Sigma1 Regulates Lipid Droplet-mediated Redox Homeostasis Required for Prostate Cancer Proliferation

UNLABELLED: Lipid droplets (LD) are dynamic organelles that serve as hubs of cellular metabolic processes. Emerging evidence shows that LDs also play a critical role in maintaining redox homeostasis and can mitigate lipid oxidative stress. In multiple cancers, including prostate cancer, LD accumulation is associated with cancer aggressiveness, therapy resistance, and poor clinical outcome. Prostate cancer arises as an androgen receptor (AR)-driven disease. Among its myriad roles, AR mediates the biosynthesis of LDs, induces autophagy, and modulates cellular oxidative stress in a tightly regulated cycle that promotes cell proliferation. The factors regulating the interplay of these metabolic processes downstream of AR remain unclear. Here, we show that Sigma1/SIGMAR1, a unique ligand-operated scaffolding protein, regulates LD metabolism in prostate cancer cells. Sigma1 inhibition triggers lipophagy, an LD selective form of autophagy, to prevent accumulation of LDs which normally act to sequester toxic levels of reactive oxygen species (ROS). This disrupts the interplay between LDs, autophagy, buffering of oxidative stress and redox homeostasis, and results in the suppression of cell proliferation in vitro and tumor growth in vivo. Consistent with these experimental results, SIGMAR1 transcripts are strongly associated with lipid metabolism and ROS pathways in prostate tumors. Altogether, these data reveal a novel, pharmacologically responsive role for Sigma1 in regulating the redox homeostasis required by oncogenic metabolic programs that drive prostate cancer proliferation. SIGNIFICANCE: To proliferate, cancer cells must maintain productive metabolic and oxidative stress (eustress) while mitigating destructive, uncontrolled oxidative stress (distress). LDs are metabolic hubs that enable adaptive responses to promote eustress. Targeting the unique Sigma1 protein can trigger distress by disrupting the LD-mediated homeostasis required for proliferation

Multiphoton microscopy and ultrafast spectroscopy: Imaging meets quantum (MUSIQ) roadmap

In April 2019 the EU Marie Skłodowska-Curie Actions (MSCA) Innovative Training Networks (ITN) MUSIQ officially started. The network brought together a unique team of world-leading academics and industrial partners at the forefront of optical micro-spectroscopy and ultrafast laser technology developments merged with fundamental studies of coherent light-matter interaction phenomena, development of quantitative image analysis tools beyond state-of-the-art, and biomedical/pharmaceutical real-world applications. The unique vision of MUSIQ has been to develop and apply the next-generation optical microscopy technologies exploiting quantum coherent nonlinear phenomena. This Roadmap has been written collectively by the MUSIQ early-stage researchers and their supervisors. It provides a summary of the achievements within MUSIQ to date, with an outlook towards future directions

