Abstract The MITF transcription factor and the RAS/RAF/MEK/ERK pathway are two interconnected main players in melanoma. Understanding how MITF activity is regulated represents a key question since its dynamic modulation is involved in the phenotypic plasticity of melanoma cells and their resistance to therapy. By investigating the role of ARAF in NRAS-driven melanoma through mass spectrometry experiments followed by a functional siRNA-based screen, we unexpectedly identified MITF as a direct ARAF partner. Interestingly, this interaction is conserved among the RAF protein kinase family since the formation of BRAF/MITF and CRAF/MITF complexes was also observed in the cytosol of NRAS-mutated melanoma cells. The interaction occurs through the kinase domain of RAF proteins and is correlated with their kinase activity level. RAF/MITF complexes modulate MITF nuclear localization by inducing an accumulation of MITF in the cytoplasm, thus negatively controlling its transcriptional activity. Taken together, our study highlights a new level of regulation between two major mediators of melanoma progression, MITF and the MAPK/ERK pathway, which appears more complex than previously anticipated.
Significance Intracellular transporters are key actors in cell biological processes. Their disruption causes major physiological defects. Intracellular ion transporters are usually thought to control luminal conditions in organelles; meanwhile, their potential action on cytosolic ion homeostasis is still a black box. The case of a plant Chloride Channel (CLC) is used as a model to uncover the missing link between the regulation of conditions inside the vacuole and inside the cytosol. The development of an original live imaging workflow to simultaneously measure pH and anion dynamics in the cytosol reveals the importance of an Arabidopsis thaliana CLC, AtCLCa, in cytosolic pH homeostasis. Our data highlight an unsuspected function of endomembrane transporters in the regulation of cytosolic pH.
Objectives The RNA epitranscriptomic modification known as N6 -methyladenosine (m 6 A) represents a novel mechanism of gene regulation that is poorly understood in human autoimmune diseases. Our research explores the role of this RNA m 6 A modification in salivary gland epithelial cells (SGEC) and its impact on the pathogenesis of Sjögren’s disease (SjD). Methods SGECs from SjD patients and controls were analysed for m 6 A writers METTL3 and METTL14 expression using RNA-seq, quantitative PCR and immunohistochemistry. Functional assays assessed the impact of METTL3 knockdown or pharmacological inhibition on proinflammatory gene expression and immune cell interactions (using transwell and coculture systems). Mechanistic studies examined METTL3-mediated m 6 A modifications in double-stranded RNA (dsRNA) formation through immunofluorescence. Unsupervised clustering identified patterns of interferon activation in salivary glands and their correlation with m 6 A writers. Results METTL3 and METTL14 were elevated in SGEC from SjD patients in comparison to controls. Paradoxically, inhibiting METTL3 increased proinflammatory gene expression, enhancing SGEC’s ability to attract immune cells and activate B cells. Conversely, inhibiting the eraser FTO had the opposite effect. METTL3-mediated m 6 A modifications prevented dsRNA formation and IFN signalling activation. SGEC from SjD showed insufficient METTL3 upregulation compared with controls in response to inflammatory triggers, indicating a limited capacity to regulate the inflammatory response. SjD patients with elevated disease activity and higher interferon signature exhibit reduced METTL3 expression. Conclusions Impairment of m 6 A modifications in SGEC in response to inflammatory triggers favour the formation of dsRNA, potentially amplifying the interferon loop and contributing to SjD pathogenesis.
In plants, ions fluxes are involved in several physiological processes such as nutrient uptake, signaling pathways, cell elongation and stomata movements. However, little information is available about the intracellular anionic concentrations and anion fluxes among plant cell compartments. In the last years some promising anion concentration sensors have been developed (ClopHensor, Arosio et al. 2010) but have never been used in plant cells. The ClopHensor sensor (Figure 1) is a genetically encoded non‐FRET based sensor developed in mammals cells. It is based on a modified GFP (E²GFP) that is sensitive to chloride and nitrate concentration, and coupled to a DsRed. The DsRed fluorescent protein is used as an internal control for the expression of the construction, because its fluorescence is insensitive to anions concentration and pH, and clearly separated in term of spectral properties. The E²GFP fluorescent protein, excited at 488 nm, is sensitive to pH and anions concentration. But when it excited at 458 nm, the fluorescent signal is only sensitive to anion concentration. Thus, we are able to obtain simultaneously the intracellular pH and anion concentration maps in the cytosol by calculating ratios of fluorescence emission intensity. To do that we used a scanning confocal microscope equipped with an argon laser which provides these laser lines. In the present collaboration work, Arabidopsis thaliana transgenic lines stably expressing ClopHensor in the cytosol were generated by Elsa Demes (phD student). We are currently performing the in vivo characterization of the ClopHensor sensor in Arabidopsis cells. Indeed, we are establishing the relationship between the fluorescent signal, and pH or anion concentration values, by generating in cellulo calibration curves. This work will lead to the creation of both pH and anions maps to study those parameters at the cell level.
