Showing papers by "Elisa D’Este published in 2020"
TL;DR: A general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal is reported, which was used for wash-free, multicolour, live-cell confocal and STED microscopy.
Abstract: Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from a low cell permeability and an unspecific background signal. Here we report a general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal. Conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide does not affect the spectroscopic properties of the fluorophore, but allows us to rationally tune the dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell-permeable spirolactam. Furthermore, the equilibrium generally shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1,000-fold. Using this simple design principle, we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy. It is difficult to develop suitable fluorescent probes for live-cell nanoscopy, but a general strategy is now reported that can transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signals. Using this approach, probes in a variety of colours were developed for different cellular targets and used for wash-free, multicolour, live-cell confocal and STED microscopy.
196 citations
TL;DR: Super-resolution microscopy and in vivo imaging compatible fluorescent probes are described, which reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics in islets and brain and provide insight into class B GPCR distribution and dynamic dynamics both in vitro and in vitro.
Abstract: The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
77 citations
TL;DR: A novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine with a charged sulfonate with the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches.
Abstract: Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.
30 citations
TL;DR: It is found that disruption of Cmtm6-expression in Schwann cells causes a substantial increase of axonal diameters but does not impair myelin biogenesis, radial sorting or integrity of axons, which optimizes nerve function.
Abstract: The velocity of nerve conduction is moderately enhanced by larger axonal diameters and potently sped up by myelination of axons. Myelination thus allows rapid impulse propagation with reduced axonal diameters; however, no myelin-dependent mechanism has been reported that restricts radial growth of axons. By label-free proteomics, STED-microscopy and cryo-immuno electron-microscopy we here identify CMTM6 (chemokine-like factor-like MARVEL-transmembrane domain-containing family member-6) as a myelin protein specifically localized to the Schwann cell membrane exposed to the axon. We find that disruption of Cmtm6-expression in Schwann cells causes a substantial increase of axonal diameters but does not impair myelin biogenesis, radial sorting or integrity of axons. Increased axonal diameters correlate with accelerated sensory nerve conduction and sensory responses and perturbed motor performance. These data show that Schwann cells utilize CMTM6 to restrict the radial growth of axons, which optimizes nerve function. Myelinating cells differentially myelinate axons of different diameters, however whether they can also restrict radial axonal growth remained unclear. Here, the authors show that CMTM6 in Schwann cells restricts axon diameters, affecting sensory nerve conduction and behavioral performance.
28 citations
24 Jan 2020
TL;DR: In this article, a far-red fluorescent GLP1R antagonistic peptide label is presented, which produces intense and specific membrane labeling throughout live and fixed tissue, and provides insight into class B GPCR distribution and dynamics both in vitro and in vivo.
Abstract: The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645 , a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645 . Using LUXendin645 and LUXendin651 , we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo. Glucagon-like peptide-1 receptor is an important regulator of appetite and glucose homeostasis. Here the authors describe super-resolution microscopy and in vivo imaging compatible fluorescent probes, which reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics in islets and brain.
26 citations
TL;DR: It is shown that jasplakinolide-based fluorescent probes bind not only to actin filaments, but also to G-actin, which enables imaging highly dynamic actin structures and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity.
Abstract: The actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes, such as SiR-actin, suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We analyzed a series of new probes composed of jasplakinolide and modified rhodamine fluorophores and found that rhodamine positional isomerism has a profound effect on probe performance. The probes based on the 6'-carboxy-carbopyronine scaffold are considerably less susceptible to efflux and allow efficient staining without efflux pump inhibitors. They can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We show that jasplakinolide-based fluorescent probes bind not only to actin filaments, but also to G-actin, which enables imaging highly dynamic actin structures. We demonstrate an excellent performance of the new probes in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.
15 citations
TL;DR: The results suggest that in small, low-activity synapses the secretory pathway components are tightly clustered in the synaptic area, presumably to enable rapid local responses, while bigger synapses utilise secretory machinery components from larger, more diffuse areas.
