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Biology subjects

Tramier, M.

Publications and source records attributed to Tramier, M..

11 recordsLinked to original sources

Semi supervised GAN for smart microscopy, fast and data efficient cell cycle classification

Modern optical microscopes are fully motorised; however, transforming them into truly smart systems requires real-time adjustment of acquisition settings in response to detected objects and dynamic biological events. At the core are classification algorithms that commonly depend on customised softwares and are generally designed for narrowly-defined biological applications. In addition, they often require substantial annotated datasets for effective training. We introduce a semi-supervised generative adversarial network (SGAN) for robust cell-cycle stage classification under low-resource conditions, adaptable to diverse cellular structures. The framework combines unlabelled microscopy images with synthetically generated samples to mitigate limited annotation, while preserving stable performance even when the unlabelled subset is class-imbalanced. Tested on the Mitocheck dataset, which features five mitosis classes, the model achieved 93{+/-}2% accuracy using only 80 labelled per class and 600 unlabelled images. The proposed algorithm is generic and can be readily adapted to new labeling schemes, classification targets, cell lines, or microscopy modalities through transfer learning. SGAN is well suited for integration into automated microscopes, enabling efficient and adaptable image analysis across diverse biological and microscopy applications.

bioinformatics↗

Selective targeting of RalA with intrabodies impairs Triple Negative Breast Cancer metastasis

This study successfully developed and validated isoform-specific intrabodies targeting the highly homologous Ral oncoproteins, key effectors in cancer progression. Phage display was used to isolate single-chain variable fragment (scFv) clones that recognize specifically RalA (C1-A, G5-A), RalB (F6-B), or both paralogs (A12-AB). Using lentiviral transduction, these intrabodies were stably expressed as GFP fusions in murine breast cancer 4T1 cells. The anti-RalA clones C1-A and A12-AB demonstrated clear colocalization with RalA, confirming their binding activity inside the cell. We further confirmed their activity in cells by analyzing Ral-dependent pathways. All intrabodies targeting RalA (C1-A, G5-A, A12-AB) but not the one specific to RalB inhibited mitochondrial fission, a known RalA function. All clones, except the pan-Ral one, altered the endo-lysosome pathway by decreasing lysosome number. Furthermore, the RalA-specific C1-A clone reduced lysosomes size, and uniquely and strongly reduced extracellular vesicle secretion, highlighting its distinct inhibitory potential. In an orthotopic Triple-Negative Breast Cancer (TNBC) mouse model, The C1-A RalA specific clone significantly but weakly reduced primary tumor growth, but exerted a powerful anti-metastatic effect, dramatically reducing lung metastases, with a complete abolition of metastases observed in 2/5 mice. In summary, these potent, isoform-specific Ral intrabodies act as effective intracellular inhibitors, successfully modulating RalA-specific functions in cells and offering a promising therapeutic strategy for significantly suppressing tumor growth and metastasis in vivo.

cancer biology↗

The Roboscope: Smart and Fast Microscopy for Generic Event-Driven Acquisition

Automation of fluorescence microscopy is a challenge for capturing rare or transient events in biology and medicine. It relies on smart devices that integrate and interpret the observed data, and react to the targeted biological event. We report on the Roboscope, a novel autonomous microscope combining sequence interruption and deep learning integration, allowing generic event-driven acquisitions. This system distinguishes itself by its adaptability to various experiments, quick capture of dynamic events, and minimal data greediness - training with less than 100 images per class. The Roboscopes capability is demonstrated in non-synchronized cells by capturing the metaphase, a 20-minute event happening once per day or less. Conversely, double thymidine-block synchronisation, despite occurring during DNA replication, may perturb mitotic-spindle mechanics. The Roboscopes versatility and efficiency offer significant advancements to tackle the current challenges of cell biology, spreading out advanced microscopy methods to fundamental research as well as high content screening and precision medicine.

bioengineering↗

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells

Although several mechanisms of autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to its activation, the ATG4B protease primes the key autophagy player LC3B. Given the lack of reporters to follow this event in living cells, we developed a Forsters Resonance Energy Transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine/tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image allows to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are pools of unprimed LC3B upon ATG4B downregulation, and that the priming of the biosensor is abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Moreover, we screened for commercially-available ATG4B inhibitors, and we illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Finally, we uncovered the CDK1-dependent regulation of the ATG4B-LC3B axis at mitosis. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.

