Biology subjects

Bertolin, G.

Publications and source records attributed to Bertolin, G..

13 recordsLinked to original sources

bioRxiv2026

TERT interacts with TFAM to activate mitochondrial fragmentation and auto/mitophagy

Previous studies have demonstrated that Telomerase Reverse Transcriptase (TERT) is localized inside mitochondria, where it associates with mitochondrial DNA (mtDNA), thereby influencing replication and transcription. Using a combination of quantitative fluorescence microscopy and biochemistry, we demonstrate that TERT binds indirectly to mtDNA through the interaction with mitochondrial transcription factor A (TFAM) to induce mitochondrial fragmentation. These findings reveal a novel mitochondrial interaction involving TERT and highlight a role for TERT and TFAM in the regulation of auto/mitophagy. This interaction is an important factor for mitochondrial function and contributes to the remodelling of mitochondrial respiratory capacity.

cell biology↗

bioRxiv2026

Mitochondrial inner membrane interactors of Aurora kinase A/AURKA and PHB2 shape organelle metabolic heterogeneity.

Mitochondria support tumor growth by flexibly rewiring their own activity. Yet, how signaling networks organize this metabolic diversity across and within cells remains poorly understood. Here, we uncover a platform of inner mitochondrial membrane proteins that couple Aurora kinase A (AURKA) and the mitophagy receptor Prohibitin-2 (PHB2) to the local control of ATP production and organelle architecture. Using proximity interactomics and live-cell FRET/FLIM microscopy, we identify NDUFA9, ATP5F1A/B, SAMM50, and SLC25A13 as shared AURKA/PHB2 interactors positioned at respiratory chain and cristae-maintaining sites. We show that AURKA overexpression profoundly rewires the interactomes of NDUFA9 and ATP5F1A without disrupting their nanoscale proximity in the inner membrane, and reshapes mitochondrial morphology while preserving these signaling hubs. The AURKA/PHB2-targeting compound HMBB restores NDUFA9 and ATP5F1A interactomes, indicating that these complexes act as key nodes for AURKA-dependent metabolic adaptation at the population level. Finally, single-cell FRET microscopy coupled to super-resolution imaging reveals that SLC25A13 is required to sustain AURKA-induced metabolic heterogeneity within individual cancer cells. Our work links a multifunctional kinase, a mitophagy receptor, and respiratory complexes into a common inner membrane platform that spatially drives mitochondrial heterogeneity, with implications for metabolism-focused anticancer strategies.

cell biology↗

bioRxiv2026

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↗

bioRxiv2025

Combining FRET and super-resolution microscopy reveals kinase activation and mitochondrial activity at the nanoscale

Protein kinases are key regulators of intracellular signaling, showing activities in specific subcellular compartments and in micro- or nano-domains. Genetically encoded biosensors based on Forsters Resonance Energy Transfer (FRET) are powerful tools to track kinase dynamics. Yet, they are typically limited by spatial resolution and remain challenging to implement with super-resolution microscopy approaches that require specific fluorophores or optical setups. Aurora kinase A (AURKA), a multifunctional serine/threonine kinase, has recently emerged as a critical regulator of mitochondrial physiology. However, visualising AURKA activation and activity in living cells with sub-diffraction precision remains a challenge. Here, we introduce BioSenSRRF, a versatile approach that combines conventional FRET biosensors with Super-Resolution Radial Fluctuations (SRRF) microscopy. BioSenSRRF requires no modification of existing probes, can be implemented using standard microscopy setups, and is supported by a publicly available ImageJ framework to streamline image processing and data analysis. Using this pipeline, we imaged AURKA activation at mitochondria and its activity in regulating mitochondrial ATP levels. We uncover that AURKA activation and activity are compartmentalised into distinct mitochondrial domains containing the mitochondrial ATP synthase. These subdomains depend on the catalytic activity of AURKA, and they can be altered using previously validated AURKA inhibitors. Finally, we demonstrate that the cancer-associated polymorphism F31I enhances AURKA activation and ATP production on ATP synthase-enriched subdomains, underscoring its pathological relevance. Altogether, BioSenSRRF provides a broadly accessible framework to enhance the spatial resolution of genetically encoded biosensors. This strategy opens new avenues for dissecting the subcellular organisation of protein complexes and their contribution to physiology and disease states.

