Aurélie Dupont

Université Grenoble Alpes · Laboratoire Interdisciplinaire de Physique (LIPhy)

We present an optical method that combines confocal microscopy with position modulation to perform axial tracking and topographic imaging of fluorescent surfaces. Using a remote focusing system, the confocal observation volume is oscillated in the axial direction. The resulting modulation of the detected signal is used as a feedback to precisely co...

Viral glycoprotein-mediated membrane fusion is an essential step for productive infection of host cells by enveloped viruses; however, due to its rarity and challenges in detection, little is known about the details of fusion events at the single particle level. Here, we have developed dual-color foamy viruses (FVs) composed of eGFP-tagged prototyp...

Förster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. This sensitivity is regularly achieved in single-molecule experiments in vitro but is still challenging in biological materials. Despite many efforts, quantitative FRET in living samples is either restricted to specific instrumen...

The nucleoprotein filament (NPF) is the fundamental element of homologous recombination (HR), a major mechanism for the repair of double-strand DNA breaks in the cell. The NPF is made of the damaged DNA strand surrounded by recombinase proteins, and its sensitivity to base-pairing mismatches is a crucial feature that guarantees the fidelity of the...

Foerster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. This sensitivity is regularly achieved in single-molecule experiments in vitro but is still challenging in biological materials. Despite many efforts, quantitative FRET in living samples is either restricted to specific instrume...

AMP-activated protein kinase AMPK senses and regulates cellular energy state. AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. Based on engineer...

Cells are able to sense and react to their physical environment by translating a mechanical cue into an intracellular biochemical signal that triggers biological and mechanical responses. This process, called mechanotransduction, controls essential cellular functions such as proliferation and migration. The cellular response to an external mechanic...

Cells are able to sense and react to their physical environment by translating a mechanical cue into an intracellular biochemical signal that triggers biological and mechanical responses. This process, called mechanotransduction, controls essential cellular functions such as proliferation and migration. The cellular response to an external mechanic...

Cells are able to sense and react to their physical environment by translating a mechanical cue into an intracellular biochemical signal that triggers biological and mechanical responses. This process, called mechanotransduction, controls essential cellular functions such as proliferation and migration. The cellular response to an external mechanic...

How a nano-searcher finds its nano-target is a general problem in non-equilibrium statistical physics. It becomes vital when the searcher is a damaged DNA fragment trying to find its counterpart on the intact homologous chromosome. If the two copies are paired, that intact homologous sequence serves as a template to reconstitute the damaged DNA seq...

How a nano-searcher finds its nano-target is a general problem in non-equilibrium statistical physics. It becomes vital when the searcher is a damaged DNA fragment trying to find its counterpart on the intact homologous chromosome. If the two copies are paired, that intact homologous sequence serves as a template to reconstitute the damaged DNA seq...

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for ea...

Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in thre...

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative gl...

The technical advances of single particle tracking (SPT) have come with an increasing demand for such techniques to follow single biomolecules as they accomplish their function in living cells. From the single particle trajectory, quantitative mechanistic information can be obtained that are unattainable in classical ensemble methods. Traditionally...

It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake proce...

Movie. Comparison of syncytia formation in PFV or SFVmac infected cells. HeLa cells were incubated with double-tagged PFV Gag-eGFP (non-spiked) and either PE Ch or SE Ch containing viral particles at low temperature (~10°C). After warming the cells to 37°C, z-stacks with 13 planes were recorded every 15 minutes for a total of 7.5 h using wide-field...

Movie. Uptake and trafficking of incoming wild-type PFV particles in host cells. HeLa cells were incubated with double-tagged PFV Gag-eGFP (non-spiked) and PE Ch containing viral particles at low temperature (~10°C). After warming the cell to 37°C, z-stacks with 10 planes were recorded every 15 minutes for a total of 24 h using wide-field live-cell...

