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The AURKA biosensor rescues the phenotype induced by AURKA deficiency and it is active at discrete subcellular locations.: (a) (Left panels) Immunofluorescent micrographs of non-transfected, GFP-AURKA and GFP-AURKA-mCherry stable U2OS cells silenced for endogenous AURKA and synchronized in metaphase. The mitotic spindle and centrosomal defects in cells following AURKA depletion and the corresponding rescue by GFP-AURKA and by GFP-AURKA-mCherry were detected by labelling the mitotic spindle with TUBA1A and the centrosomes with TUBG1. DNA was stained with DAPI. (Right panel) Quantification of the proportion of cells with centrosomal defects in the three cell lines transfected with a control- or an AURKA-specific siRNA. n=100 cells per condition scored in each of three independent experiments. (b) Representative fluorescence (GFP channel) and corresponding lifetime images of GFP-AURKA and GFP-AURKA-mCherry U2OS stable cell lines not synchronized (left panels) or synchronized in mitosis (right panels) and illustrating the presence of both proteins at the centrosome, in the cytosol and at mitotic spindles. Graphs: corresponding quantification of the lifetime of EGFP in the two cell lines and in the indicated subcellular compartments. Centrosomes were labelled with CETN1-iRFP670. n=30–40 cells per condition from three independent experiments. (c) Quantification of the lifetime of EGFP GFP-AURKA and GFP-AURKA-mCherry cells synchronized at mitosis and treated with dimethylsulfoxide (DMSO) or with MLN8237. n=30–40 cells per condition from three independent experiments. (d) (Left panels) Fluorescence (GFP channel) and lifetime images of GFP-AURKA and GFP-AURKA-mCherry cells transfected with an AURKA-specific siRNA and synchronized at mitosis. (Right panel) Quantification of EGFP lifetime in GFP-AURKA or in GFP-AURKA-mCherry cells transfected with a control- or an AURKA-specific siRNA and synchronized at mitosis; n=30–40 cells per condition from three independent experiments. Data represent means±s.e.m. Pseudocolour scale: pixel-by-pixel lifetime. Scale bar, 10 μm. ***P<0.001 against the corresponding ‘GFP-AURKA’ condition in a–d; aP<0.01 against the corresponding ‘GFP-AURKA-mCherry’ condition at the centrosome in b and the corresponding MLN8327 condition in c. NS, not significant. Statistical tests: two-way ANOVA in a,c and d; Student’s t-test in b.
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Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster’s resonance energy transfer...
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Citations
... Therefore, the conformational change in the protein can be studied by monitoring the laser intensity and threshold, so as to realize the study on spatiotemporal regulation and function of proteins. 59 See the supplementary material for the up-to-date FP lasers' parameters, the qualitative analysis of the redshift between the laser and the fluorescence spectrum, and the threshold energy derivation from the threshold energy density. ...
Biolasers show considerable potential in the biomedical field. Fluorescent protein (FP) is a type of biomaterial with good luminescence efficiency that can be used as the luminescent gain medium in biolasers. Due to the higher cell/tissue permeability, lower cell phototoxicity, and relatively less background fluorescence than other fluorescent proteins, the red fluorescent protein is more suitable in biological applications. MCherry is the most extensively used high-quality red fluorescent protein because of its short maturation time and stable luminescence properties. In this study, using mCherry and microbubble cavity, we realize a highly stable mCherry fluorescent protein laser. The laser resonator achieves a quality factor of 108, which is the highest Q factor among the currently available FP lasers. Moreover, this laser exhibits a low threshold of 1.15 μJ/mm2, which can effectively protect the luminescent material from being damaged by pump light. The prepared laser shows excellent stability in a wide pH range with good photobleaching resistance and can be stored at 4 °C for 30 days. Also, the laser can serve as a high-sensitivity molecular concentration detector with mCherry as biomarker, owing to its lasing threshold behavior.
... For control conditions, cells were stimulated with dimethyl sulfoxide (DMSO) to a final concentration of ≤0.1%. The siRNA against AURKA was synthesized and purchased from Eurogenetec (sequence: 5'-AUGCCCUGUCUUACUGUCA-3'), as previously described [39], while siRNAs against ATP5F1A (SI04989873), ATP5F1B (SI02626722), and Allstars negative control (SI03650318) were purchased from Qiagen. SiRNA transfections were done using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) following the manufacture's protocol. ...
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 functionally interacts with the mitochondrial Complex V core subunits ATP5F1A and ATP5F1B. Altering the AURKA/ATP5F1A/ATP5F1B 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/ATP5F1A/ATP5F1B 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/ATP5F1A/ATP5F1B nexus to lower cancer cell metabolism and proliferation.
