Curr Opin Genet Dev

The need for quantification and real time visualization of developmental processes has called for increasingly sophisticated imaging techniques. Among them, multiphoton microscopy reveals itself to be an extremely versatile tool owing to its unique ability to combine fluorescent imaging, laser ablation, and higher harmonic generation. Furthermore, recent advances in femtosecond lasers and optical parametric oscillators (OPO) are now opening doors for imaging at unprecedented wavelengths centered in the tissue transparency window. This Review describes promising multiphoton approaches using OPO and the growing number of useful applications of non-linear microscopy in the field of developmental biology. Basic characteristics associated with these techniques are described along with the main experimental challenges when applied to embryo imaging

Facteurs structuraux et physicochimiques déterminant la localisation cellulaire de photosensibilisateurs et leur capacité à photo-perméabiliser des membranes (étude sur des membranes modèles, des protéines plasmatiques et des cellules en culture)

Excités par la lumière, les photosensibilisateurs (PS) génèrent des espèces transitoires, oxydantes qui altèrent les constituants cellulaires proches. De plus, les PS présentent une affinité sélective envers les tissus prolifératifs. Ces deux propriétés sont à la base d applications thérapeutiques notamment dans le traitement de tumeurs. Le but de ce travail est d établir des corrélations entre les paramètres structuraux des PS, leur accumulation spécifique dans les tissus cancéreux et leur efficacité à photodégrader des membranes lipidiques. Dans la première partie nous étudions par fluorescence l effet du pH sur les interactions de la chlorine e6 (Ce6), un photosensibilisateur carboxylique, avec les protéines plasmatiques et les vésicules lipidiques. L affinité de la Ce6 pour l albumine diminue, alors que celle pour les lipoprotéines de basse densité (LDL) et les membranes lipidiques augmente lorsque le pH diminue. L internalisation cellulaire par endocytose et la localisation sub-cellulaire de la Ce6, étudiées sur des fibroblastes, sont interprétées en termes de vectorisation par les LDL et de dynamique d interaction avec les membranes lipidiques étudiée par stopped flow . Ces résultats éclairent la sélectivité des PS pour les tumeurs. En seconde partie, nous étudions la peroxydation induite par trois chlorines de structures différentes (Ce6, m-THPC, TPCS2a). La m-THPC et la TPCS2a sont respectivement de 69% et 39% plus efficaces pour oxyder le linoléate de méthyle que la Ce6. Par contre, la perméation photoinduite de la membrane est plus importante avec les chlorines chargées. La position membranaire du PS pourrait jouer un rôle déterminant dans ces processusPARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins

AbstractThe uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (∼ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake

Tetrapyrrole-photosensitizers vectorization and plasma LDL: A physico-chemical approach

International audienc

Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membranes

Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed

Dynamin 2 homozygous mutation in humans with a lethal congenital syndrome

Heterozygous mutations in dynamin 2 (DNM2) have been linked to dominant Charcot-Marie-Tooth neuropathy and centronuclear myopathy. We report the first homozygous mutation in the DNM2 protein p.Phe379Val, in three consanguineous patients with a lethal congenital syndrome associating akinesia, joint contractures, hypotonia, skeletal abnormalities, and brain and retinal hemorrhages. In vitro membrane tubulation, trafficking and GTPase assays are consistent with an impact of the DNM2p.Phe379Val mutation on endocytosis. Although DNM2 has been previously implicated in axonal and muscle maintenance, the clinical manifestation in our patients taken together with our expression analysis profile during mouse embryogenesis and knockdown approaches in zebrafish resulting in defects in muscle organization and angiogenesis support a pleiotropic role for DNM2 during fetal development in vertebrates and humans