Biology subjects

Rupert, J.

Publications and source records attributed to Rupert, J..

4 recordsLinked to original sources

bioRxiv2023

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimens fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a com-prehensive framework of live-cell spectroscopy methodologies - compatible with imaging - to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the pro-posed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation - inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes - not limited to liquid-liquid phase transition - and we anticipate their widespread application in future life-science research.

biophysics↗

bioRxiv2023

Gemcitabine plus nab-paclitaxel preserves skeletal and cardiac mass and function in a murine model of pancreatic cancer cachexia

More than 85% of patients with pancreatic ductal adenocarcinoma (PDAC) suffer from cachexia, a debilitating syndrome characterized by the loss of muscle and fat and remains an unmet medical need. While chemotherapy remains an effective treatment option, it can also induce weight and muscle loss in patients with cancer. Gemcitabine combined with nab paclitaxel (GnP) is a first line treatment option for patients with PDAC but GnPs effect on cachexia has not been comprehensively investigated. We interrogated the effects of GnP in a murine model of pancreatic cancer cachexia. Mice were orthotopically implanted with the cachexia inducing pancreatic cell line (KPC) and were administered GnP or vehicle. The controls underwent sham surgery. We defined GnP effects on cachexia and tumor burden by evaluating muscle and cardiac mass and function, fat mass, bone morphometry, and hematology measurements. We completed RNA sequencing and deep proteome profiling in skeletal and cardiac muscle. KPC+GnP reduced tumor burden over 50% and increased survival compared to KPC. KPC vehicle group had more than 15% muscle mass loss and decreased left ventricular mass, this was not present in KPC+GnP when compared to controls. RNA Seq and deep proteomics analyses suggested that muscle and cardiac dysfunction pathways activated in KPC group were either reversed or decreased in KPC+GnP. In all, our data suggests that GnP protects against muscle and cardiac wasting in an experimental model of PDAC cachexia. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/536434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1d394f7org.highwire.dtl.DTLVardef@1d8fdaforg.highwire.dtl.DTLVardef@f709d4org.highwire.dtl.DTLVardef@a668e0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2022

A Theoretical Model reveals RNA sequestration in Alpha Synuclein Aggregates

Nucleic acids can act as potent modulators of protein aggregation, and RNA is able to either hinder or facilitate protein assembly depending on the molecular context. Here we used a computational approach to characterize the physico-chemical properties of regions involved in amyloid aggregation. In different experimental datasets we observed that, while the core is hydrophobic and highly ordered, external regions, more disordered, display a distinct tendency to interact with nucleic acids. To validate our predictions, we performed aggregation assays with -synuclein (aS140), a non-nucleic acid binding amyloidogenic protein, and a mutant truncated at the acidic C-terminus (aS103) that is predicted to sequester RNA. For both aS140 and aS103 we observed acceleration of the aggregation upon RNA addition with a significantly stronger effect for aS103. Due to the favorable electrostatics, we observed enhanced nucleic-acid sequestration ability for aS103 that entrapped a larger amount of RNA. Overall, our research suggests that RNA sequestration is a rather common phenomenon linked to protein aggregation and constitutes a gain-of-function mechanism to be further investigated. STATEMENT OF SIGNIFICANCEOur study indicates that aggregation confers RNA-binding ability to non-RNA-binding proteins such as alpha synuclein. The sequestration of RNA upon protein aggregation might alter RNA homeostasis and impact multiple biochemical cascades.

biochemistry↗

bioRxiv2021

New lessons on TDP-43 from the killifish N. furzeri

Frontotemporal dementia and amyotrophic lateral sclerosis are fatal and incurable neurodegenerative diseases linked to the pathological aggregation of the TDP-43 protein. This is an essential DNA/RNA binding protein involved in transcription regulation, pre-RNA processing and RNA transport. Having suitable animal models to study the mechanisms of TDP-43 aggregation is crucial to develop treatments against disease. We have previously demonstrated that the killifish Nothobranchius furzeri offers the unique advantage as a model system that it is an organism with compressed lifespan and a conserved ageing phenotype that develops within months, making this organism the available shortest-lived vertebrate with a clear ageing phenotype. Here, we show that the two paralogs of TDP-43 from the killifish N. furzeri share high sequence homology with the human protein and recapitulate its cellular and biophysical behaviour. We prove that, during ageing, N. furzeri TDP-43 spontaneously forms insoluble intracellular TDP-43 aggregates that have amyloid characteristics and colocalize with the stress granule core protein G3BP. Our results propose this organism as a valuable model of TDP-43-related pathologies and show that even minute differences between the human and N. furzeri proteins may help to shed new light onto the role of TDP-43 in RNA recognition and granule formation.

neuroscience↗