Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α‐synuclein aggregates into inclusions throughout the central and peripheral nervous ...system. Mounting evidence suggests that intercellular propagation of α‐synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co‐cultured neurons, here we show that α‐synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α‐synuclein fibrils are able to seed soluble α‐synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α‐synuclein aggregates in lysosomes dispose of this material by hijacking TNT‐mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies.
Synopsis
Misfolded α‐synuclein fibrils propagate in cell culture by transferring between neurons through tunneling nanotubes (TNTs) inside lysosomes, indicating a possible role of TNTs and lysosomes in the spreading and propagation of Parkinson's pathology.
α‐synuclein fibrils are targeted to cell lysosomes for degradation.
α‐synuclein fibrils enhance formation of TNTs between neighbouring cells, possibly though increasing oxidative stress.
Lysosomes overloaded with α‐synuclein fibrils transfer from donor cells to neighbouring (acceptor) cells inside TNTs connecting the two populations.
Once in acceptor cells, α‐synuclein fibrils are able to seed the aggregation of endogenous soluble cytosolic α‐synuclein, conceivably by escaping lysosomes.
Lysosomes containing α‐synuclein fibrils can transfer between primary neurons via tunneling nanotubes.
In this paper, we present a method for simultaneously tracking thousands of targets in biological image sequences, which is of major importance in modern biology. The complexity and inherent ...randomness of the problem lead us to propose a unified probabilistic framework for tracking biological particles in microscope images. The framework includes realistic models of particle motion and existence and of fluorescence image features. For the track extraction process per se, the very cluttered conditions motivate the adoption of a multiframe approach that enforces tracking decision robustness to poor imaging conditions and to random target movements. We tackle the large-scale nature of the problem by adapting the multiple hypothesis tracking algorithm to the proposed framework, resulting in a method with a favorable tradeoff between the model complexity and the computational cost of the tracking procedure. When compared to the state-of-the-art tracking techniques for bioimaging, the proposed algorithm is shown to be the only method providing high-quality results despite the critically poor imaging conditions and the dense target presence. We thus demonstrate the benefits of advanced Bayesian tracking techniques for the accurate computational modeling of dynamical biological processes, which is promising for further developments in this domain.
The progress of digital pathology in recent years has been an opportunity for the development of automated image analysis algorithms for quantitative measurements and computer aided diagnosis. With ...those new methods comes the need for high staining quality and reproducibility, as image analysis tools are typically more sensible to slight stain variations than trained pathologists. This article presents a method for the automated analysis of cytology slides stains specifically adapted to the challenges encountered in digital cytopathology. In particular, the variety of cell types in cytology slides, the 3D distribution of the cellular material, the presence of superposed cells and the need for independent analysis of sub‐cellular compartments are addressed. The proposed method is applied to the quantification of staining variations for quality control, resulting from changes in the staining protocol such as reagent immersion time or a reagent change. Another demonstrated application is the selection of staining protocol parameters that maximize the visible details in nucleus. Finally the analysis pipeline is also used to compare different stain normalization algorithms on digital cytology slides. Code available at: https://gitlab.com/vitadx/articles/automated_staining_analysis.
Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that ...appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin‐like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1‐dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP‐induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.
Synopsis
This study provides evidence that septins, conserved components of the cytoskeleton, are involved in Drp1‐mediated mitochondrial fission.
Septin depletion causes mitochondrial elongation in mammalian cells and in the nematode C. elegans.
Septin 2 directly interacts with the mitochondrial fission factor Drp1 and regulates its mitochondrial association.
This study provides evidence that septins, conserved components of the cytoskeleton, are involved in Drp1‐mediated mitochondrial fission.
This paper reviews the state-of-the-art in denoising methods for biological microscopy images and introduces a new and original sparsity-based algorithm. The proposed method combines total variation ...(TV) spatial regularization, enhancement of low-frequency information, and aggregation of sparse estimators and is able to handle simple and complex types of noise (Gaussian, Poisson, and mixed), without any a priori model and with a single set of parameter values. An extended comparison is also presented, that evaluates the denoising performance of the thirteen (including ours) state-of-the-art denoising methods specifically designed to handle the different types of noises found in bioimaging. Quantitative and qualitative results on synthetic and real images show that the proposed method outperforms the other ones on the majority of the tested scenarios.
Abstract
The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the ...cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.
To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse ...transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings 1. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.
G-protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic ...targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses type 1 (HIV-1). We used TIRF microscopy and a statistical method to track and classify the motion of different receptor subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on β-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.
Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent ...infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human–parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three‐dimensional (3D)‐intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria‐like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D‐intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.
The human colon is home to multiple pathogens and is often the sole target for intestinal diseases. To overcome ethical and methodological limitations of research on intestinal infections, we built a a 3D‐scaffold with cells and collagen reproducing important characteristics of the intestine. This device with other cutting‐edge technologies have made it possible to successfully examine tissue interaction with Entamoeba histolytica, the agent of amebiasis. We highlighted novel regulatory mechanisms used by this parasite to modulate immune responses and survival within the human intestine.
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