Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Eye movements in response to different cognitive activities measured by eyetracking: a prospective study on some of the neurolinguistics programming theories.

The eyes are in constant movement to optimize the interpretation of the visual scene by the brain. Eye movements are controlled by complex neural networks that interact with the rest of the brain. The direction of our eye movements could thus be influenced by our cognitive activity (imagination, internal dialogue, memory, etc.). A given cognitive activity could then cause the gaze to move in a specific direction (a brief movement that would be instinctive and unconscious). Neuro Linguistic Programming (NLP), which was developed in the 1970s by Richard Bandler and John Grinder (psychologist and linguist respectively), issued a comprehensive theory associating gaze directions with specific mental tasks. According to this theory, depending on the visual path observed, one could go back to the participant's thoughts and cognitive processes. Although NLP is widely used in many disciplines (communication, psychology, psychotherapy, marketing, etc), to date, few scientific studies have examined the validity of this theory. Using eye tracking, this study explores one of the hypotheses of this theory, which is one of the pillars of NLP on visual language. We created a protocol based on a series of questions of different types (supposed to engage different brain areas) and we recorded by eye tracking the gaze movements at the end of each question while the participants were thinking and elaborating on the answer. Our results show that 1) complex questions elicit significantly more eye movements than control questions that necessitate little reflection, 2) the movements are not random but are oriented in selected directions, according to the different question types, 3) the orientations observed are not those predicted by the NLP theory. This pilot experiment paves the way for further investigations to decipher the close links between eye movements and neural network activities in the brain.

A specific molecular signature in SARS-CoV-2-infected kidney biopsies.

Acute kidney injury is one of the most important complications in patients with COVID-19 and is considered a negative prognostic factor with respect to patient survival. The occurrence of direct infection of the kidney by SARS-CoV-2, and its contribution to the renal deterioration process, remain controversial issues. By studying 32 renal biopsies from patients with COVID-19, we verified that the major pathological feature of COVID-19 is acute tubular injury (ATI). Using single-molecule fluorescence in situ hybridization, we showed that SARS-CoV-2 infected living renal cells and that infection, which paralleled renal angiotensin-converting enzyme 2 expression levels, was associated with increased death. Mechanistically, a transcriptomic analysis uncovered specific molecular signatures in SARS-CoV-2-infected kidneys as compared with healthy kidneys and non-COVID-19 ATI kidneys. On the other hand, we demonstrated that SARS-CoV-2 and hantavirus, 2 RNA viruses, activated different genetic networks despite triggering the same pathological lesions. Finally, we identified X-linked inhibitor of apoptosis-associated factor 1 as a critical target of SARS-CoV-2 infection. In conclusion, this study demonstrated that SARS-CoV-2 can directly infect living renal cells and identified specific druggable molecular targets that can potentially aid in the design of novel therapeutic strategies to preserve renal function in patients with COVID-19.

Polymer simulations guide the detection and quantification of chromatin loop extrusion by imaging.

Genome-wide chromosome conformation capture (Hi-C) has revealed the organization of chromatin into topologically associating domains (TADs) and loops, which are thought to help regulate genome functions. TADs and loops are understood as the result of DNA extrusion mediated by the cohesin complex. However, despite recent efforts, direct visualization and quantification of this process in single cells remains an open challenge. Here, we use polymer simulations and dedicated analysis methods to explore if, and under which conditions, DNA loop extrusion can be detected and quantitatively characterized by imaging pairs of fluorescently labeled loci located near loop or TAD anchors in fixed or living cells. We find that under realistic conditions, extrusion can be detected and the frequency of loop formation can be quantified from fixed cell images alone, while the lifetime of loops and the speed of extrusion can be estimated from dynamic live-cell data. Our delineation of appropriate imaging conditions and the proposed analytical methods lay the groundwork for a systematic quantitative characterization of loop extrusion in fixed or living cells.

Deep learning in image-based phenotypic drug discovery.

Modern drug discovery approaches often use high-content imaging to systematically study the effect on cells of large libraries of chemical compounds. By automatically screening thousands or millions of images to identify specific drug-induced cellular phenotypes, for example, altered cellular morphology, these approaches can reveal 'hit' compounds offering therapeutic promise. In the past few years, artificial intelligence (AI) methods based on deep learning (DL) [a family of machine learning (ML) techniques] have disrupted virtually all image analysis tasks, from image classification to segmentation. These powerful methods also promise to impact drug discovery by accelerating the identification of effective drugs and their modes of action. In this review, we highlight applications and adaptations of ML, especially DL methods for cell-based phenotypic drug discovery (PDD).

Versatile CRISPR-Based Method for Site-Specific Insertion of Repeat Arrays to Visualize Chromatin Loci in Living Cells.