... The emission wavelengths were 525-565 nm for tdLanYFP, and 650-720 nm for LAMP2-Alexa Fluor 647. For FLIM analyses, images were acquired with a time-gated custom-made setup based on a spinning disk microscope as described in [71]. Aquamarine was used as a FRET donor in all experiments, and excited at 440 ± 10 nm with a supercontinuum picosecond pulsed laser source. ...
Although several mechanisms of macroautophagy/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 MAP1LC3B/LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster's 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 makes it possible 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 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 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.Abbreviations: Aqua: aquamarine; ATG: autophagy related; AURKA: aurora kinase A; BafA1: bafilomycin A1; CDK1: cyclin dependent kinase 1; DKO: double knockout; FLIM: fluorescence lifetime imaging microscopy; FP: fluorescence protein; FRET: Förster's resonance energy transfer; GABARAP: GABA type A receptor-associated protein; HBSS: Hanks' balanced salt solution; KO: knockout; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSC: NSC 185058; PE: phosphatidylethanolamine; SKO: single knockout; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type; ZPCK: Z-L-phe chloromethyl ketone.
... Spatiotemporal regulation of AURKA's activity is multifactorial. A growing body of evidences suggests the following two main pathways that active AURKA: (i) the phosphorylation of conserved Thr288 residing on the activation segment or the (ii) interaction with co-factor proteins (e.g., TPX2) that induces the structural rearrangement competent for the phosphotransfer activity [37][38][39]88,[90][91][92][93][94][95]. Although the phosphorylation of Thr288 and TPX2 binding synergistically activate AURKA in vitro [91,96], two distinct pathways seem to work independently in an intracellular environment [23,[97][98][99]. ...
Aurora kinase A (AURKA), which is a member of serine/threonine kinase family, plays a critical role in regulating mitosis. AURKA has drawn much attention as its dysregulation is critically associated with various cancers, leading to the development of AURKA inhibitors, a new class of anticancer drugs. As the spatiotemporal activity of AURKA critically depends on diverse intra- and inter-molecular factors, including its interaction with various protein cofactors and post-translational modifications, each of these pathways should be exploited for the development of a novel class of AURKA inhibitors other than ATP-competitive inhibitors. Several lines of evidence have recently shown that redox-active molecules can modify the cysteine residues located on the kinase domain of AURKA, thereby regulating its activity. In this review, we present the current understanding of how oxidative modifications of cysteine residues of AURKA, induced by redox-active molecules, structurally and functionally regulate AURKA and discuss their implications in the discovery of novel AURKA inhibitors.
... For control conditions, cells were stimulated with dimethyl sulfoxide (DMSO) to a final concentration of ≤0.1%. The siRNA against AURKA was synthesized and purchased from Eurogenetec (sequence: 5'-AUGCCCUGUCUUACUGUCA-3'), as previously described (Bertolin et al., 2016) ...
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.
... Taken together, our results indicate that mutation in R 371 prevents the active, degradable conformation of AURKA. An autoinhibitory state involving interaction between the kinase domain and the N-terminus of AURKA has previously been described (Zhang et al, 2007;Bai et al, 2014), and studies with a FRET-based conformational sensor confirm that the relative configuration of N-and C-terminus is altered upon activation of the kinase (Bertolin et al, 2016). Because interaction with APC/C FZR1 occurs through an N-terminal degron motif, we propose that the Q 45 xxL D-box is "buried" in the inactive conformation, which may be the previously described autoinhibited state mediated through interaction between N-and C-terminal domains (Fig 5B). ...
Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the anaphase-promoting complex (APC/C FZR1 ) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the “A-box” in addition to a reported canonical D-box degron in the C-terminus. Here, we find that the reported C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, the N-terminal intrinsically disordered region of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL short linear interacting motif of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA also depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA.
... Fluorophore incorporation is also restricted to peptide regions that will tolerate modification by a donor and acceptor probe. However, the growing number of studies using FRET to monitor conformational changes associated with folding, function, enzyme activation, posttranslational modification, and oligomerization, support a broader applicability for this approach [56][57][58][59][60][61] . ...
Genetic mutations cause a wide spectrum of human disease by disrupting protein folding, both during and after synthesis. Transient de-novo folding intermediates therefore represent potential drug targets for pharmacological correction of protein folding disorders. Here we develop a FRET-based high-throughput screening (HTS) assay in 1,536-well format capable of identifying small molecules that interact with nascent polypeptides and correct genetic, cotranslational folding defects. Ribosome nascent chain complexes (RNCs) containing donor and acceptor fluorophores were isolated from cell free translation reactions, immobilized on Nickel-NTA/IDA beads, and imaged by high-content microscopy. Quantitative FRET measurements obtained from as little as 0.4 attomole of protein/bead enabled rapid assessment of conformational changes with a high degree of reproducibility. Using this assay, we performed a pilot screen of ~ 50,000 small molecules to identify compounds that interact with RNCs containing the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) harboring a disease-causing mutation (A455E). Screen results yielded 133 primary hits and 1 validated hit that normalized FRET values of the mutant nascent peptide. This system provides a scalable, tractable, structure-based discovery platform for screening small molecules that bind to or impact the folding of protein substrates that are not amenable to traditional biochemical analyses.