Movie. Uptake and trafficking of fusion-deficient PFV particles in host cells. HeLa cells were incubated with double-tagged PFV Gag-eGFP (non-spiked) and PE Ch iCS containing viral particles at low temperature (~10°C). After warming the cell to 37°C, z-stacks with 12 planes were recorded every 15 minutes for a total of 24 h using wide-field live-ce...

Figure. HeLa cell with representative particle number during confocal time-lapsed analysis of colocalized capsid and Env signal in live cells. HeLa cells were incubated with PFV PE-Ch at ~ 10°C for 10–15 min, warmed to 37°C and imaged for 90 minutes. One z-slice from a representative cell containing a total of ~50 particles is shown 22 min after re...

Figure. Influence of various drugs on host cell transduction by retroviral vectors pseudotyped with different glycoproteins. HeLa cells were preincubated with the appropriate drugs (1 h, 37°C) in two different concentrations (as indicated) prior to exposure to PFV or SFVmac Env containing FVs or VSV-G enveloped HIV pseudoparticles for 4 h in the pr...

Movie. Gag tethering to host cell chromatin during mitosis. HeLa cells were incubated with double-tagged PFV Gag-eGFP (non-spiked) and either PE Ch or PE Ch iCS containing viral particles at low temperature (~10°C). After warming the cells to 37°C, z-stacks with 12 planes were recorded every 15 minutes for a total of 6 h using wide-field live-cell...

Movie. FV uptake and trafficking in Bafilomycin A1 treated cells. Hela cells pretreated with 60 nm BAF were incubated with double-tagged PFV Gag-eGFP (non-spiked) and either PE Ch, SE Ch or SE Ch iCS containing viral particles at low temperature (~10°C) in the presence of the drug. After warming the cell to 37°C, z-stacks with 13 planes were recor...

Three-dimensional tracking of single-walled carbon nanotubes (SWNT) with an orbital tracking microscope is demonstrated. We determine the viscosity regime (above 250 cP) at which the rotational diffusion coefficient can be used for length estimation. We also demonstrate SWNT tracking within live HeLa cells and use these findings to spatially map co...

Promising as a gene carrier, the Foamy virus (FV) is an atypical retrovirus that shares similarities with HIV and the hepatitis B virus. Despite numerous biochemical studies, its entry pathway remains unclear, namely whether entry occurs through fusion at the plasma membrane or after endocytosis. To investigate this issue, dual-color fluorescent vi...

Single particle tracking (SPT) in biological systems is a quickly growing field. Many new technologies are being developed providing new tracking capabilities, which also lead to higher demands and expectations for SPT. Following a single biomolecule as it performs its function provides quantitative mechanistic information that cannot be obtained i...

HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles...

The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5 degrees . Our results combined with earlier structural in...

In this thesis, we have studied the mechanics and thermodynamics of a crucial process for repairing DNA breaks: homologous recombination. Some preleminary experiments on the observation of stretched DNA during homologous recombination have shed light on the complexity of this process and the difficulty to observe it in fluorescence. We have then de...

Dans cette thèse, nous avons étudié les aspects mécanique et thermodynamique de la recombinaison homologue, un processus crucial de réparation de l'ADN. Des travaux préliminaires d'observation de molécules d'ADN étirées lors de la recombinaison homologue ont mis à jour la complexité de ce processus et la difficulté de l'observer en fluorescence. No...

Gold islands, vapor deposited on silicon and quartz by microsphere lithography patterning, are used to nucleate arrays of ZnO nanorods. ZnO is grown on approximately 0.32 microm2 Au islands by carbothermal reduction in a tube furnace. Scanning electron microscopy (SEM) and energy dispersive atomic X-ray spectroscopy (EDS) confirm that the gold effe...

The kinesin is a dimeric molecular motor, which processively moves along Q.1 the microtubule in a sequence of 8 nm steps. The stepping dynamics needs to Q.2 be clarified: there are controversial reports about the existence of substructures in the mechanical step and the step timescale. We present observations of the kinesin steps in the absence of...

Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein–DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and a...