Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis.

Loss-of-function (LOF) methods such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing provide unparalleled power for studying the biological function of genes of interest. However, a major concern is non-specific targeting, which involves depletion of transcripts other than those intended. Little work has been performed to characterize the off-target effects of these common LOF methods at the whole-transcriptome level. Here, we experimentally compared the non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible off-target effects in gene expression, with CRISPRi also exhibiting strong clonal effects. As an illustrative example, we evaluated the performance of each method for determining the role of an uncharacterized long noncoding RNA (lncRNA). Several LOF methods successfully depleted the candidate lncRNA but yielded different sets of differentially expressed genes as well as a different cellular phenotype upon depletion. Similar discrepancies between methods were observed with a protein-coding gene (Ch-TOG/CKAP5) and another lncRNA (MALAT1). We suggest that the differences between methods arise due to method-specific off-target effects and provide guidelines for mitigating such effects in functional studies. Our recommendations provide a framework with which off-target effects can be managed to improve functional characterization of genes of interest.

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level.

The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2 or M phase but eventually advanced through mitosis into the next interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development.

STED microscopy: A simplified method for liver sinusoidal endothelial fenestrae analysis.

BACKGROUND INFORMATION: Liver sinusoidal endothelial cells (LSECs) possess fenestrae, open transcellular pores with an average diameter of 100 nm. These fenestrae allow for the exchange between blood and hepatocytes. Alterations in their number or diameter in liver diseases have important implications for hepatic microcirculation and function. Although decades of studies, fenestrae are still observed into fixed cells and we have poor knowledge of their dynamics. RESULTS: Using stimulated emission depletion (STED) super-resolution microscopy, we have established a faster and simplest method to observe and quantify fenestrae. Indeed, using cytochalasin D, an actin depolymerising agent known to promote fenestrae formation, we measure the increase of fenestrae number. We adapted this methodology to develop an automated method to study fenestrae dynamics. Moreover, with two-colour STED analysis, we have shown that this approach could be useful to study LSECs fenestrae molecular composition. CONCLUSIONS: Our approach demonstrates that STED microscopy is suitable for LSEC fenestrae study. SIGNIFICANCE: This new way of analysing LSEC fenestrae will allow for expedited investigation of their dynamics, molecular composition and functions to better understand their function in liver pathophysiology.

Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Single Particle Tracking reveals two distinct environments for CD4 receptors at the surface of living T lymphocytes.

We investigated the lateral diffusion of the HIV receptor CD4 at the surface of T lymphocytes at 20°C and 37°C by Single Particle Tracking using Quantum Dots. We found that the receptors presented two major distinct behaviors that were not equally affected by temperature changes. About half of the receptors showed a random diffusion with a diffusion coefficient increasing upon raising the temperature. The other half of the receptors was permanently or transiently confined with unchanged dynamics on raising the temperature. These observations suggest that two distinct subpopulations of CD4 receptors with different environments are present at the surface of living T lymphocytes.

A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division.

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.

Which Elements to Build Co-localization Workflows? From Metrology to Analysis.

Co-localization analysis is one of the main interests of users entering a facility with slides in hands and nice analysis perspectives in mind. While being available through most, if not all, analysis software, co-localization tools are mainly perceived as black boxes, fed with images, that will, hopefully, return (the expected) numbers.In this chapter, we will aim at deconstructing existing generic co-localization workflows, extracting elementary tools that may be reused and recombined to generate new workflows. By differentiating work cases, identifying co-localization reporters and the metrics others have been using, we aim at providing the audience with the elementary bricks and methods to build their really own co-localization workflows. A special emphasis is given on the preparatory phase where the acquisition system is assessed, using basic metrological tests.

Actin Depolymerization in Dedifferentiated Liver Sinusoidal Endothelial Cells Promotes Fenestrae Re-Formation.

Liver sinusoidal endothelial cells (LSECs) possess fenestrae, which are key for the exchange between blood and hepatocytes. Alterations in their number or diameter have important implications for hepatic function in liver diseases. They are lost early in the development of hepatic fibrosis through a process called capillarization. In this study, we aimed to demonstrate whether in vitro dedifferentiated LSECs that have lost fenestrae are able to re-form these structures. Using stimulated emission depletion super-resolution microscopy in combination with transmission electron microscopy, we analyzed fenestrae formation in a model mimicking the capillarization process in vitro. Actin is known to be involved in fenestrae regulation in differentiated LSECs. Using cytochalasin D, an actin-depolymerizing agent, we demonstrated that dedifferentiated LSECs remain capable of forming fenestrae. Conclusion: We provide a new insight into the complex role of actin in fenestrae formation and in the control of their size and show that LSEC fenestrae re-formation is possible, suggesting that this process could be used during fibrosis regression to try to restore exchanges and hepatocyte functions.

Synthetic lethality between androgen receptor signalling and the PARP pathway in prostate cancer.

Emerging data demonstrate homologous recombination (HR) defects in castration-resistant prostate cancers, rendering these tumours sensitive to PARP inhibition. Here we demonstrate a direct requirement for the androgen receptor (AR) to maintain HR gene expression and HR activity in prostate cancer. We show that PARP-mediated repair pathways are upregulated in prostate cancer following androgen-deprivation therapy (ADT). Furthermore, upregulation of PARP activity is essential for the survival of prostate cancer cells and we demonstrate a synthetic lethality between ADT and PARP inhibition in vivo. Our data suggest that ADT can functionally impair HR prior to the development of castration resistance and that, this potentially could be exploited therapeutically using PARP inhibitors in combination with androgen-deprivation therapy upfront in advanced or high-risk prostate cancer.Tumours with homologous recombination (HR) defects become sensitive to PARPi. Here, the authors show that androgen receptor (AR) regulates HR and AR inhibition activates the PARP pathway in vivo, thus inhibition of both AR and PARP is required for effective treatment of high risk prostate cancer.