... An auto-inhibitory state involving interaction between the kinase domain and the Nterminus of AURKA has previously been described (Y. Zhang et al. 2007;Bai et al. 2014) and studies with a FRET-based conformational sensor confirm that the relative configuration of N-and C-terminus is altered upon activation of the kinase (Bertolin et al. 2016). Since interaction with APC/C-FZR1 occurs through an N-terminal degron motif, we propose that the Q45xxL D-box is 'buried' in the inactive conformation, which may be the previously described auto-inhibited state mediated through interaction between N-and C-terminal domains ( Figure 5B). ...
Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and association with cancer, and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the Anaphase-Promoting Complex (APC/C FZR1 ) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the ‘A-box’ in addition to a reported canonical D-box degron in the C-terminus. Here we find that the proposed C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, as previously reported in vitro , the N-terminal intrinsically disordered region (IDR) of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL Short Linear Interacting Motif (SLiM) of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA additionally depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA.
Summary blurb
AURKA degron motifs are redefined to show that the so-called N-terminal ‘A-box’ is in fact a D-box, and the so-called ‘D-box’ in the C-terminus is not a degron but a motif critical for the active, degradable conformation of AURKA
... Subcellular compartment-specific signaling can be analyzed using Förster or fluorescence resonance energy transfer (FRET) sensors fused to signal peptides. FRET sensors have been developed to analyze multiple signaling pathways, including those involving AURKA [214], AKT [139,215], cyclic adenosine monophosphate (AMP) [216,217], and calcium [218]. For example, an AURKA FRET sensor composed of AURKA within an eGFP and mCherry donor-acceptor fluorophore pair was based on the conformational change exhibited by AURKA upon autophosphorylation of Thr288 [214]. ...
... FRET sensors have been developed to analyze multiple signaling pathways, including those involving AURKA [214], AKT [139,215], cyclic adenosine monophosphate (AMP) [216,217], and calcium [218]. For example, an AURKA FRET sensor composed of AURKA within an eGFP and mCherry donor-acceptor fluorophore pair was based on the conformational change exhibited by AURKA upon autophosphorylation of Thr288 [214]. Phosphopeptide-binding domains (PBDs) have also been employed in the development of FRET biosensors to visualize kinase activity [219]. ...
Dysregulation of kinase signaling is associated with various pathological conditions, including cancer, inflammation, and autoimmunity; consequently, the kinases involved have become major therapeutic targets. While kinase signaling pathways play crucial roles in multiple cellular processes, the precise manner in which their dysregulation contributes to disease is dependent on the context; for example, the cell/tissue type or subcellular localization of the kinase or substrate. Thus, context-selective targeting of dysregulated kinases may serve to increase the therapeutic specificity while reducing off-target adverse effects. Primary cilia are antenna-like structures that extend from the plasma membrane and function by detecting extracellular cues and transducing signals into the cell. Cilia formation and signaling are dynamically regulated through context-dependent mechanisms; as such, dysregulation of primary cilia contributes to disease in a variety of ways. Here, we review the involvement of primary cilia-associated signaling through aurora A and AKT kinases with respect to cancer, obesity, and other ciliopathies.
... FRET biosensors were previously generated by flanking AURKA with donor-acceptor FRET pairs. FRET increase allowed to demonstrate that AURKA was activated at centrosomes both at mitosis and during the G1 phase 34,35 . We flanked AURKA with mTurquoise2 and pFAST. ...
... FLIM-FRET. FLIM analyses were performed with a time-gated custom-built system coupled to a Leica DMI6000 microscope (Leica) with a CSU-X1 spinning disk module (Yokogawa) and a 63 ×/1.4 NA oil immersion objective as described elsewhere 34 . To calculate fluorescence lifetime, five temporal gates with a step of 2 ns each allowed the sequential acquisition of five images covering a total delay time spanning from 0 to 10 ns. ...
... To calculate fluorescence lifetime, five temporal gates with a step of 2 ns each allowed the sequential acquisition of five images covering a total delay time spanning from 0 to 10 ns. The five images were used to calculate the pixel-bypixel mean fluorescence lifetime as described elsewhere 34,61 . Lifetime measurements and calculations were performed using the Inscoper software (Inscoper). ...
Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.
