ibidi	60101
OptiMem	Life Technologies	51985-042
anti-CD3	Cambridge Bioscience	317315
anti-CD28	BD Bioscience / Insight Biotechnology	555725
PBS	Life Technologies	20012-019
Lens oil
Name	Company	Catalog number	Comments
Equipment:
Gene Pulsar Xcell System	BioRad	165-2660
Cuvette (0.4cm)	BioRad	165-2088
Centrifuge (5810R)	Eppendorf	5805 000.327
Centrifuge (Heraeus)	Biofuge pico	75003235
6 well plate	Corning	3516
Stage-top incubator	Tokai Hit	INUH-TIZSH
Nikon N-SIM microscope	Nikon	Contact company
Nikon Analyse software	Nikon	Contact company
Incubator (190D)	LEEC	Contact company