Biology subjects

Corradi, E.

Publications and source records attributed to Corradi, E..

7 recordsLinked to original sources

bioRxiv2025

Electric field intensity modulates keratocyte migration without altering turning dynamics

Cell migration is a cornerstone of biological systems, enabling organisms to adapt to environmental stimuli and maintain homeostasis. Disruptions in this process can lead to functional impairment or system failure. In many cases, cells do not move randomly; instead, they migrate directionally in response to external cues, allowing them to perform essential biological functions. This directed movement is especially important in processes such as morphogenesis, cancer invasion, and wound healing. To unravel the complexities of directional cell migration, investigating natural guiding stimuli is crucial. Among these, electrical fields stand out as precise and physiologically relevant stimulus. Using a platform designed to apply programmable electric fields, the SCHEEPDOG device; we applied controlled electric field of varying intensities to keratocytes and quantitatively analyzed their migratory behavior. Our findings reveal that electric field stimulation not only induces robust directional migration but also enhances migration speed in an intensity-dependent manner. Additionally, cells initially moving in random directions gradually align with the field vector, with higher intensities accelerating the alignment. Intriguingly, while both speed and alignment time can be modulated through stimulation, the overall shape of migration trajectories remains unchanged. In other terms, for cells initially moving to the opposite direction of the field, the alignment is accompanied by making a turn and the size and shape of this turn is not affected by the magnitude of the electrical stimulation. Together, these results demonstrate that electrical stimulation can tune the speed and directional alignment of keratocyte migration without altering turning dynamics. These findings contribute to a deeper understanding of electrotaxis and offers new insights into how biophysical cues regulate cell migration in both physiological and pathological contexts.

biophysics↗

bioRxiv2025

Using DNA origami to study nanoscale organization of plasma membranes

Plasma membrane (PM) lipids and proteins are organized into nanoscale regions called nanodomains, which regulate essential cellular processes by controlling local membrane organization. Despite advances in super-resolution microscopy and single particle tracking, the small size and temporal instability of nanodomains make them difficult to study in living cells. To overcome these challenges, we built fluorescent DNA origami probes that insert into the PM via lipid anchors displayed on the cell. The number and spatial distribution of anchors between the origami and the cell surface were precisely defined by the origami, enabling nanometer-scale sampling of the cell surface. Inserting these DNA origami particles into the membrane with lipid anchors allowed them to passively diffuse across the membrane, and we tracked their movement using single particle tracking to survey the PM landscape. By varying the number and spatial arrangement of lipid anchors connecting the DNA origami to the cell surface, we showed that immobilization of DNA origami particles requires simultaneous interactions with multiple nanodomains. Disruption of the actin cytoskeleton reduced immobilization, confirming its role in supporting nanodomain stability. Moreover, transient mechanical stretching of cells led to reversible increases in DNA origami mobility, indicating that mechanical force can reversibly regulate PM nanodomain organization. Altogether, we present a novel membrane-integrated DNA origami approach that provides mechanistic insights into PM nanodomain architecture and dynamics in living cells.

cell biology↗

bioRxiv2023

BIRC3: A Prognostic Predictor and Novel Therapeutic Target in TMZ-Resistant Glioblastoma Tumors

