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Photoacoustic effect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis.

ABSTRACT: The photoacoustic effect is generated when a variable light interacts with a strongly light-absorbing material. In water, it may produce hot bubbles and shock waves that could affect the integrity of nearby cellular membranes, opening transient pores (photoporation). In this study, we have evaluated the effect of pulsed laser-irradiated carbon nanoparticles (cNP) on model membranes and on Chinese hamster ovary (CHO) cells. Fluorescence lifetime measurements of calcein-loaded liposomes support the notion that the photoacoustic effect causes transient openings in membranes, allowing diffusion fluxes driven by gradient concentrations. With CHO cells, we have shown that this effect can induce either intracellular delivery of calcein, or release of cellular compounds. The latter process has been recorded live with multiphoton excitation microscopy during pulsed infrared laser irradiation. Calcein loading and cell viability were assayed by flow cytometry, measuring necrotic cells as well as those in early apoptosis. To further assess long-term cell recovery after the rather harsh treatment, cells were reseeded and their behaviour recorded for 48?h. These extended studies on cell viability show that pulsed laser cNP photoporation may be considered an adequate intracellular delivery technique only if employed with soft irradiation conditions (below 50?mJ/cm2).

SUBMITTER: Galisteo-Gonzalez F

PROVIDER: S-EPMC6962462 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Photoacoustic effect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis.

Galisteo-González Francisco F Monasterio Bingen G BG Gil David D Valle Mikel M Goñi Félix M FM

Scientific reports 20200115 1

The photoacoustic effect is generated when a variable light interacts with a strongly light-absorbing material. In water, it may produce hot bubbles and shock waves that could affect the integrity of nearby cellular membranes, opening transient pores (photoporation). In this study, we have evaluated the effect of pulsed laser-irradiated carbon nanoparticles (cNP) on model membranes and on Chinese hamster ovary (CHO) cells. Fluorescence lifetime measurements of calcein-loaded liposomes support th ...[more]

PMID: 31941922

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Project description:BackgroundHuman APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.MethodologyHerein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.ConclusionsAll three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.

Dataset Information

Photoacoustic effect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis.

Publications

Photoacoustic effect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis.

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