Nonlinear imaging of molecular organization in 3D in biological media

La microscopie polarimétrique par fluorescence et génération de seconde harmonique est une technique utile pour obtenir des données multidimensionnelles sur des échantillons biologiques au niveau moléculaire. Pour les molécules fluorescentes liées de manière rigide à des biomolécules structurelles plus grandes, telles que les sondes lipidiques orientées et les structures fibreuses des protéines, la mesure de l'orientation de l'étiquette fluorescente peut rendre compte de la structure biologique sous-jacente. Ces marqueurs d'orientation, lorsqu'ils sont mesurés dans un ensemble localisé au foyer du microscope, ont une émission polarisée anisotrope due à leur orientation dipolaire. De même, les sous-unités protéiques répétitives dans les fibres de collagène ont un dipôle induit par deux photons (génération de seconde harmonique) sensible à l'orientation de ces sous-unités. L'absorption à deux photons pour les sondes fluorescentes permet d'améliorer la résolution axiale pour l'imagerie 3D, ainsi que de réduire la diffusion des grandes longueurs d'onde dans les tissus plus épais. Ces avantages de la microscopie à deux photons étaient intéressants pour mettre en œuvre une nouvelle méthode de détection polarisée pour mesurer l'orientation moléculaire. La plupart des techniques polarisées produisent une projection 2D de l'orientation moléculaire 3D dans l'échantillon, ce qui introduit des biais dans les lectures d'orientation résultantes. Cette thèse applique un concept de mesure de l'orientation dipolaire 3D d'une molécule unique à des mesures d'ensemble avec excitation à 2 photons dans un microscope à balayage laser. Tout d'abord, je démontre la théorie transposable d'une détection simultanée de 4 polarisations émises avec un filtrage d'intensité sur 2 de ces polarisations de la technique de molécule unique à l'ensemble à un dipôle excité par TPF ou SHG. J'examine les biais dans les différents calculs de géométrie d'illumination, ainsi que les effets du bruit de Poisson de la détection sur le calcul de l'orientation moléculaire. Ensuite, j'ai construit un microscope modulaire à 2 photons et discute des défis de l'étalonnage des composants sensibles à la polarisation à une variété de longueurs d'onde. Je démontre ensuite l'imagerie d'orientation moléculaire 3D de sondes d'orientation fluorescentes dans des membranes lipidiques dans des membranes modèles et des cellules vivantes, ainsi que liées à la F-actine dans des cellules en culture cellulaire 2D et 3D. Enfin, je montre une mesure sans marquage de l'orientation 3D des sous-unités moléculaires du collagène en utilisant l'imagerie SHG 4polaire. Les applications potentielles de cette nouvelle technique 4polaire couplée à des mesures d'ensemble multiphotoniques sont l'obtention d'informations moléculaires dans des échantillons biologiques 3D tels que la structure du collagène dans les tissus, l'hétérogénéité des membranes lipidiques dans la culture cellulaire 3D, et l'interrogation des rôles de l'actine pour les cellules interagissant avec un environnement 3DPolarimetric fluorescence and second harmonic generation microscopy is a useful technique providing multi-dimensional data about biological samples at the molecular level. Using fluorescent tags rigidly linked to structural biomolecules such as orientational lipid probes, and protein fibre dyes, measuring the fluorescent tags' orientations can report on the underlying biological structure. These orientational tags when measured in a localized ensemble at the focus of the microscope have an anisotropic polarized emission due to their dipole orientation. Similarly, repetitive protein subunits in collagen fibres have a 2-photon induced dipole (second harmonic generation, or SHG) sensitive to the orientation of these subunits. Two-photon absorption for fluorescent probes,(TPF) allows for improved axial resolution for 3D imaging, as well as reduced scattering of longer wavelengths in thicker tissues. These 2 photon advantages to microscopy were of interest to implement a novel polarized detection method to measure molecular orientation. Most polarized microscopy results in a 2D projection of the 3D molecular orientation in the sample, introducing biases to the resulting orientation readouts. This thesis applies a concept for single molecule 3D dipole orientation measurement to ensemble measurements with 2-photon excitation in a laser scanning microscope. First, I demonstrate the translatable theory of a simultaneous detection of 4 emitted polarizations with an amplitude mask at the Fourier plane of 2 of these polarizations from single molecule technique to ensemble of TPF or SHG excited dipoles (4polar imaging). I examine the biases in different illumination geometry calculations, as well as the effects of Poisson noise from the detection on the molecular orientation calculation. Next, I constructed a modular 2-photon microscope and discuss the challenges of calibrating polarization-sensitive components at a variety of wavelengths. I then demonstrate 3D molecular orientational imaging of fluorescent orientational probes in lipid membranes in model membranes and live cells, as well as bound to F-actin in cells in 2D and 3D cell culture. Finally I show a label-free measurement of the 3D orientation of molecular subunits of collagen using SHG 4polar imaging. The prospective applications of this novel 4polar technique coupled with multi-photon ensemble measurements are for gaining molecular information in 3D biological samples such as collagen structure in tissue, lipid membrane heterogeneity in 3D cell culture, and roles of actin for cells interacting with a 3D environmen

Creating the Melancholic Artist

I trace the connection between representations of melancholy in Western Art and the concept of the melancholic artist, beginning with Albrecht Dürer\u27s Melencholia I, continuing with Francisco Goya\u27s The Sleep of Reason Produces Monsters, and concluding with to modern and post-modern art works

Influence of the excitation polarization on single molecule 3D orientation imaging

International audienceSingle Molecule Orientation and Localization Microscopy (SMOLM) is gaining an increasing interest in the community of localization microscopy, due to the capability to monitor orientational information in addition to spatial reconstruction. In many cases, molecule’s orientations are not random and their linker to a protein of interest is sufficiently rigid to be able to report orientation information from this protein. While several strategies exist to report single molecule orientation based on polarization splitting and point spread function engineering, the effect of the incident polarization has been often neglected by supposing its effect embedded in the retrieved angular parameters, or by supposing the excitation to be isotropic or the rotational diffusion to be very fast. In this work we quantify the amount of possible bias brought in SMOLM readout due to the incident polarization effect, using analytical derivations. We illustrate this effect experimentally on single molecules attached to a surface in presence of wobbling

Nonlinear imaging of 3D molecular orientation in cells and tissues by polarization splitting detection.