BackgroundGlioblastoma (GB) is an incurable malignant tumor of the central nervous system, with a poor prognosis. Robust molecular biomarkers associated with therapeutic response or survival are still lacking in GB. Previously, using NADH-fluorescence lifetime imaging (NADH-FLIM), as a new drug screening precision medicine ex-vivo approach, we categorized patient-derived vital tumors into TMZ responder (Resp) and non-responder (Non-Resp) groups, revealing differentially expressed genes. MethodsExpanding on our previous study, we assessed TMZ response in a larger cohort of primary and recurrent ex-vivo live GB tumors (n=33) using NADH-FLIM. Transcriptome analysis was performed to characterize TMZ Resp and Non-Resp cases, and in-silico and functional cellular investigations were conducted to explore the efficacy of potential biomarkers. ResultsGenes dysregulated in the previous study showed consistent expression patterns. BIRC3, a potent apoptosis inhibitor, was significantly upregulated in TMZ-resistant samples. BIRC3 expression complemented MGMT status as a prognostic factor in multiple TCGA cohorts. BIRC3 functioned as a prognostic factor of survival also in separate European private glioblastoma cohorts. The BIRC3 antagonist, AZD5582, in combination with TMZ, effectively reversed TMZ resistance by restoring apoptosis in glioblastoma cell lines and patient-derived organoids. ConclusionsBIRC3 holds promise as a prognostic biomarker and predictor of TMZ response in GB. Assessing BIRC3 expression could aid in stratifying patients for combined TMZ and AZD5582 therapy. Our study highlights the potential of functional precision medicine and BIRC3 assessment as a standard tool in glioblastoma clinical oncology, improving outcomes. KEYPOINTSO_LIBIRC3, previously overlooked, identified through dynamic precision medicine using TMZ perturbation of glioblastoma tissue as a robust prognostic factor. C_LIO_LIThe gene BIRC3 is an independent prognostic factor associated with shorter survival and TMZ resistance, rigorously validated across various case studies and datasets, including two expansive European case studies. C_LIO_LIProposal of anti-BIRC3 drug, AZD5582, shows promise as a novel therapeutic option to overcome TMZ resistance in GB tumors, providing hope for improved outcomes and personalized treatment strategies for patients with limited treatment options C_LI IMPORTANCE OF THE STUDYGlioblastoma (GB), an aggressive cancer type with a bleak prognosis, lacks dependable biomarkers for treatment prediction. Few markers like MGMT promoter methylation, IDH1 mutation, TERT gene mutations, and EGFR amplification are known, but their predictive consistency varies. Temozolomide (TMZ) resistance, seen in over 50% of GB patients, complicates matters. BIRC3, an apoptosis-inhibiting gene, displays heightened expression in TMZ-resistant tumors. Our study examined BIRC3 in GB patient samples, finding it an independent prognostic factor linked to shorter survival and TMZ resistance. Our research builds upon Wang et al.s 2016 and 2017 findings, delving deeper through TCGA data and European case studies. BIRC3s consistent prominence suggests its significance, with functional experiments confirming its role. We assessed AZD5582, targeting BIRC3, which, when combined with TMZ, curtailed cell growth and induced apoptosis. Notably, AZD5582 countered TMZ resistance in patient-derived GB-EPXs, except for low BIRC3 cases. Our precision medicine approach enhances personalized therapies and outcomes, highlighting BIRC3s potential as a prognostic marker and AZD5582 as a new therapy for TMZ-resistant GB.

cancer biology↗

bioRxiv2023

Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We now develop a method based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Using the highly conserved Xenopus miR-181a family as a proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting pre-miRNA apical region elicit a precursor-selective inhibition of mature miRNA-5p production. The levels of miRNAs released from the 3'-arm of these precursors are not reduced, suggesting that our approach is also arm-selective. Overall, we show that this strategy can be successfully applied in vivo to achieve high target selectivity to study identical or highly similar miRNAs stemming from different precursors.

molecular biology↗

bioRxiv2021

SHOT-R: A next generation algorithm for particle kinematics analysis

Analysis of live-imaging experiments is crucial to decipher a plethora of cellular mechanisms within physiological and pathological contexts. Kymograph, i.e. graphical representations of particle spatial position over time, and single particle tracking (SPT) are the currently available tools to extract information on particle transport and velocity. However, the spatiotemporal approximation applied in particle trajectory reconstruction with those methods intrinsically prevents an accurate analysis of particle kinematics and of instantaneous behaviours. Here, we present SHOT-R, a novel numerical method based on polynomial reconstruction of 4D (3D+time) particle trajectories. SHOT-R, contrary to other tools, computes bona fide instantaneous and directional velocity, and acceleration. Thanks to its high order continuous reconstruction it allows, for the first time, kinematics analysis of co-trafficked particles. Overall, SHOT-R is a novel, versatile, and physically reliable numerical method that achieves all-encompassing particle kinematics studies at unprecedented accuracy on any live-imaging experiment where the spatiotemporal coordinates can be retrieved.

cell biology↗

bioRxiv2021

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is important also for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current methods fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. Additionally, we demonstrated the protocol to be effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids.

cell biology↗

bioRxiv2018

Precursor miRNAs are trafficked along axons associated with vesicles and locally processed to regulate growth cone steering

Various species of non-coding RNAs (ncRNAs) are enriched in subcellular compartments but the mechanisms orchestrating their delocalization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked, anchored to CD63-positive vesicles, to distal axons along microtubules. Upon exposure to the chemotropic cue Sema3A, pre-miRNAs are processed specifically within axons into newly synthesized mature miRNAs, which, in turn, silence the basal translation of TUBB3 but not of APP. At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly synthesized miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodelling of subcellular compartments.\n\nHighlightsO_LIPrecursor miRNAs are actively transported along axons to the growth cone tethered to CD63-positive vesicles\nC_LIO_LISema3A but not Slit2 induces the local biogenesis of specific miRNAs within axons\nC_LIO_LIMature miRNAs are important for growth cone responsiveness ex vivo and the establishment of functional connections in vivo\nC_LIO_LINewly synthesized miRNAs inhibit the basal translation of TUBB3 but not APP upon Sema3A exposure\nC_LI

cell biology↗