Biology subjects

Vitiello, E.

Publications and source records attributed to Vitiello, E..

9 recordsLinked to original sources

bioRxiv2026

Human Choroid Plexus Organoids Progress to Postnatal-like Maturation and Reveal a Closing Window of Developmental Plasticity

The choroid plexus (ChP) produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier, yet the developmental programme driving its maturation in humans remains poorly understood. Using a 15-timepoint single-cell RNA sequencing atlas of human ChP organoids benchmarked against human tissue, we resolve an ordered differentiation process comprising cell cycle exit, ciliogenesis and ion transporter expression. Maturation culminates in a transcriptional state with features of human adult ChP, establishing the first in vitro ChP model to reach postnatal-like milestones. Centrosome proteomics identified previously uncharacterised, stage-specific regulators of ciliogenesis. To probe developmental plasticity, we perturbed ChP organoids with valproic acid (VPA), a clinically used antiepileptic and established teratogen. Early VPA-mediated disruption of multiciliogenesis and secretion recovered upon drug withdrawal, whereas late treatment washout resulted in persistent impairment of mature transporters. Together, these findings resolve the developmental programme by which the ChP reaches functionally mature identity and identify a stage-dependent plasticity window during its maturation.

developmental biology↗

bioRxiv2026

LINC00205 acts as a multivalent scaffold promoting FUSP525L recruitment in Amyotrophic Lateral Sclerosis stress granules

Stress granules (SG) are dynamic, membrane-less ribonucleoprotein assemblies that orchestrate cellular stress responses and rapidly disassemble upon stress relief. In Amyotrophic Lateral Sclerosis (ALS), mutations in RNA-binding proteins such as Fused in Sarcoma (FUS) impair SG dynamics, promoting the formation of aberrant and persistent granules. Although long non-coding RNAs (lncRNAs) are emerging as regulators of ribonucleoprotein organization, their mechanistic role in SG architecture and pathological remodeling remains largely unexplored. Here, we identify LINC00205 as a critical RNA regulator of pathological SG dynamics in FUSP525L-associated ALS. Using Neuroblastoma cells and human iPSC-derived Motor Neurons (MN), we show that LINC00205 is enriched in SG upon oxidative stress and directly interacts with mutant FUSP525L. Knock-out of LINC00205 selectively reduces the formation of FUSP525L-containing SG and restores physiological SG disassembly kinetics, without affecting normal SG or FUS expression levels. Mechanistically, LINC00205 acts as a multivalent RNA scaffold, directly binding mRNAs specifically enriched in pathological SG, such as PLCXD3 and PIK3CA, as well as the RNA helicase DHX36, which is preferentially associated with FUSP525L-containing SG. We demonstrate that LINC00205 promotes their specific recruitment into pathological SG, thereby contributing to the aberrant features of these assemblies. Together, our findings uncover an active lncRNA-driven mechanism that shapes the molecular composition of aberrant ALS-related SG and regulates their persistence, establishing lncRNAs as key organizers of RNA-protein assemblies under stress and providing a conceptual framework for modulating pathological condensates.

molecular biology↗

bioRxiv2026

Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation

Neurons depend on tightly regulated positioning of cellular components to maintain long-distance signaling, but the mechanisms guiding specific mRNAs to distant regions remain unclear. Here we show that late endosomes function as selective RNA carriers in human motor neurons and uncover the molecular logic guiding their loading. Using APEX2-mediated proximity labeling, we identify the external transcriptome of RAB7A-positive endosomes and find a specific population of mRNAs enriched for endosomal, axonal and synaptic functions. We find that mRNAs enriched in RAB7A endosomes contain evolutionarily conserved 5'UTRs which act as localization signals, and that the RNA-binding protein GEMIN5 interacts with these regions to promote endosomal RNA recruitment. Finally, we show that in ALS-associated conditions there is a conspicuous loss of endosome-associated transcripts and the mislocalization of GEMIN5 from endosomes. These findings uncover fundamental principles of RNA compartmentalization and highlight endosomal mRNA loading as a vulnerable axis in neuronal homeostasis.

molecular biology↗

bioRxiv2025

Co-evolution of a near-infrared aptamer:dye system for live-cell super-resolution RNA imaging

