Orestis Faklaris

Institut Jacques Monod | IJM · ImagoSeine Imaging Facility

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently...

The chromosome translocations generating PAX3FOXO1 and PAX7FOXO1 chimeric proteins are the primary hallmarks of the paediatric cancer, Alveolar Rhabdomyosarcoma (ARMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant trans...

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright, a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono- and two-photon m...

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright: a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon mi...

Elucidating protein functions and molecular organisation requires to localise precisely single or aggregated molecules and analyse their spatial distributions. We develop a statistical method SODA (Statistical Object Distance Analysis) that uses either micro- or nanoscopy to significantly improve on standard co-localisation techniques. Our method c...

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders as well as diseases including obesity, diabetes and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenin...

Nuclear Pore Complex (NPC) is of paramount importance for cellular processes since it is the unique gateway for molecular exchange through the nucleus. Unraveling the modifications of the NPC structure in response to physiological cues, also called nuclear pore plasticity, is key to the understanding of the selectivity of this molecular machinery....

G protein-coupled receptors are key proteins in the regulation of most of the physiological responses. Their conformations are generally oscillating between inactive and active forms leading to the activation of no, a few, or many signaling pathways. Although receptors can spontaneously adopt these various conformations, their interactions with lig...

Controlling nucleus localization is crucial for a variety of cellular functions. In the Drosophila oocyte, nuclear asymmetric positioning is essential for the reorganization of the microtubule (MT) network that controls the polarized transport of axis determinants. A combination of quantitative three-dimensional live imaging and laser ablation-medi...

Supplementary Figures

Ablation between the anterior cortex and the nucleus does not affect its position. Jupiter-GFP labels the MTs.

Ablation between the nucleus and the posterior cortex shifts the nucleus posteriorly. Jupiter-GFP labels the MTs.

Ablation between the nucleus and the anterior cortex does not impair the nuclear migration in 62.5% of cases. The nuclear envelope is labelled by Fs(2)Ket-GFP (green) and the plasma membrane by PHPLC-?1-RFP (red).

Nuclear displacement along the LPM. The nucleus (green) is labelled by Fs(2)Ket-GFP and the plasma membrane (red) with PHPLC-?1-RFP.

Targeting two photon-mediated pulses directly onto the nucleus does not affect its eventual migration. The nuclear envelope is labelled by Fs(2)Ket-GFP (green) and the plasma membrane by PHPLC-?1-RFP (red).

Ablation between the nucleus and the posterior cortex impairs the nuclear migration in 58.3% of cases. The nuclear envelope is labelled by Fs(2)Ket-GFP (green) and the plasma membrane by PHPLC-?1-RFP (red).

Centrosomes coalesce in a compact structure that migrates in close association with the nucleus in 92 % of the cases. The nuclear envelope is revealed by Mud-GFP (green) and the centrosomes by Asl-tdTomato (red).

Mud-GFP shows an asymmetric distribution around the nuclear envelope during th migration of the nucleus.

Knocking down Mud transcripts by RNAi decreases the nuclear velocity. The nucleus is revealed by EB1-GFP.

Fluorescence intensity measurement of Jupiter-GFP during nuclear migration. Jupiter-GFP distribution is displayed as a heat-map look up table.

Ablation all around the nucleus prior to the onset of its migration inhibits subsequent displacement to the antero-dorsal cortex. The nuclear envelope is labelled by Fs(2)Ket-GFP (green) and the plasma membrane by PHPLC-?1-RFP (red).

Nuclear displacement along the APM. The nucleus (green) is labelled by Fs(2)Ket-GFP and the plasma membrane (red) with PHPLC-?1-RFP.

MT nucleation at the nuclear envelope after MT depolymerisation by colcemid, followed by local UV-laser mediated colcemid inactivation. MTs are decorated with EB1-GFP.

The deformation of the nuclear envelope is not aligned with the actual displacement of the migrating nucleus. Fs(2)Ket-GFP decorates the nuclear envelope and Jupiter-GFP labels the oocyte cytoplasm.

The oocyte nucleus fails to migrate upon MT-depolymerisation with colcemid. EB1-GFP (green) labels the (+) ends of MTs, and Asl-tdTomato (red) labels the centrosomes.

Nuclear displacement straight through the cytoplasm (STAD). The nucleus (green) is labelled by Fs(2)Ket-GFP and the plasma membrane (red) by PHPLC-?1-RFP.

Centrosomes display a more scattered pattern in the vicinity of the nucleus in 8 % of the cases. The nucleus is revealed by Fs(2)Ket-GFP (green) and the centrosomes by Asl-tdTomato (red).

Knocking down Asl transcripts by RNAi decreases nuclear velocity. The nucleus is revealed by EB1-GFP.

MT nucleation after MT depolymerisation by colcemid, followed by local UV-laser mediated colcemid inactivation. MTs are decorated with Jupiter-GFP.

Identifying the interacting partners and the dynamics of the molecular networks constitutes the key point in understanding cellular processes. Different methods often based on energy transfer strategies have been developed to examine the molecular dynamics of protein complexes. However, these methods suffer a couple of drawbacks: a single complex c...

The promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are stress-responsive domains where many partner proteins accumulate. Here, we clarify the basis for NB formation and identify stress-induced partner sumoylation as the primary NB function. NB nucleation does not rely primarily on intermolecular interactions between...

Diamond nanocrystals containing Nitrogen-Vacancy (NV) color centers have been used in recent years as fluorescent probes for near-field and cellular imaging. In this work we report that an infrared (IR) pulsed excitation beam can quench the photoluminescence of NV color center in a diamond nanocrystal (size < 50 nm) with an extinction ratio as high...

Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement appa...

GPCR oligomerization has been a matter of intense research these last years. FRET and BRET methods have paved the way to a generalized concept of potential GPCR oligomerization in artificial systems (transfected cell lines). More recently, the use of fluorescent ligands compatible with time-resolved FRET studies has opened the possibility of GPCR o...

The concept of oligomerization of G protein-coupled receptor (GPCR) opens new perspectives regarding physiological function regulation. The capacity of one GPCR to modify its binding and coupling properties by interacting with a second one can be at the origin of regulations unsuspected two decades ago. Although the concept is interesting, its vali...

The identification of new drugs exhibiting reduced adverse side-effects constitutes a great challenge for the next decade. Various steps are needed to screen for good ligand candidates and one of them is the evaluation of their binding properties. New strategies based on fluorescence measurement constitute excellent alternatives to the traditional...

A bunch of loners: Fluorescent nanodiamonds (FNDs) noncovalently conjugated with bovine serum albumin (BSA) or α-lactalbumin exhibited good dispersibility in a buffer with only minor or no agglomeration. They are useful as photostable fluorescent markers in cells for superresolution imaging by STED (see confocal fluorescence image of an FND-labeled...

Nitrogen-Vacancy (NV) color centers in diamond nanoparticles (NanoDiamonds, NDs) have a great number of applications, in particular in quantum information processing and as cellular fluorescent labels. In this work we compare the photoluminescence properties of a single NV color center embedded in a 30 nm diameter nanodiamond to that of a single or...

S olid-state nanoparticles hold great promises for biomedical applications, notably thanks to the possibility to combine biological and inorganic materials with the prospect to develop innovative di-agnostic and therapeutic tools. Among them, nanoparticles such as quantum dots, 1,2 gold nanobeads, 3 or silicon beads 4 are used to label a biomolecul...

This thesis work studies the use of Photoluminescent NanoDiamonds (PNDs) for bio-imaging applications. Nanodiamonds are photoluminescent thanks to embedded nitrogen-vacancy (NV) color centers. The thesis is divided in two parts. The first part concerns the study of the optical properties of NV color centers in nanodiamonds. After optimization of th...

We introduce a point-like scanning single-photon source that operates at room temperature and offers an exceptional photostability (no blinking, no bleaching). This is obtained by grafting in a controlled way a diamond nanocrystal (size around 20 nm) with single nitrogen-vacancy color-center occupancy at the apex of an optical probe. As an applicat...

In order to achieve an efficient biolabeling, a reliable marker is needed. Organic dyes and fluorophores are widely used, but they lack of photophysical stability. Alternative systems are Quantum dots (QDs). Although very efficient for multicolour staining and no photobleaching, they suffer of photoblinking and may be cytotoxic in long term. In con...

Long-term observations of photoluminescence at the single-molecule level were until re-cently very difficult, due to the photobleaching of organic fluorophore molecules. Al-though inorganic semiconductor nanocrystals can overcome this difficulty showing very low photobleaching yield, they suffer from photoblinking. A new marker has been re-cently i...

PACS: 78.67.Bf 07.79.Fc 33.50.Àj Keywords: Nanodiamonds Nitrogen-vacancy (NV) colour centres Near-field scanning optical microscopy Fluorescent probes Active tips a b s t r a c t We present results showing the potential of diamond nanoparticles with size o50 nm as photoluminescent nanoprobes for serving as stable point-like emitters attached at the...

Nitrogen-Vacancy (NV) color centers in diamond nanoparticles (NanoDiamonds, NDs) have a great number of applications, in particular in quantum information processing and as cellular fluorescent labels. In this work we compare the photoluminescence properties of a single NV color center embedded in a 30 nm diameter nanodiamond to that of a single or...

Diamond nanoparticles are promising photoluminescent probes for tracking intracellular processes, due to embedded, perfectly photostable color centers. In this work, the spontaneous internalization of such nanoparticles (diameter 25 nm) in HeLa cancer cells is investigated by confocal microscopy and time-resolved techniques. Nanoparticles are obser...

We present results showing the potential of diamond nanoparticles with size <50 nm as photoluminescent nanoprobes for serving as stable point-like emitters attached at the tip apex of a near-field optical microscope to achieve enhanced spatial resolution.

Diamond nanocrystals containing highly photoluminescent color centers are attractive, nonclassical, and near-field light sources. For near-field applications, the size of the nanocrystal is crucial, since it defines the optical resolution. Nitrogen-vacancy (NV) color centers are efficiently created by proton irradiation and annealing of a nanodiamo...

Diamond nanocrystals containing highly photoluminescent color centers are attractive, nonclassical, and near-field light sources. For near-field applications, the size of the nanocrystal is crucial, since it defines the optical resolution. Nitrogen-vacancy (NV) color centers are efficiently created by proton irradiation and an-nealing of a nanodiam...

The present work reports our attempt in developing diamond nanoparticles with size<50 nm as photoluminescent nanoprobes for biological applications like biomolecule tracking in living cell.

We present preliminary results showing the potential of diamond nanoparticles with size < 50 nm as photoluminescent nanoprobes (i) for biological applications like biomolecule tracking in living cell and (ii) for serving as stable point-like emitters attached at the tip apex of a near-field optical microscope to achieve enhanced spatial resolution.

Ce travail de thèse porte sur l'utilisation des NanoDiamants Photoluminescents (NDPs) pour des applications en bio-imagerie. Les nanodiamants (NDs) sont photoluminescents grâce à la présence de centres colorés azote-lacune (NV) dans leur maille cristalline. Le manuscrit est divisé en deux parties. La première concerne l'étude des propriétés optique...