Actin cytoskeleton differently regulates cell surface organization of GPI-anchored proteins in polarized epithelial cells and fibroblasts.

The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological activities. These GPI-APs occupy distinct cellular functions such as enzymes, receptors, and adhesion molecules, and they are implicated in several vital cellular processes. Thus, unraveling the mechanisms and regulators of their membrane organization is essential. In polarized epithelial cells, GPI-APs are enriched at the apical surface, where they form small cholesterol-independent homoclusters and larger heteroclusters accommodating multiple GPI-AP species, all confined within areas of approximately 65-70 nm in diameter. Notably, GPI-AP homoclustering occurs in the Golgi apparatus through a cholesterol- and calcium-dependent mechanism that drives their apical sorting. Despite the critical role of Golgi GPI-AP clustering in their cell surface organization and the importance of cholesterol in heterocluster formation, the regulatory mechanisms governing GPI-AP surface organization, particularly in the context of epithelial polarity, remain elusive. Given that the actin cytoskeleton undergoes substantial remodeling during polarity establishment, this study explores whether the actin cytoskeleton regulates the spatiotemporal apical organization of GPI-APs in MDCK cells. Utilizing various imaging techniques (number and brightness, FRET/FLIM, and dSTORM coupled to pair correlation analysis), we demonstrate that the apical organization of GPI-APs, at different scales, does not rely on the actin cytoskeleton, unlike in fibroblastic cells. Interestingly, calcium chelation disrupts the organization of GPI-APs at the apical surface by impairing Golgi GPI-AP clustering, emphasizing the existence of an interplay among Golgi clustering, apical sorting, and surface organization in epithelial cells. In summary, our findings unveil distinct mechanisms regulating the organization of GPI-APs in cell types of different origins, plausibly allowing them to adapt to different external signals and different cellular environments in order to achieve specialized functions.

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

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.

The RNA-binding proteins CELF1 and ELAVL1 cooperatively control the alternative splicing of CD44.

CD44 mRNA contains nine consecutive cassette exons, v2 to v10. Upon alternative splicing, several isoforms are produced with different impacts on tumor biology. Here, we demonstrate the involvement of the RNA-binding proteins CELF1 and ELAVL1 in the control of CD44 splicing. We show by FRET-FLIM that these proteins directly interact in the nucleus. By combining RNAi-mediated depletion and exon array hybridization in HeLa cells, we observe that the exons v7 to v10 of CD44 are highly sensitive to CELF1 and ELAVL1 depletion. We confirm by RT-PCR that CELF1 and ELAVL1 together stimulate the inclusion of these exons in CD44 mRNA. Finally, we show in eight different tumor types that high expression of CELF1 and/or ELAVL1 is correlated with the inclusion of CD44 variable exons. These data point to functional interactions between CELF1 and ELAVL1 in the control of CD44 splicing in human cancers.

Neural network fast-classifies biological images through features selecting to power automated 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 by making acquisition decisions based on the images. It calls for analysing them on the fly. Biology further imposes training on a reduced data set due to cost and time to prepare the samples and have the data sets annotated by experts. We propose 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 to understand the contribution of each feature in decision and execution time. We found that the non-linear-classifier random forests outperformed Fisher's linear discriminant. More importantly, the most discriminant and time-consuming features could be excluded without significant accuracy loss, 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 eight phases of the cell cycle, using 12 feature groups and operating a consumer market ARM-based embedded system. 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 optimizing these algorithms for smart microscopy.

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging.

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.

tdLanYFP, a Yellow, Bright, Photostable, and pH-Insensitive Fluorescent Protein for Live-Cell Imaging and Förster Resonance Energy Transfer-Based Sensing Strategies.

Yellow fluorescent proteins (YFPs) are widely used as optical reporters in Förster 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 pH values. Here, we characterize the yellow fluorescent protein tdLanYFP, derived from the tetrameric protein from the cephalochordate Branchiostoma 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 subdiffraction resolution using STED nanoscopy and is compatible with the use of spectromicroscopies in single-molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pH values. Finally, we show that tdLanYFP is a valuable FRET partner either as a 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.

Label-free microscopy of mitotic chromosomes using the polarization orthogonality breaking technique.