Abstract BACKGROUND Glioblastoma (GB) is one of the deadliest types of human cancer. Despite a very aggressive treatment regime, including resection of the tumor, radiation, and chemotherapy, the recurrence rate is more than 90%. Recurrence is mostly caused by the regrowth of highly invasive and resistant cells that have spread from the tumor bulk and are not removed by resection. To develop an effective therapeutic approach, we need to better understand the underlying molecular and cellular mechanisms of GB chemoradiation resistance and tumor spreading. MATERIAL AND METHODS To dynamically follow the changes occurring in GB post-therapy and investigate its relationship with vascular microenvironment, we employed multiple bulk and single-cell RNA-Seq analyses, phosphoproteome, in vitro and in vivo real-time imaging, organotypic cultures and functional assays, digital pathology, and spatial transcriptomics on patient material or preclinical models of GB. RESULTS We demonstrated that chemoradiation and the brain vasculature induce a transition to a functional cell state, which we named VC-Resist. This cell state is midway through the transcriptomic axis between proneural and mesenchymal GB cells and is closer to the AC/MES-like state. Better cell survival, G2M-arrest, activation of senescence/stemness pathways make this GB cell state more resistant to therapy. Notably, these persister GB cells are highly vessel co-opting, allowing homing to the perivascular niche, which, in turn, increases their transition to this cell state and resistance to therapy. Molecularly, the transition to the VC-Resist cell state is driven by FGF-FGFR1 signaling, which leads to the activation of DNA damage repair and YAP1 pathways. CONCLUSION These findings demonstrate that the perivascular niche and GB cell plasticity jointly generate a vicious loop that leads to resistance to therapy and brain infiltration during GB recurrence. SUPPORT This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 (Grant Agreement No. 805225), the INSERM-CNRS ATIP-Avenir grant, the NanoTheRad grant from Paris-Saclay University, Fondation ARC pour la recherche sur le cancer, Campus France and Canceropole Ile-de-France (2022-1-EMERG-06-ICR-1).
ABSTRACT The SUMO-targeted ubiquitin ligase (STUbL) family is involved in multiple cellular processes via a wide range of mechanisms to maintain genome stability. One of the evolutionarily conserved functions of STUbL is to promote changes in the nuclear positioning of DNA lesions, targeting them to the nuclear periphery. In Schizossacharomyces pombe, the STUbL Slx8 is a regulator of SUMOylated proteins and promotes replication stress tolerance by counteracting the toxicity of SUMO conjugates. In order to study the dynamic dialectic between ubiquitinylation and SUMOylation in the nuclear space of the S. pombe genome, we analyzed Slx8 localization. Unexpectedly, we did not detect replication stress-induced Slx8 foci. However, we discovered that Slx8 forms a single nuclear focus, enriched at the nuclear periphery, which marks both clustered centromeres at the spindle pole body and the silent mating-type region. The formation of this single Slx8 focus requires the E3 SUMO ligase Pli1, poly-SUMOylation and the histone methyl transferase Clr4 that is responsible for the heterochromatin histone mark H3-K9 methylation. Finally, we established that Slx8 promotes centromere clustering and gene silencing at heterochromatin domains. Altogether, our data highlight evolutionarily conserved and functional relationships between STUbL and heterochromatin domains to promote gene silencing and nuclear organization.
Abstract The MITF transcription factor and the RAS/RAF/MEK/ERK pathway are two interconnected main players in melanoma. Understanding how MITF activity is regulated represents a key question since its dynamic modulation is involved in the phenotypic plasticity of melanoma cells and their resistance to therapy. By investigating the role of ARAF in NRAS-driven mouse melanoma through mass spectrometry experiments followed by a functional siRNA-based screen, we unexpectedly identified MITF as a direct ARAF partner. Interestingly, this interaction is conserved among the RAF protein kinase family since BRAF/MITF and CRAF/MITF complexes were also observed in the cytosol of NRAS-mutated mouse melanoma cells. The interaction occurs through the kinase domain of RAF proteins. Importantly, endogenous BRAF/MITF complexes were also detected in BRAF-mutated human melanoma cells. RAF/MITF complexes modulate MITF nuclear localization by inducing an accumulation of MITF in the cytoplasm, thus negatively controlling its transcriptional activity. Taken together, our study highlights a new level of regulation between two major mediators of melanoma progression, MITF and the MAPK/ERK pathway, which appears more complex than previously anticipated.
Abstract Nuclear pores complexes (NPCs) are genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and acting as docking sites for DNA repair. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function occurs independently of SUMO chains formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect RDR dynamics in distinct ways. Our work probes the mechanisms by which the NPC environment ensures optimal RDR. Highlights ● Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, in a SUMO chain-dependent manner ● Proteasome-associated NPCs foster the progression of restarted DNA synthesis, in a SUMO chain-independent manner ● The nucleoporin Nup60 promotes the spatial sequestration of Ulp1 at the nuclear periphery ● Ulp1 and proteasome activities are differently required for optimal recombination-mediated fork restart.
Here, we present a protocol for multivariate quantitative-image-based cytometry (QIBC) analysis by fluorescence microscopy of asynchronous adherent cells. We describe steps for the preparation, treatment, and fixation of cells, sample staining, and imaging for QIBC. We then detail image analysis with our open source Fiji script developed for QIBC and present multiparametric data visualization. Our QIBC Fiji script integrates modern artificial-intelligence-based tools, applying deep learning, for robust automated nuclei segmentation with minimal user adjustments, a major asset for efficient QIBC analysis. For complete details on the use and execution of this protocol, please refer to Besse et al. (2023).
ABSTRACT Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-end-binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana. However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution stimulated emission depletion (STED) microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules. This article has an associated First Person interview with the first author of the paper.