cell biology↗

tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

Yellow fluorescent proteins (YFP) are widely used as optical reporters in Forster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. Here, we characterize the yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol-1.L.cm-1, it is, to our knowledge, the brightest dimeric fluorescent protein available. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and in live cells. As a consequence, tdLanYFP allows imaging of cellular structures with sub-diffraction resolution using STED nanoscopy and is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP is valuable FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFP a very attractive yellow fluorescent protein for long-term or single-molecule live cell imaging including FRET experiments at acidic pH.

biophysics↗

Neural network fast-classifies biological images using features selected after their random-forests-importance to power smart microscopy.

Artificial intelligence is nowadays used for cell detection and classification in optical microscopy, during post-acquisition analysis. The microscopes are now fully automated and next expected to be smart, to make acquisition decisions based on the images. It calls for analysing them on the fly. Biology further imposes training on a reduced dataset due to cost and time to prepare the samples and have the datasets annotated by experts. We propose here a real-time image processing, compliant with these specifications by balancing accurate detection and execution performance. We characterised the images using a generic, high-dimensional feature extractor. We then classified the images using machine learning for the sake of understanding the contribution of each feature in decision and execution time. We found that the non-linear-classifier random forests outperformed Fishers linear discriminant. More importantly, the most discriminant and time-consuming features could be excluded without any significant loss in accuracy, offering a substantial gain in execution time. It suggests a feature-group redundancy likely related to the biology of the observed cells. We offer a method to select fast and discriminant features. In our assay, a 79.6 {+/-} 2.4 % accurate classification of a cell took 68.7 {+/-} 3.5 ms (mean {+/-} SD, 5-fold cross-validation nested in 10 bootstrap repeats), corresponding to 14 cells per second, dispatched into 8 phases of the cell cycle using 12 feature-groups and operating a consumer market ARM-based embedded system. Interestingly, a simple neural network offered similar performances paving the way to faster training and classification, using parallel execution on a general-purpose graphic processing unit. Finally, this strategy is also usable for deep neural networks paving the way to optimising these algorithms for smart microscopy.

bioinformatics↗

Mitochondrial Aurora kinase A induces mitophagy by interacting with MAP1LC3 and Prohibitin 2

Epithelial and haematologic tumours often show the overexpression of the serine/threonine kinase AURKA. Recently, AURKA was shown to localise at mitochondria, where it regulates mitochondrial dynamics and ATP production. Here we define the molecular mechanisms of AURKA in regulating mitochondrial turnover by mitophagy. When overexpressed, AURKA induces the disappearance of Outer Mitochondrial Membrane proteins by the proteasome. Then, AURKA triggers the degradation of Inner Mitochondrial Membrane (IMM)/matrix proteins by interacting with core components of the autophagy pathway. On the IMM, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2. This complex is necessary to trigger mitophagy in a PARK2/Parkin-independent manner. The formation of the tripartite complex is induced by the phosphorylation of PHB2 on Ser39, which is required for MAP1LC3 to interact with PHB2. Last, treatment with the PHB2 ligand Xanthohumol blocks AURKA-induced mitophagy by destabilising the tripartite complex. This treatment also restores normal ATP production levels. Altogether, these data provide evidence for a previously undetected role of AURKA in promoting mitophagy through the interaction with PHB2 and MAP1LC3. This work paves the way to the use of function-specific pharmacological inhibitors to counteract the effects of the overexpression of AURKA in cancer.

cell biology↗

Optimised FRET pairs and quantification approaches to detect the activation of Aurora kinase A at mitosis