Hi-C and related sequencing-based techniques have brought a detailed understanding of the 3D genome architecture and the discovery of novel structures such as topologically associating domains (TADs) and chromatin loops, which emerge from cohesin-mediated DNA extrusion. However, these techniques require cell fixation, which precludes assessment of chromatin structure dynamics, and are generally restricted to population averages, thus masking cell-to-cell heterogeneity. By contrast, live-cell imaging allows to characterize and quantify the temporal dynamics of chromatin, potentially including TADs and loops in single cells. Specific chromatin loci can be visualized at high temporal and spatial resolution by inserting a repeat array from bacterial operator sequences bound by fluorescent tags. Using two different types of repeats allows to tag both anchors of a loop in different colors, thus enabling to track them separately even when they are in close vicinity. Here, we describe a versatile cloning method for generating many repeat array repair cassettes in parallel and inserting them by CRISPR-Cas9 into the human genome. This method should be instrumental to studying chromatin loop dynamics in single human cells.

FISH-quant v2: a scalable and modular tool for smFISH image analysis.

Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling quantitative studies of gene expression and its regulatory mechanisms. Today, these methods are applicable at a large scale, which in turn come with a need for adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular tool accessible for both experts and non-experts. Our user-friendly package allows the user to segment nuclei and cells, detect isolated RNAs, decompose dense RNA clusters, quantify RNA localization patterns and visualize these results both at the single-cell level and variations within the cell population. This tool was validated and applied on large-scale smFISH image data sets, revealing diverse subcellular RNA localization patterns and a surprisingly high degree of cell-to-cell heterogeneity.

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging.

Intermediate filaments (IFs) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49-nanometer axial repeat both in cells and in vitro. As unit-length filaments (ULFs) were measured at ~59 nanometers, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical, and structural properties in cells.

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner.

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75.

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context.

Super-resolution visualization and modeling of human chromosomal regions reveals cohesin-dependent loop structures.

BACKGROUND: The 3D organization of the chromatin fiber in cell nuclei plays a key role in the regulation of gene expression. Genome-wide techniques to score DNA-DNA contacts, such as Hi-C, reveal the partitioning of chromosomes into epigenetically defined active and repressed compartments and smaller "topologically associated" domains. These domains are often associated with chromatin loops, which largely disappear upon removal of cohesin. Because most Hi-C implementations average contact frequencies over millions of cells and do not provide direct spatial information, it remains unclear whether and how frequently chromatin domains and loops exist in single cells. RESULTS: We combine 3D single-molecule localization microscopy with a low-cost fluorescence labeling strategy that does not denature the DNA, to visualize large portions of single human chromosomes in situ at high resolution. In parallel, we develop multi-scale, whole nucleus polymer simulations, that predict chromatin structures at scales ranging from 5 kb up to entire chromosomes. We image chromosomes in G1 and M phase and examine the effect of cohesin on interphase chromatin structure. Depletion of cohesin leads to increased prevalence of loose chromatin stretches, increased gyration radii, and reduced smoothness of imaged chromatin regions. By comparison to model predictions, we estimate that 6-25 or more purely cohesin-dependent chromatin loops coexist per megabase of DNA in single cells, suggesting that the vast majority of the genome is enclosed in loops. CONCLUSION: Our results provide new constraints on chromatin structure and showcase an affordable non-invasive approach to study genome organization in single cells.

Single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.

Clustering and reverse transcription of HIV-1 genomes in nuclear niches of macrophages.

In order to replicate, human immunodeficiency virus (HIV-1) reverse-transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time, but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early viral replication cycle in macrophages, the natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA (vDNA) and viral RNA, in which multiple viral genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6, and localize to nuclear speckles. Surprisingly, these viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where reverse transcription is pharmacologically suppressed and in untreated cells. We demonstrate that following temporary inhibition, reverse transcription can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear reverse transcription can result in transcription-competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle and may have implications for addressing HIV-1 persistence.

A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting.

Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For ß-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

instaGRAAL: chromosome-level quality scaffolding of genomes using a proximity ligation-based scaffolder.

Hi-C exploits contact frequencies between pairs of loci to bridge and order contigs during genome assembly, resulting in chromosome-level assemblies. Because few robust programs are available for this type of data, we developed instaGRAAL, a complete overhaul of the GRAAL program, which has adapted the latter to allow efficient assembly of large genomes. instaGRAAL features a number of improvements over GRAAL, including a modular correction approach that optionally integrates independent data. We validate the program using data for two brown algae, and human, to generate near-complete assemblies with minimal human intervention.

How the Genome Folds: The Biophysics of Four-Dimensional Chromatin Organization.

The genetic information that instructs transcription and other cellular functions is carried by the chromosomes, polymers of DNA in complex with histones and other proteins. These polymers are folded inside nuclei five orders of magnitude smaller than their linear length, and many facets of this folding correlate with or are causally related to transcription and other cellular functions. Recent advances in sequencing and imaging-based techniques have enabled new views into several layers of chromatin organization. These experimental findings are accompanied by computational modeling efforts based on polymer physics that can provide mechanistic insights and quantitative predictions. Here, we review current knowledge of the main levels of chromatin organization, from the scale of nucleosomes to the entire nucleus, our current understanding of their underlying biophysical and molecular mechanisms, and some of their functional implications.