We report how a recently developed polarization imaging technique, implementing micro-wave photonics and referred to as orthogonality-breaking (OB) imaging, can be adapted on a classical confocal fluorescence microscope, and is able to provide informative polarization images from a single scan of the cell sample. For instance, the comparison of the images of various cell lines at different cell-cycle stages obtained by OB polarization microscopy and fluorescence confocal images shows that an endogenous polarimetric contrast arizes with this instrument on compacted chromosomes during cell division.

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. AURKA triggers the degradation of Inner Mitochondrial Membrane/matrix proteins by interacting with core components of the autophagy pathway. On the inner mitochondrial membrane, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2, which triggers 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 and restores normal ATP production levels. Altogether, these data provide evidence for a 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.

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells.

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded Förster's Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context.

Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.

Automated screening of AURKA activity based on a genetically encoded FRET biosensor using fluorescence lifetime imaging microscopy.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust tool to measure Förster Resonance Energy Transfer (FRET) between two fluorescent proteins, mainly when using genetically-encoded FRET biosensors. It is then possible to monitor biological processes such as kinase activity with a good spatiotemporal resolution and accuracy. Therefore, it is of interest to improve this methodology for future high content screening purposes. We here implement a time-gated FLIM microscope that can image and quantify fluorescence lifetime with a higher speed than conventional techniques such as Time-Correlated Single Photon Counting (TCSPC). We then improve our system to perform automatic screen analysis in a 96-well plate format. Moreover, we use a FRET biosensor of AURKA activity, a mitotic kinase involved in several epithelial cancers. Our results show that our system is suitable to measure FRET within our biosensor paving the way to the screening of novel compounds, potentially allowing to find new inhibitors of AURKA activity.

Imaging Mitochondrial Functions: from Fluorescent Dyes to Genetically-Encoded Sensors.

Mitochondria are multifunctional organelles that are crucial to cell homeostasis. They constitute the major site of energy production for the cell, they are key players in signalling pathways using secondary messengers such as calcium, and they are involved in cell death and redox balance paradigms. Mitochondria quickly adapt their dynamics and biogenesis rates to meet the varying energy demands of the cells, both in normal and in pathological conditions. Therefore, understanding simultaneous changes in mitochondrial functions is crucial in developing mitochondria-based therapy options for complex pathological conditions such as cancer, neurological disorders, and metabolic syndromes. To this end, fluorescence microscopy coupled to live imaging represents a promising strategy to track these changes in real time. In this review, we will first describe the commonly available tools to follow three key mitochondrial functions using fluorescence microscopy: Calcium signalling, mitochondrial dynamics, and mitophagy. Then, we will focus on how the development of genetically-encoded fluorescent sensors became a milestone for the understanding of these mitochondrial functions. In particular, we will show how these tools allowed researchers to address several biochemical activities in living cells, and with high spatiotemporal resolution. With the ultimate goal of tracking multiple mitochondrial functions simultaneously, we will conclude by presenting future perspectives for the development of novel genetically-encoded fluorescent biosensors.

Insights into the non-mitotic functions of Aurora kinase A: more than just cell division.

AURKA is a serine/threonine kinase overexpressed in several cancers. Originally identified as a protein with multifaceted roles during mitosis, improvements in quantitative microscopy uncovered several non-mitotic roles as well. In physiological conditions, AURKA regulates cilia disassembly, neurite extension, cell motility, DNA replication and senescence programs. In cancer-like contexts, AURKA actively promotes DNA repair, it acts as a transcription factor, promotes cell migration and invasion, and it localises at mitochondria to regulate mitochondrial dynamics and ATP production. Here we review the non-mitotic roles of AURKA, and its partners outside of cell division. In addition, we give an insight into how structural data and quantitative fluorescence microscopy allowed to understand AURKA activation and its interaction with new substrates, highlighting future developments in fluorescence microscopy needed to better understand AURKA functions in vivo. Last, we will recapitulate the most significant AURKA inhibitors currently in clinical trials, and we will explore how the non-mitotic roles of the kinase may provide new insights to ameliorate current pharmacological strategies against AURKA overexpression.

Optimized FRET Pairs and Quantification Approaches To Detect the Activation of Aurora Kinase A at Mitosis.

Genetically encoded Förster's Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signaling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must be optimized 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-color 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.

Chromatin condensation fluctuations rather than steady-state predict chromatin accessibility.