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In‐depth polarisation resolved SHG microscopy in biological tissues using iterative wavefront optimisation

International audienceAbstract Polarised nonlinear microscopy has been extensively developed to study molecular organisation in biological tissues, quantifying the response of nonlinear signals to a varying incident linear polarisation. Polarisation Second harmonic Generation (PSHG) in particular is a powerful tool to decipher sub‐microscopic modifications of fibrillar collagen organisation in type I and III collagen‐rich tissues. The quality of SHG imaging is however limited to about one scattering mean free path in depth (typically 100 micrometres in biological tissues), due to the loss of focus quality, induced by wavefront aberrations and scattering at even larger depths. In this work, we study how optical depth penetration in biological tissues affects the quality of polarisation control, a crucial parameter for quantitative assessment of PSHG measurements. We apply wavefront shaping to correct for SHG signal quality in two regimes, adaptive optics for smooth aberration modes corrections at shallow depth, and wavefront shaping of higher spatial frequencies for optical focus correction at larger depths. Using nonlinear SHG active nanocrystals as guide stars, we quantify the capabilities of such optimisation methods to recover a high‐quality linear polarisation and investigate how this approach can be applied to in‐depth PSHG imaging in tissues, namely tendon and mouse cranial bone

Effects of Solution Structure on the Folding of Lysozyme Ions in the Gas Phase

The fidelity between the structures of proteins in solution and protein ions in the gas phase is critical to experiments that use gas-phase measurements to infer structures in solution. Here we generate ions of lysozyme, a 129-residue protein whose native tertiary structure contains four internal disulfide bonds, from three solutions that preserve varying extents of the original native structure. We then use cation-to-anion proton-transfer reactions (CAPTR) to reduce the charge states of those ions in the gas phase and ion mobility to probe their structures. The collision cross section (Ω) distributions of each CAPTR product depends to varying extents on the original solution, the charge state of the product, and the charge state of the precursor. For example, the Ω distributions of the 6+ ions depend strongly on the original solutions conditions and to a lesser extent on the charge state of the precursor. Energy-dependent experiments suggest that very different structures are accessible to disulfide-reduced and disulfide-intact ions, but similar Ω distributions are formed at high energy for disulfide-intact ions from denaturing and from aqueous conditions. The Ω distributions of the 3+ ions are all similar but exhibit subtle differences that depend more strongly on the original solutions conditions than other factors. More generally, these results suggest that specific CAPTR products may be especially sensitive to specific elements of structure in solution

Folding of Protein Ions in the Gas Phase after Cation-to-Anion Proton-Transfer Reactions

The structure and folding of a protein in solution depends on noncovalent interactions within the protein and those with surrounding ions and molecules. Decoupling these interactions in solution is challenging, which has hindered the development of accurate physics-based models for structure prediction. Investigations of proteins in the gas phase can be used to selectively decouple factors affecting the structures of proteins. Here, we use cation-to-anion proton-transfer reactions (CAPTR) to reduce the charge states of denatured ubiquitin ions in the gas phase, and ion mobility to probe their structures. In CAPTR, a precursor charge state is selected (P) and reacted with monoanions to generate charge-reduced product ions (C). Following each CAPTR event, denatured ubiquitin ions (13+ to 6+) yield products that rapidly isomerize to structures that have smaller collision cross sections (Ω). The Ω values of CAPTR product ions depend strongly on C and very weakly on P. Pre- and post-CAPTR activation was then used to probe the potential-energy surfaces of the precursor and product ions, respectively. Post-CAPTR activation showed that ions of different P fold differently and populate different regions of the potential-energy surface of that ion. Finally, pre-CAPTR activation showed that the structures of protein ions can be indirectly investigated using ion mobility of their CAPTR product ions, even for subtle structural differences that are not apparent from ion mobility characterization of the activated precursor ions. More generally, these results show that CAPTR strongly complements existing techniques for characterizing the structures and dynamics of biological molecules in the gas phase