A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease

Therapeutic targeting of the PLK1-PRC1-axis triggers cell death in genomically silent childhood cancer.

Chromosomal instability (CIN) is a hallmark of cancer1. Yet, many childhood cancers, such as Ewing sarcoma (EwS), feature remarkably 'silent' genomes with minimal CIN2. Here, we show in the EwS model how uncoupling of mitosis and cytokinesis via targeting protein regulator of cytokinesis 1 (PRC1) or its activating polo-like kinase 1 (PLK1) can be employed to induce fatal genomic instability and tumor regression. We find that the EwS-specific oncogenic transcription factor EWSR1-FLI1 hijacks PRC1, which physiologically safeguards controlled cell division, through binding to a proximal enhancer-like GGAA-microsatellite, thereby promoting tumor growth and poor clinical outcome. Via integration of transcriptome-profiling and functional in vitro and in vivo experiments including CRISPR-mediated enhancer editing, we discover that high PRC1 expression creates a therapeutic vulnerability toward PLK1 inhibition that can repress even chemo-resistant EwS cells by triggering mitotic catastrophe.Collectively, our results exemplify how aberrant PRC1 activation by a dominant oncogene can confer malignancy but provide opportunities for targeted therapy, and identify PRC1 expression as an important determinant to predict the efficacy of PLK1 inhibitors being used in clinical trials.This work was mainly supported by a grant from the German Cancer Aid (DKH-70114111). In addition, the laboratory of T.G.P.G. was supported by the LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative, the ‘Mehr LEBEN für krebskranke Kinder—Bettina-Bräu-Stiftung’, the Matthias-Lackas Foundation, the Dr. Leopold and Carmen Ellinger Foundation, the Boehringer-Ingelheim Foundation, the Wilhelm Sander-Foundation (2016.167.1), the Barbara and Hubertus Trettner Foundation, the Dr. Rolf M. Schwiete Foundation, the Friedrich-Baur Foundation, the German Cancer Aid (DKH-70112257 and DKH-111886), the Gert und Susanna Mayer Foundation, the Barbara und Wilfried Mohr Foundation, the SMARCB1 association, and the Deutsche Forschungsgemeinschaft (DFG-391665916). J.L. was supported by a scholarship of the Chinese Scholarship Council (CSC), and a grant of the German Cancer Aid (DKH-70114111). M.D. was by a scholarship of the ‘Deutsche Stiftung für junge Erwachsene mit Krebs‘, J.M. by a scholarship of the Kind-Philipp-Foundation, and C.M.F., M.K. and T.L.B.H. by scholarships from the German Cancer Aid. The laboratory of J.A. was supported by grants from the Instituto de Salud Carlos III (PI16CIII/00026; DTS18CIII/00005), Asociación Pablo Ugarte, ASION, Fundación Sonrisa de Alex, Asociación Todos somos Iván y Asociación Candela Riera. Freely available clipart used for design of parts of figures was kindly provided by Servier Medical Art (https://smart.servier.com/).S

Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

<p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p

Patterned illumination single molecule localization microscopy (piSMLM): user defined blinking regions of interest cellSTORM - Cost-effective Super-Resolution on a Cellphone using dSTORM

Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale

„GFP-Walking“: Artifical Construct Conversions caused by Simultaneous Co-Transfection analysed by Spatially and Intensity Resolved Planeometric Microscopy (SIRPM)

Several GFP variants have been developed for multicolor labeling in vivo. Here we report that simultaneous co- transfection of fluorescent protein chimeras can give falsepositive results caused by the conversion of spectral properties. The cDNA of the cysteine protease cathepsin B (CB) tagged with the enhanced yellow fluorescent protein (eYFP) and the cDNA of the histone H2A was tagged with the enhanced cyan fluorescent protein (eCFP) and were cotransfected into lung carcinoma cells. Stable clones with converted fluorescence properties were established by G418 selection and prooven on the DNA sequence level by genomic PCR. Thus, conversion is based on homologous recombination/repair/replication processes that occur between the nucleotide sequences of the fluorescent proteins. To quantify the abundance of conversion high-throughput spatially and intensity resolved planeometric microscopy (SIRPM) was applied: The fluorescent nuclei imaged with an epifluorescence microscope were segmented according to their spatial and intensity properties in both the eCFP and eYFP channels and the conversion rate was calculated from the respective number of fluorescent nuclei. Under standard transfection conditions, approximately 8% of cells produce false-positive results, but, depending on the conditions, up to 26% of the cells permanently express altered fusion proteins. The conversion is independent of transfection methods or cell types. Generally, this compromises the interpretation of results obtained by dual- colour cotransfection using auto-fluorescent proteins. Consecutive transfection or low sequence similarities, however, avoided recombination. The appearance of conversion facilitates exchanges of spectral properties in fusion proteins, the creation of libraries, or the assembly of DNA fusion constructs in vivo. The detailed quantification of the conversion rate allows the investigation of recombination/repair/ replication processes in general

The Effects of an Informational Intervention on Attention to Anti-Vaccination Content on YouTube

The spread of misinformation related to health, especially vaccination, is a potential contributor to myriad public health problems. This misinformation is frequently spread through social media. Recently, social media companies have intervened in the dissemination of misinformation regarding vaccinations. In the current study we focus on YouTube. Recognizing the extent of the problem, YouTube implemented an informational modification that affected many videos related to vaccination beginning in February 2019. We collect original data and analyze the effects of this intervention on video viewership. We find that this informational intervention reduced traffic to the affected videos, both overall, and in comparison to a carefully-matched set of control videos that did not receive the informational modification

Visualisation interactive de données volumiques texturées pour la détection supervisée de failles en imagerie sismique

L'interprétation des images sismiques tridimensionnelles est une étape clé de l'exploration pétrolière. Les enjeux de cette activité sont de produire un modèle des différentes structures, telles que les failles, incluses au sein des données sismiques. Les objectifs multiples de cette thèse, menée en partenariat avec le groupe TOTAL, sont de concevoir, d'implémenter et de valider des techniques innovantes de visualisation et d'interaction aidant à la détection et à la modélisation des failles, supervisée par un interprétateur. Bien que spécifiquement développés à destination d'un contexte opéraionnel particulier, les outils proposés s'appliquent plus généralement à l'ensemble des données volumiques texturées ainsi qu'à la segmentation manuelle de structures tridimentionnelles. Ils s'inscrivent dans un cadre théorique et méthodologique nécessairement pluridisciplinaire qui concerne des domaines aussi divers que la spychologie expérimentale, l'imagerie cognitive, la réalité virtuelle ou encore l'interaction homme-machine.BORDEAUX1-BU Sciences-Talence (335222101) / SudocSudocFranceF