Biology subjects

Esposito, A.

Publications and source records attributed to Esposito, A..

5 recordsLinked to original sources

bioRxiv2018

Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited1. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -92, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes3,4. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.

cancer biology↗

bioRxiv2018

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

Two decades of high-paced innovation have improved the spatial resolution of fluorescence microscopy to enable molecular resolution combined with the low-invasiveness and specificity characteristic of optical microscopy. However, fluorescence microscopy also enables scientists and clinicians to map and quantitate the physico-chemical properties (e.g., analyte concentration, enzymatic activities and protein-protein interactions) of biological samples. But the optimization of the biochemical resolving power in fluorescence microscopy is not as well-developed compared to its spatial resolution. Typical techniques rely on the observation of individual properties of fluorescence thus limiting the opportunities for sensing and multiplexing. Aiming to overcome existing limitations, we demonstrate a new imaging paradigm -- Hyper Dimensional Imaging Microscopy (HDIM) -- that enables the orthogonal properties of fluorescence emission (excited state lifetime, polarization and spectra) in biological samples to be quantified simultaneously and efficiently. Therefore, akin to how multi-dimensional separation in mass-spectroscopy and multi-dimensional spectra in NMR impacted proteomics and structural biology, we envisage that HDIM spectra of unprecedented dimensionality will impact the fields of systems biology and medical diagnostics by maximizing the biochemical resolving power of fluorescence microscopy.

biophysics↗

bioRxiv2018

Cooperation of partially-transformed clones: the invisible force behind the early stages of carcinogenesis

Most tumours exhibit significant heterogeneity and are best described as communities of cellular populations competing for resources. Growing experimental evidence also suggests, however, that cooperation between cancer clones is important as well for the maintenance of tumour heterogeneity and tumour progression. However, a role for cell communication during the earliest steps in oncogenesis is not well characterised despite its vital importance in normal tissue and clinically manifest tumours. By modelling the interaction between the mutational process and cell-to-cell communication in three-dimensional tissue architecture, we show that non-cell-autonomous mechanisms of carcinogenesis could support and accelerate pre-cancerous clonal expansion through the cooperation of different, non- or partially- transformed mutants. We predict the existence of a cell-autonomous time-horizon, a time before which cooperation between cell-to-cell communication and DNA mutations might be one of the most fundamental forces shaping the early stages of oncogenesis. The understanding of this process could shed new light on the mechanisms leading to clinically manifest cancers.

cancer biology↗

bioRxiv2018

Checkpoint non-fidelity induces a complex landscape of lineage fitness after DNA damage

DNA damage in proliferating mammalian cells causes death1, senescence2 or continued survival, via checkpoints that monitor damage and regulate cell cycle progression, DNA repair and fate determination3. Cell cycle checkpoints facilitate tumour suppression by preventing the generation of proliferating mutated cells4, particularly by blocking passage of DNA lesions into replication and mitosis5. While checkpoint non-fidelity permits cells to carry genomic aberrations into subsequent cell cycle phases6, its long-term consequences on lineages descendant from damaged cells remains poorly characterised. Devising methods for microscopy-based lineage tracing, we unexpectedly demonstrate that transient DNA damage to single living cells bearing a negligent checkpoint induces heterogenous cell-fate outcomes in their descendant generations removed from the initial insult. After transiently damaged cells undergo an initial arrest, pairs of descendant cells without obvious cell-cycle abnormalities either divide or die in a seemingly stochastic way. Progeny of transiently damaged cells may die generations afterwards, creating considerable variability of lineage fitness that promotes overall persistence in a mutagenic environment. Descendants of damaged cells frequently form micronuclei, activating immunogenic signalling. Our findings reveal previously unrecognized, heterogenous effects of cellular DNA damage that manifest long afterwards in descendant cells. We suggest that these heterogenous descendant cell-fate responses may function physiologically to ensure the elimination and immune clearance of damaged cell lineages, but pathologically, may enable the prolonged survival of cells bearing mutagenic damage.

cancer biology↗

bioRxiv2018

mTOR-dependent phosphorylation controls TFEB nuclear export

The transcriptional activation of catabolic processes during starvation is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient re-feeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data reveal that modulation of TFEB nuclear export via phosphorylation plays a major role in the modulation of TFEB localization and activity.

cell biology↗