Abstract: Neurons are highly asymmetric cells that span long distances and need to react promptly to local demands. Consequently, neuronal secretory pathway elements are distributed throughout neurites, specifically in post-synaptic compartments, to enable local protein synthesis and delivery. Whether and how changes in local synaptic activity correlate to post-synaptic secretory elements is still unclear. To assess this, we used STED nanoscopy and automated quantitative image analysis of post-synaptic markers of the endoplasmic reticulum, ER-Golgi intermediate compartment, trans-Golgi network, and spine apparatus. We found that the distribution of these proteins was dependent on pre-synaptic activity, measured as the amount of recycling vesicles. Moreover, their abundance correlated to both pre- and post-synaptic markers of synaptic strength. Overall, the results suggest that in small, low-activity synapses the secretory pathway components are tightly clustered in the synaptic area, presumably to enable rapid local responses, while bigger synapses utilise secretory machinery components from larger, more diffuse areas.
8 citations
TL;DR: It is demonstrated that spine size can follow different autoregressive integrated moving average (ARIMA) models and that shape- and size-changes are not correlated, and speculated that such a balance might be functionally beneficially to allow a spine to quickly reconfigure itself after LTP induction.
Abstract: It is known that dendritic spines change their size and shape spontaneously and sometimes to a large degree, but the function of this remains unclear. Here, we quantify these changes using time-series analysis of confocal data and demonstrate that spine size can follow different autoregressive integrated moving average (ARIMA) models and that shape- and size-changes are not correlated. We capture this behavior with a biophysical model, based on the spines9 actin dynamics, and find the presence of 1/f noise. When investigating its origins, the model predicts that actin in the dendritic spines self-organizes into a critical state, which creates a fine balance between static actin filaments and free monomers. We speculate that such a balance might be functionally beneficially to allow a spine to quickly reconfigure itself after LTP induction.
4 citations
TL;DR: A chimeric approach was utilized to pinpoint regions within the K.v7.3 C-terminal tail with an impact upon AIS localization raising the possibility that the two domains contribute to the regulation of Kv7 channel numbers and nanoscale organization at the AIS.
Abstract: The voltage-gated Kv7.2/Kv7.3 potassium channel is a critical regulator of neuronal excitability. It is strategically positioned at the axon initial segment (AIS) of neurons, where it effectively inhibits repetitive action potential firing. While the selective accumulation of Kv7.2/Kv7.3 channels at the AIS requires binding to the adaptor protein ankyrin G, it is currently unknown if additional molecular mechanisms contribute to the localization and fine-tuning of channel numbers at the AIS. Here, we utilized a chimeric approach to pinpoint regions within the Kv7.3 C-terminal tail with an impact upon AIS localization. This strategy identified two domains with opposing effects upon the AIS localization of Kv7.3 chimeras expressed in cultured hippocampal neurons. While a membrane proximal domain reduced AIS localization of Kv7.3 chimeras, helix D increased and stabilized chimera AIS localization. None of the identified domains were required for AIS localization. However, the domains modulated the relative efficiency of the localization raising the possibility that the two domains contribute to the regulation of Kv7 channel numbers and nanoscale organization at the AIS.
4 citations
TL;DR: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
4 citations
TL;DR: SBG-conjugated fluorophores across the visible spectrum are reported, which cleanly label SNAP-fused proteins in the plasma membrane of living cells and are demonstrated to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches.
Abstract: Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.
DOI•
24 Jan 2020
TL;DR: This paper aims to demonstrate the efforts towards in-situ applicability of the EMMARM, as to provide real-time information about concrete mechanical properties such as E-modulus and compressive strength.
Abstract: Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); Grant(s): 715884
TL;DR: It is found that the best performing probes are based on 6-carboxy-carbopyronine scaffold, which show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity.
Abstract: Actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We addressed this issue by constructing a series of probes which employ modified rhodamine fluorophores. We found that the best performing probes are based on 6-carboxy-carbopyronine scaffold. These probes show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We demonstrate their excellent performance in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.