spot-variation-fluorescence-correlation-spectroscopy-for-analysis

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Aix Marseille Univ

In the present paper, an experimental feasibility study on the detection of long-range intermolecular interactions through three-dimensional molecular diffusion in solution is performed. This follows recent theoretical and numerical analyses reporting that long-range electrodynamic forces between biomolecules could be identified through deviations from Brownian diffusion. The suggested experimental technique was fluorescence correlation spectroscopy (FCS). By considering two oppositely charged molecular species in aqueous solution, namely, lysozymes and fluorescent dye molecules (Alexa488), the diffusion coefficient of the dyes has been measured for different values of the concentration of lysozyme, that is, for different average distances between the oppositely charged molecules. For our model, long-range interactions are of electrostatic origin, suggesting that their action radius can be varied by changing the ionic strength of the solution. The experimental outcomes clearly prove the detectability of long-range intermolecular interactions by means of the FCS technique. Molecular dynamics simulations provide a clear and unambiguous interpretation of the experimental results.

Detection of long-range electrostatic interactions between charged molecules by means of fluorescence correlation spectroscopy.

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.

Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

The skin protects animals from infection and physical damage. In  wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Here, by establishing a simple wounding system, we show that wounding provokes a reorganisation of plasma membrane subdomains. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerisation. We show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signalling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.

Microtubule plus-end dynamics link wound repair to the innate immune response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Article	Year
Detection of long-range electrostatic interactions between charged molecules by means of fluorescence correlation spectroscopy. Physical review. E\| PubMed ID: 28950524	2017
A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses. Methods (San Diego, Calif.)\| PubMed ID: 29454082	2018
Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation. Scientific reports\| PubMed ID: 29563576	2018
Microtubule plus-end dynamics link wound repair to the innate immune response. eLife\| PubMed ID: 31995031	2020
Author Correction: Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation. Scientific reports\| PubMed ID: 32704037	2020

Sebastien Mailfert

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