Chromatin accessibility to protein factors is critical for genome activities. However, the dynamic properties of chromatin higher-order structures 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.

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40phox, p47phox, and p67phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

The polarity-induced force imbalance in Caenorhabditis elegans embryos is caused by asymmetric binding rates of dynein to the cortex.

During asymmetric cell division, the molecular motor dynein generates cortical pulling forces that position the spindle to reflect polarity and adequately distribute cell fate determinants. In Caenorhabditis elegans embryos, despite a measured anteroposterior force imbalance, antibody staining failed to reveal dynein enrichment at the posterior cortex, suggesting a transient localization there. Dynein accumulates at the microtubule plus ends, in an EBP-2EB-dependent manner. This accumulation, although not transporting dynein, contributes modestly to cortical forces. Most dyneins may instead diffuse to the cortex. Tracking of cortical dynein revealed two motions: one directed and the other diffusive-like, corresponding to force-generating events. Surprisingly, while dynein is not polarized at the plus ends or in the cytoplasm, diffusive-like tracks were more frequently found at the embryo posterior tip, where the forces are higher. This asymmetry depends on GPR-1/2LGN and LIN-5NuMA, which are enriched there. In csnk-1(RNAi) embryos, the inverse distribution of these proteins coincides with an increased frequency of diffusive-like tracks anteriorly. Importantly, dynein cortical residence time is always symmetric. We propose that the dynein-binding rate at the posterior cortex is increased, causing the polarity-reflecting force imbalance. This mechanism of control supplements the regulation of mitotic progression through the nonpolarized dynein detachment rate.

Aurora kinase A localises to mitochondria to control organelle dynamics and energy production.

Many epithelial cancers show cell cycle dysfunction tightly correlated with the overexpression of the serine/threonine kinase Aurora A (AURKA). Its role in mitotic progression has been extensively characterised, and evidence for new AURKA functions emerges. Here, we reveal that AURKA is located and imported in mitochondria in several human cancer cell lines. Mitochondrial AURKA impacts on two organelle functions: mitochondrial dynamics and energy production. When AURKA is expressed at endogenous levels during interphase, it induces mitochondrial fragmentation independently from RALA. Conversely, AURKA enhances mitochondrial fusion and ATP production when it is over-expressed. We demonstrate that AURKA directly regulates mitochondrial functions and that AURKA over-expression promotes metabolic reprogramming by increasing mitochondrial interconnectivity. Our work paves the way to anti-cancer therapeutics based on the simultaneous targeting of mitochondrial functions and AURKA inhibition.

Bioconjugated fluorescent organic nanoparticles targeting EGFR-overexpressing cancer cells.

The field of optical bioimaging has considerably flourished with the advent of sophisticated microscopy techniques and ultra-bright fluorescent tools. Fluorescent organic nanoparticles (FONs) have thus recently appeared as very attractive labels for their high payload, absence of cytotoxicity and eventual biodegradation. Nevertheless, their bioconjugation to target specific receptors with high imaging contrast is scarcely performed. Moreover, assessing the reality of bioconjugation represents high challenges given the sub-nanomolar concentrations resulting from the commonly adopted nanoprecipitation fabrication process. Here, we describe how the combination of a magnetic shell allows us to easily generate red-emitting FONs conjugated with the epidermal growth factor ligand (EGF), a small protein promoting cancer cell proliferation by activating the EGF receptor (EGFR) pathway. Dual color fluorescence correlation spectroscopy combined with immunofluorescence is originally harnessed in its time trace mode to unambiguously demonstrate covalent attachment between the FON and EGF at sub-nanomolar concentrations. Strong asymmetric clustering of EGF-conjugated FONs is observed at the membrane of MDA-MB-468 human breast cancer cells overexpressing EGF receptors using super-resolution fluorescence microscopy. Such high recruitment of EGF-conjugated FONs is attributed to their EGF multivalency (4.7 EGF per FON) which enables efficient EGFR activation and subsequent phosphorylation. The large hydrodynamic diameter (DH ∼ 301 nm) of EGF-conjugated FONs prevents immediate engulfment of the sequestered receptors, which provides very bright and localized spots in less than 30 minutes. The reported bioconjugated nanoassemblies could thus serve as ultra-bright probes of breast cancer cells with EGFR-overexpression that is often associated with poor prognosis.