Despite their advantageous properties for live-cell imaging and super-resolution microscopy, high-performance silicon rhodamine (SiR) near-infrared (NIR) probes are still rarely employed in RNA imaging via fluorescent light-up aptamers (FLAPs). Here, we developed the SiRiuS:SiR-5 system through a combined evolutionary approach: evolving the aptamer via fluorescence-activated cell sorting (FACS), along with targeted mutations, truncations, and rational design, and improvement of the dye by systematic chemical derivatization. This resulted in an aptamer:dye pair with high fluorogenicity and photostability, specifically optimized for visualization of RNAs in mammalian live cells. Our system demonstrates strong fluorescence enhancement in live-cell imaging, enabling time resolved imaging of dynamic processes such as stress-granule formation. Notably, we validate its application in STED super-resolution microscopy, establishing it as a powerful NIR imaging platform for RNA structures below the refraction limit. Its orthogonality to existing FLAPs operating in the yellow-orange spectrum further broadens its versatility for exploring complex RNA dynamics in live cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/677088v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11051eeorg.highwire.dtl.DTLVardef@7309d6org.highwire.dtl.DTLVardef@8ac3e8org.highwire.dtl.DTLVardef@43eafd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Live-cell imaging of circular and long non-coding RNAs associated to FUS pathological aggregates by Pepper fluorescent RNA

Lately, important advancements in visualizing RNAs in fixed and live cells have been achieved. While mRNA imaging techniques are well-established, the development of effective methods for studying non-coding RNAs (ncRNAs) in living cells are still challenging but necessary, as they cover a variety of function and of intracellular localization, including highly dynamic processes like phase-transition, still poorly studied in vivo. Addressing this issue, we tagged two exemplary ncRNAs with the innovative fluorescent RNA (fRNA) Pepper. Specifically, we show circ-HDGFRP3 interaction with p-bodies, we recapitulate its recruitment in pathological FUS aggregates in a dynamic fashion and we super-resolve its distribution in such aggregates via Structured Illumination Microscopy. Moreover, we tracked the long non-coding RNA (lncRNA) nHOTAIRM1, a motor neurons-specific constituent of stress granules (SG), monitoring its behavior throughout the oxidative-stress response in physiological and pathological conditions. Overall, as fRNAs development advances, our work shows a successful use of Pepper for the monitoring of complex processes, as phase-transition, of paradigmatic molecules like circular RNAs (circRNAs) and lncRNAs with super-resolution potential in living mammalian cells.

molecular biology↗

bioRxiv2023

ALS-associated FUS mutation reshapes the RNA and protein composition and dynamic of Stress Granules.

Stress Granules (SG) formation is a cellular protection mechanism, constituting a storage for untranslated mRNAs and RNA-binding proteins (RBPs); however, these condensates can turn into pathological aggregates, related to the onset of neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS). This transition towards cytotoxic inclusions is triggered by ALS-causative mutations in the RBP FUS, which lead to its cytoplasmic mis-localization and accumulation in SG. Here, we describe the SG transcriptome in a neural context and describe several features for RNA recruitment in SG. We demonstrate that SG dynamics and RNA content are strongly modified by the incorporation of mutant FUS, switching to a more unstructured, AU-rich SG transcriptome. Moreover, we show that mutant FUS, together with its protein interactors and their target RNAs, are responsible for the reshaping of the mutant SG transcriptome with alterations that can be linked to neurodegeneration. Therefore, our data give a comprehensive view of the molecular differences between physiological and pathological SG in ALS conditions, showing how FUS mutations impact the RNA and protein population of these condensates.

molecular biology↗

bioRxiv2023

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimens fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a com-prehensive framework of live-cell spectroscopy methodologies - compatible with imaging - to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the pro-posed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation - inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes - not limited to liquid-liquid phase transition - and we anticipate their widespread application in future life-science research.

biophysics↗

bioRxiv2021

Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behaviour. Although the contribution of ECM compliance to the control of cell migration or division has been extensively studied, little has been reported regarding infectious processes. We have studied how mechanical properties of the ECM impact invasion of cells by the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identified OPTN as a protein regulated by ECM stiffness whose function is required for bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. We showed that OPTN knockdown cells display enhanced Rac1 activation, strong mechanochemical adhesion signalling and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Despite such features, OPTN knockdown cells displayed defective traction force buildup associated with limited cellular invasion by UTI89. Together, our data indicate that OPTN, through a new role in mechanobiology, supports CNF1-producing uropathogenic E. coli invasion and links HACE1-mediated ubiquitylation of Rac1 to ECM mechanical properties and integrin mechanotransduction.

cell biology↗

bioRxiv2019

Spatial Asymmetries of the Prophase Nucleus Regulate Dynein Recruitment to Ensure Mitotic Fidelity

During prophase, centrosomes need to separate and position to correctly assemble the mitotic spindle. This process occurs through the action of molecular motors, cytoskeletal networks and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. Here we show that during prophase the centrosomes-nucleus axis reorients, so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. This centrosomes-nucleus configuration depends on mechanical cues generated by mitotic chromosome condensation on the prophase nucleus. We further show these mechanosensitive cues act through SUN1/2 and NudE+NudEL to enable the polarized loading of Dynein on the NE. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation. We propose that chromosome segregation fidelity depends on the mechanical properties of the prophase nucleus that facilitate spindle assembly by regulating NE-Dynein localization.

cell biology↗