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

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.

Targeting angiogenesis has been and continues to be an attractive therapeutic modality in glioblastoma (GBM) patients. However, GBM rapidly becomes refractory to anti-VEGF therapies. Myeloid cell infiltration is an important determinant of tumor progression. Given that VEGF is a modulator of the innate immune response we sought to analyze the dynamics of this response in a mouse model of GBM undergoing anti-VEGF therapy.

Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice.

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

Sophie Brustlein

CNRS,
Inserm,
Centre d'Immunologie de Marseille-Luminy,
CNRS, Inserm, Centre d'Immunologie de Marseille-Luminy

Aix Marseille Univ

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

Sophie Brustlein

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}CNRS,Inserm,Centre d'Immunologie de Marseille-Luminy,CNRS, Inserm, Centre d'Immunologie de Marseille-Luminy

Aix Marseille Univ

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

CNRS,
Inserm,
Centre d'Immunologie de Marseille-Luminy,
CNRS, Inserm, Centre d'Immunologie de Marseille-Luminy