Genetically-encoded Forsters Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signalling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must be optimised to monitor multiple biochemical activities simultaneously and in single cells. In addition, their sensitivity must be increased to follow their activation even when the abundance of the biosensor is low. We describe here the development of a second generation of Aurora kinase A/AURKA biosensors. First, we adapt the original AURKA biosensor -GFP-AURKA-mCherry- to multiplex FRET by using dark acceptors as ShadowG or ShadowY. Then, we use the novel superYFP acceptor protein to measure FRET by 2-colour Fluorescence Cross-Correlation Spectroscopy, in cytosolic regions where the abundance of AURKA is extremely low and undetectable with the original AURKA biosensor. These results pave the way to the use of FRET biosensors to follow AURKA activation in conjunction with substrate-based activity biosensors. In addition, they open up the possibility of tracking the activation of small pools of AURKA and its interaction with novel substrates, which would otherwise remain undetectable with classical biochemical approaches.

cell biology↗

The RNA-binding proteins CELF1 and ELAVL1 cooperatively control alternative splicing

ELAVL1 and CELF1 are RNA-binding proteins that are involved in alternative splicing control. To explore their functional relationship, we looked for mRNAs that are differentially spliced following the depletion of CELF1, ELAVL1, or both. We found that these proteins control the usage of their target exons in the same direction. Double depletion has a greater effect than individual depletions, showing that CELF1 and ELAVL1 exert additive control. To confirm these results, we carried out RT-PCR on the alternative cassette exons of several mRNAs, including CD44, WNK1, PHACTR2, MICAL3, SPTBN1, and PPP3CB. Using FRET, we found that CELF1 and ELAVL1 directly interact in cell nuclei. We demonstrated that the combined levels of CELF1 and ELAVL1 are a valuable biomarker in several cancers, even when their individual levels may yield very limited information. CD44 alternative splicing probably accounts in part for the effects of CELF1 and ELAVL1 levels on patient survival. These data point to strong functional interactions between CELF1 and ELAVL1 in the control of mRNA isoform production, resulting in significant impacts on human pathology.

molecular biology↗

Chromatin Condensation Fluctuations Rather Than Steady-State Predict Chromatin Accessibility

Chromatin accessibility to protein factors is critical for genome activities. Dynamic changes in nucleosomal DNA compaction and higher order chromatin structures are expected to allow specific sites to be accessible to regulatory factors and the transcriptional machinery. However, the dynamic properties of chromatin that regulate its accessibility are poorly understood. Here, we took advantage of the microenvironment sensitivity of the fluorescence lifetime of EGFP-H4 histone incorporated in chromatin to map in the nucleus of live cells the dynamics of chromatin condensation and its direct interaction with a tail acetylation recognition domain (the double bromodomain module of human TAFII250, dBD). We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation. Fluctuations are spontaneous, yet their amplitudes are affected by their sub-nuclear localization and by distinct and competing mechanisms dependent on histone acetylation, ATP, and both. Moreover, we show that accessibility of acetylated histone H4 to dBD is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations.\n\nSignificanceIn higher eukaryotes, the structure and compaction of chromatin are considered as barriers to genome activities. Epigenetic marks such as post-translational modifications of histones can modify the structure and compaction of chromatin. The accessibility of protein factors to these epigenetic marks is therefore of paramount importance for genome activities. We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation itself. We show that accessibility of acetylated histone H4 to double bromodomains is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations.\n\nClassificationBiological Sciences, Cell Biology

cell biology↗

Dynamics Of Dynein At Microtubule Plus-Ends And The Cortex During The Division Of The C. elegans Zygote

During asymmetric cell division, dynein generates forces, which position the spindle to reflect polarity and ensure correct daughter cell fates. The transient cortical localization of dynein raises the question of its targeting. We found that it accumulates at the microtubule plus-ends like in budding yeast, indirectly hitch-hiking on EBP-2EB1 likely via dynactin. Importantly, this mechanism, which modestly accounts for cortical forces, does not transport dynein, which displays the same binding/unbinding dynamics as EBP-2EB1. At the cortex, dynein tracks can be classified as having either directed or diffusive-like motion. Diffusive-like tracks reveal force-generating dyneins. Their densities are higher on the posterior tip of the embryos, where GPR-1/2LGN concentrate, but their durations are symmetric. Since dynein flows to the cortex are non-polarized, we suggest that this posterior enrichment increases dynein binding, thus accounts for the force imbalance reflecting polarity, and supplements the regulation of mitotic progression via the non-polarized detachment rate.

biophysics↗