cell biology↗

bioRxiv2024

Sex-specific loss of mitochondrial membrane integrity and mass in the auditory brainstem of a mouse model of Fragile X syndrome

Sound sensitivity is one of the most common sensory complaints for people with autism spectrum disorders (ASDs). How and why sounds are perceived as overwhelming by affected people is unknown. To process sound information properly, the brain requires high activity and fast processing, as seen in areas like the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem. Recent work has shown dysfunction in mitochondria, which are the primary source of energy in cells, in a genetic model of ASD, Fragile X syndrome (FXS). Whether mitochondrial functions are also altered in sound-processing neurons, has not been characterized yet. To address this question, we imaged the MNTB in a mouse model of FXS. We stained MNTB brain slices from wild-type and FXS mice with two mitochondrial markers, TOMM20 and PMPCB, located on the Outer Mitochondrial Membrane and in the matrix, respectively. These markers allow exploration of mitochondrial subcompartments. Our integrated imaging pipeline reveals significant sex-specific differences between genotypes. Colocalization analyses between TOMM20 and PMPCB reveal that the integrity of mitochondrial subcompartments is most disrupted in female FXS mice compared to female wildtype mice. We highlight a quantitative fluorescence microscopy pipeline to monitor mitochondrial functions in the MNTB from control or FXS mice and provide four complementary readouts. Our approach paves the way to understanding how cellular mechanisms important to sound encoding are altered in ASDs.

neuroscience↗

bioRxiv2024

Development of capsaicin derivatives as prohibitin ligands to modulate the Aurora kinase A/PHB2 interaction in cancer cells

Aurora kinase A/AURKA is a serine/threonine kinase overexpressed in a variety of solid and hematological malignancies. In the last decades, clinical trials aiming to counteract the overexpression of AURKA turned out to be largely unsuccessful. Meanwhile, recent discoveries pointed to new functions of AURKA at the subcellular level, including at mitochondria. At this location, AURKA induces organelle clearance by mitophagy acting in complex with the mitophagy mediator LC3, and its inner mitochondrial membrane receptor PHB2. The natural polyphenol xanthohumol was shown to act as a PHB2 ligand, altering the interaction between AURKA and PHB2 and restoring mitochondrial functions in cancer cells. However, its chemical nature prevents its broader use as an anticancer agent. Using Forsters Resonance Energy Transfer/Fluorescence Lifetime Imaging Microscopy (FRET/FLIM) in live breast cancer cells, we here explore the effects of alternative PHB ligands in altering the proximity between AURKA and PHB2. Among the already-available compounds, we found that the pungent natural product capsaicin partially alters the AURKA/PHB2 protein-protein proximity. We then synthesized 16 novel capsaicin analogs to enhance the effects of capsaicin. We found that replacing the long hydrophobic acyl moiety with a butyryl one increases the AURKA/PHB2 interaction. Among the capsaicin derivatives carrying this modification, we uncover that compounds 12 and 13 enhance the AURKA/PHB2 proximity. Molecular docking approaches corroborate FRET/FLIM data, and we visualize compounds 12 and 13 in complex with AURKA, PHB2 and LC3. We show that compounds 12 and 13 stabilize the AURKA/PHB2 interaction, and that they can bind to the inhibitory pocket of PHB2 and to the AURKA active site. Finally, we report that compound 13 specifically inhibits AURKA-dependent mitophagy, while leaving the activation of AURKA unaltered at centrosomes. Together, our data demonstrate that compound 13 is a promising PHB ligand acting on the AURKA/PHB2 interaction. Thanks to its specificity toward the mitochondrial roles of AURKA, it may provide the basis for the development of new anticancer drugs targeting the mitochondrial functions of AURKA.

cell biology↗

bioRxiv2023

Aurora kinase A/AURKA interacts with the mitochondrial ATP synthase to regulate energy metabolism and cell death

