17 research outputs found
Reconstruction of destruction â in vitro reconstitution methods in autophagy research
International audienceAutophagy is one of the most elaborative membrane remodeling systems in eukaryotic cells. Its major function is to recycle cytoplasmic material by delivering it to lysosomes for degradation. To achieve this, a membrane cisterna is formed that gradually captures cargo such as organelles or protein aggregates. The diversity of cargo requires autophagy to be highly versatile to adapt the shape of the phagophore to its substrate. Upon closure of the phagophore, a double-membrane-surrounded autophagosome is formed that eventually fuses with lysosomes. In response to environmental cues such as cytotoxicity or starvation, bulk cytoplasm can be captured and delivered to lysosomes. Autophagy thus supports cellular survival under adverse conditions. During the past decades, groundbreaking genetic and cell biological studies have identified the core machinery involved in the process. In this Review, we are focusing on in vitro reconstitution approaches to decipher the details and spatiotemporal control of autophagy, and how such studies contributed to our current understanding of the pathways in yeast and mammals. We highlight studies that revealed the function of the autophagy machinery at a molecular level with respect to its capacity to remodel membranes
Fluorescence energy transfer enhancement in aluminum nanoapertures
Zero-mode waveguides (ZMWs) are confining light into attoliter volumes,
enabling single molecule fluorescence experiments at physiological micromolar
concentrations. Among the fluorescence spectroscopy techniques that can be
enhanced by ZMWs, F\"{o}rster resonance energy transfer (FRET) is one of the
most widely used in life sciences. Combining zero-mode waveguides with FRET
provides new opportunities to investigate biochemical structures or follow
interaction dynamics at micromolar concentration with single molecule
resolution. However, prior to any quantitative FRET analysis on biological
samples, it is crucial to establish first the influence of the ZMW on the FRET
process. Here, we quantify the FRET rates and efficiencies between individual
donor-acceptor fluorophore pairs diffusing in aluminum zero-mode waveguides.
Aluminum ZMWs are important structures thanks to their commercial availability
and the large literature describing their use for single molecule fluorescence
spectroscopy. We also compare the results between ZMWs milled in gold and
aluminum, and find that while gold has a stronger influence on the decay rates,
the lower losses of aluminum in the green spectral region provide larger
fluorescence brightness enhancement factors. For both aluminum and gold ZMWs,
we observe that the FRET rate scales linearly with the isolated donor decay
rate and the local density of optical states (LDOS). Detailed information about
FRET in ZMWs unlocks their application as new devices for enhanced single
molecule FRET at physiological concentrations
Nanophotonic enhancement of the F\"orster resonance energy transfer rate on single DNA molecules
Nanophotonics achieves accurate control over the luminescence properties of a
single quantum emitter by tailoring the light-matter interaction at the
nanoscale and modifying the local density of optical states (LDOS). This
paradigm could also benefit to F\"orster resonance energy transfer (FRET) by
enhancing the near-field electromagnetic interaction between two fluorescent
emitters. Despite the wide applications of FRET in nanosciences, using
nanophotonics to enhance FRET remains a debated and complex challenge. Here, we
demonstrate enhanced energy transfer within single donor-acceptor fluorophore
pairs confined in gold nanoapertures. Experiments monitoring both the donor and
the acceptor emission photodynamics at the single molecule level clearly
establish a linear dependence of the FRET rate on the LDOS in nanoapertures.
These findings are applied to enhance the FRET rate in nanoapertures up to six
times, demonstrating that nanophotonics can be used to intensify the near-field
energy transfer and improve the biophotonic applications of FRET
Plasmonic antennas and zero mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy towards physiological concentrations
Single-molecule approaches to biology offer a powerful new vision to
elucidate the mechanisms that underpin the functioning of living cells.
However, conventional optical single molecule spectroscopy techniques such as
F\"orster fluorescence resonance energy transfer (FRET) or fluorescence
correlation spectroscopy (FCS) are limited by diffraction to the nanomolar
concentration range, far below the physiological micromolar concentration range
where most biological reaction occur. To breach the diffraction limit, zero
mode waveguides and plasmonic antennas exploit the surface plasmon resonances
to confine and enhance light down to the nanometre scale. The ability of
plasmonics to achieve extreme light concentration unlocks an enormous potential
to enhance fluorescence detection, FRET and FCS. Single molecule spectroscopy
techniques greatly benefit from zero mode waveguides and plasmonic antennas to
enter a new dimension of molecular concentration reaching physiological
conditions. The application of nano-optics to biological problems with FRET and
FCS is an emerging and exciting field, and is promising to reveal new insights
on biological functions and dynamics.Comment: WIREs Nanomed Nanobiotechnol 201
Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin
Extracellular influx of calcium or release of calcium from intracellular stores have been shown to activate mammalian TRPA1 as well as to sensitize and desensitize TRPA1 electrophilic activation. Calcium binding sites on both intracellular N- and C-termini have been proposed. Here, we demonstrate based on Forster resonance energy transfer (FRET) and bilayer patch-clamp studies, a direct calmodulin-independent action of calcium on the purified human TRPA1 (hTRPA1), causing structural changes and activation without immediate subsequent desensitization of hTRPA1 with and without its N-terminal ankyrin repeat domain (N-ARD). Thus, calcium alone activates hTRPA1 by a direct interaction with binding sites outside the N-ARD.Funding Agencies|Swedish Research CouncilSwedish Research Council [2014-3801]; Medical Faculty of Lund University - ALF [ALFSKANE-451751]; Agence Nationale de la Recherche (ANR)French National Research Agency (ANR) [ANR-17-CE09-0026-01]; European Research Council (ERC) under the European Commissions Seventh Framework Programme [278242]</p
Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components
Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of beta-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.Funding Agencies|European Commission [FP7-ICT-2011-7, ERC StG 278242]; Goran Gustafsson Foundation; Swedish Alzheimer Foundation</p
Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL
The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism
Nanophotonic Enhancement of the FoÌrster Resonance Energy-Transfer Rate with Single Nanoapertures
Tailoring the lightâmatter
interaction and the local density
of optical states (LDOS) with nanophotonics provides accurate control
over the luminescence properties of a single quantum emitter. This
paradigm is also highly attractive to enhance the near-field FoÌrster
resonance energy transfer (FRET) between two fluorescent emitters.
Despite the wide applications of FRET in nanosciences, using nanophotonics
to enhance FRET has remained a debated and complex challenge. Here
we demonstrate enhanced energy transfer within single donorâacceptor
fluorophore pairs confined in single gold nanoapertures. Experiments
monitoring both the donor and the acceptor emission photodynamics
clearly establish a linear dependence of the FRET rate on the LDOS
in nanoapertures, demonstrating that nanophotonics can be used to
intensify the near-field energy transfer. Strikingly, we observe a
significant six-fold increase in the FRET rate for large donorâacceptor
separations exceeding 13 nm. Exciting opportunities are opened to
investigate biochemical structures with donorâacceptor distances
much beyond the classical FoÌrster radius. Importantly, our
approach is fully compatible with the detection of single biomolecules
freely diffusing in water solution under physiological conditions