Spatio-temporal image correlation spectroscopy and super-resolution microscopy to quantify molecular dynamics in T cells

• Published in: Methods (San Diego, Calif.) Vol. 140-141; pp. 112 - 118
• Main Authors: Ashdown, George W., Owen, Dylan M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2018
• Subjects: Actins - metabolism; Cell Membrane - metabolism; Cortical actin; Humans; Image correlation spectroscopy; Image Processing, Computer-Assisted - methods; Immunological Synapses - immunology; Immunological Synapses - metabolism; Intravital Microscopy - instrumentation; Intravital Microscopy - methods; Jurkat Cells; Lymphocyte Activation - immunology; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Molecular Dynamics Simulation; Signal Transduction - immunology; Spectrometry, Fluorescence - instrumentation; Spectrometry, Fluorescence - methods; Structured illumination microscopy; T cell synapse; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; T cell synapse; Cortical actin; Image correlation spectroscopy; Structured illumination microscopy
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2018.01.017

[Display omitted] •Molecular-scale dynamics are important modulators of signalling pathways and processes.•Image correlation spectroscopy (ICS) permits the analysis of 2D images through time.•Combining ICS with structured illumination increases correlation robustness.•These techniques confirm the retrograde nature of F-actin within the T cell synapse.•Quantification of super-resolution data will be key for future use of these tools. Many cellular processes are regulated by the spatio-temporal organisation of signalling complexes, cytoskeletal components and membranes. One such example is at the T cell immunological synapse where the retrograde flow of cortical filamentous (F)-actin from the synapse periphery drives signalling protein microclusters towards the synapse centre. The density of this mesh however, makes visualisation and analysis of individual actin fibres difficult due to the resolution limit of conventional microscopy. Recently, super-resolution methods such as structured illumination microscopy (SIM) have surpassed this resolution limit. Here, we apply SIM to better visualise the dense cortical actin meshwork in T cell synapses formed against activating, antibody-coated surfaces and image under total-internal reflection fluorescence (TIRF) illumination. To analyse the observed molecular flows, and the relationship between them, we apply spatio-temporal image correlation spectroscopy (STICS) and its cross-correlation variant (STICCS). We show that the dynamic cortical actin mesh can be visualised with unprecedented detail and that STICS/STICCS can output accurate, quantitative maps of molecular flow velocity and directionality from such data. We find that the actin flow can be disrupted using small molecule inhibitors of actin polymerisation. This combination of imaging and quantitative analysis may provide an important new tool for researchers to investigate the molecular dynamics at cellular length scales. Here we demonstrate the retrograde flow of F-actin which may be important for the clustering and dynamics of key signalling proteins within the plasma membrane, a phenomenon which is vital to correct T cell activation and therefore the mounting of an effective immune response.