Cancer cells often hijack metabolic pathways to obtain the energy required to sustain their proliferation. Understanding the molecular mechanisms underlying cancer cell metabolism is key to fine-tune the metabolic preference of specific tumors, and potentially offer new therapeutic strategies. Here, we show that the pharmacological inhibition of mitochondrial Complex V delays the cell cycle by arresting breast cancer cell models in the G0/G1 phase. Under these conditions, the abundance of the multifunctional protein Aurora kinase A/AURKA is specifically lowered. We then demonstrate that AURKA directly interacts with the mitochondrial Complex V core subunits ATP5F1A and ATP5F1B. Altering the AURKA/ATPF1A/ATPF1B nexus is sufficient to trigger G0/G1 arrest, and this is accompanied by decreased glycolysis and mitochondrial respiration rates. Last, we discover that the roles of the AURKA/ATPF1A/ATPF1B nexus depend on the specific metabolic propensity of triple-negative breast cancer cell lines, where they correlate with cell fate. On one hand, the nexus induces G0/G1 arrest in cells relying on oxidative phosphorylation as the main source of energy. On the other hand, it allows to bypass cell cycle arrest and it triggers cell death in cells with a glycolytic metabolism. Altogether, we provide evidence that AURKA and mitochondrial Complex V subunits cooperate to maintain cell metabolism in breast cancer cells. Our work paves the way to novel anti-cancer therapies targeting the AURKA/ATPF1A/ATPF1B nexus to lower cancer cell metabolism and proliferation.

cell biology↗

bioRxiv2022

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↗

bioRxiv2021

High-resolution reconstruction and deconvolution of array detector images

Array detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high resolution image by linearly combining sub-detector images. To overcome the limitations of these techniques, we propose a new reconstruction method that takes the full stack of spatially reassigned detector signals as input. We show on both calibration slides and real data that our deconvolution method allows to achieve a better reconstruction performance in terms of resolution, image contrast, and spatial intensity homogeneity. The tested algorithms are available in an open source software.

bioinformatics↗

bioRxiv2021

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↗

bioRxiv2020

Quantitative dSTORM super-resolution microscopy localizes Aurora kinase A/AURKA in the mitochondrial matrix

Mitochondria are dynamic organelles playing essential metabolic and signaling functions in cells. Their ultrastructure has largely been investigated with electron microscopy (EM) techniques, which provided a wide range of information on how mitochondria acquire a tissue-specific shape, how they change during development, and how they are altered in disease conditions. However, quantifying protein-protein proximities using EM is extremely challenging. Super-resolution microscopy techniques as direct stochastic optical reconstruction microscopy (dSTORM) now provide a fluorescent-based alternative to EM with a higher quantitative throughput. Recently, super-resolution microscopy approaches including dSTORM led to valuable advances in our knowledge of mitochondrial ultrastructure, and in linking it with new insights in organelle functions. Nevertheless, dSTORM is currently used to image integral mitochondrial proteins only, and there is little or no information on proteins transiently present at this compartment. The cancer-related Aurora kinase A/AURKA is a protein localized at various subcellular locations, including mitochondria. After performing dSTORM, we here use the Geo-coPositioning System (GcoPS) image analysis method to quantify the degree of colocalization of AURKA with compartment-specific mitochondrial markers. We show that two-color dSTORM provides sufficient spatial resolution to visualize AURKA in the mitochondrial matrix. We conclude by demonstrating that optimizing fixation procedures is a key step to follow AURKA in the matrix. In this light, we show that a methanol-based fixation leads to a better detection of the matrix pool of AURKA than an aldehyde-based fixation. Our results indicate that dSTORM coupled to GcoPS colocalization analysis is a suitable approach to explore the compartmentalization of non-integral mitochondrial proteins as AURKA, in a qualitative and quantitative manner. This method also opens up the possibility of analyzing the proximity between AURKA and its multiple mitochondrial partners with exquisite spatial resolution, thereby allowing novel insights into the mitochondrial functions controlled by AURKA.

cell biology↗

bioRxiv2020

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↗

bioRxiv2019

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↗