Biology subjects

Guerrero, I.

Publications and source records attributed to Guerrero, I..

6 recordsLinked to original sources

bioRxiv2025

Direct cell-to-cell transport of Hedgehog morphogen is aided by the diffusible carrier Shifted/DmWif1

The establishment of morphogen gradients is fundamental during embryonic patterning. Yet, the mechanisms by which morphogens disperse remain highly controversial. In a paradigmatic example, the dispersal of the morphogen Hedgehog (Hh) is both limited by and dependent on its lipid modifications. To address this paradox, several competing transport models have been proposed, ranging from lipid-shielding solubilizing factors to transport via specialized filopodia called cytonemes. In this study, we uncover that the lipid-shielding protein DmWif1/Shifted (Shf) participates in the contact-dependent cell transfer of Hh. Using nanobody-based immobilization, we demonstrate the tight association of Shf and Hh to cytoneme membranes. We show that Shf is anchored to membranes by the Hh co-receptor Interference Hedgehog (Ihog) and that the Ihog-Shf complex is required for Hh dispersal and signaling. Together, our results unify cytoneme-mediated transport and membrane release into a single dispersal model, in which local solubilization is required for cytonemes to exchange Hh-bound Shf at cell-cell contacts between presenting and receiving cytonemes.

developmental biology↗

bioRxiv2025

Rescuing Ischemic Brain Injury by Rewiring Mitochondrial Electron Flow

Mitochondrial metabolic flux alterations are critical drivers of acute ischemia-reperfusion (IR) brain injury. Reverse electron transfer (RET), defined as the upstream flow of electrons from the quinone pool to complex I, is a major source of pathological reactive oxygen species (ROS) under stress conditions. In an in vivo brain IR model, oxygen deprivation induces the buildup of RET-supporting substrates, with glycerol 3-phosphate identified as the dominant contributor in addition to succinate. Rapid oxidation of these substrates by brain mitochondria upon reoxygenation drives massive ROS production, while also leading to over-reduction and dissociation of the complex I flavin mononucleotide (FMN) cofactor. The resulting FMN-deficient complex I becomes catalytically impaired, unable to oxidize NADH or to produce ROS. To mitigate RET and preserve complex I function, we used transgenic mice xenotopically expressing alternative oxidase (AOX). This enzyme bypasses complexes III and IV by directly oxidizing the reduced quinone pool and passing electrons onto molecular oxygen. AOX expression did not alter complex I abundance, supercomplexes assembly, or basal respiration rates, but effectively diverted electrons from the quinone pool, decreasing RET flux via complex I and limiting ROS generation during IR. For the first time we showed that AOX expression and attenuation of RET preserved complex I FMN binding, suppressed oxidative stress, and conferred neuroprotection in vivo. Our findings reveal a novel strategy for rewiring mitochondrial electron flux to mitigate initial IR brain injury, highlighting modulation of the quinone pool by AOX as a potential therapeutic strategy for IR.

biochemistry↗

bioRxiv2025

Cytoneme-mediated signalling coordinates the development of glial cells and neurons in the Drosophila eye

Effective cell communication is essential for the development and maintenance of the nervous system, where neurons and glial cells must interact closely. While cytoneme-mediated signalling is well-documented in various biological contexts, its role in coordinating neuron-glia development remains poorly understood. In this study, we investigated the function of cytonemes in neuron-glia coordination using the Drosophila eye imaginal disc as a model. This is a well-established system for examining the orchestrated development of glial and neuronal cells. Our results reveal that glial cells produce two distinct types of cytonemes based on their spatial orientation: one set extends toward nascent photoreceptors, while the other targets the morphogenetic furrow (MF). We have characterised the dynamics of glial cytonemes and demonstrated that disrupting these structures has a significant impact on glial cell migration and differentiation. This highlights the critical role of cytoneme-mediated signalling in regulating glial behaviour. Our findings also demonstrate that cytoneme function is essential for activating the Hedgehog (Hh) pathway in glial cells, with Hh ligand produced by photoreceptors. This pathway is necessary for glial differentiation, uncovering a previously unrecognised role for Hh signalling in this process. Overall, our results suggest that cytoneme-mediated Hh signalling is key to coordinating the development of both glial and neuronal populations.

developmental biology↗

bioRxiv2022

In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing

Precise genome engineering is essential for both basic and applied research. CRISPR/Cas accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we describe SEED/Harvest, an efficient knock-in method based on homology-directed (HDR) and single-strand annealing (SSA) repair pathways. HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatio-temporal control of protein labelling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knock-ins, we have developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.

genetics↗

bioRxiv2021

Improving the understanding of cytoneme-mediated morphogen gradients by in silico modeling

Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphogens gradients and to generate hypotheses that are difficult to study experimentally.

genetics↗

bioRxiv2020

Glypicans specifically regulate Hedgehog signaling through their interaction with Ihog in cytonemes

The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communication in development, tissue repair and cancer progression. These proteins can act as morphogens, inducing responses dependent on the ligand concentration in target cells located at a distance. Hh proteins are lipid modified and thereby have high affinity for membranes, which hinders the understanding of their spreading across tissues. Direct contact between cell membranes by filopodia-like structures (also known as cytonemes) could be the simplest explanation for Hh dispersal. To better understand this signaling mechanism, we have analyzed in Drosophila the interaction between the glypicans that, besides for other pathways, are necessary for Hh signaling, plus the adhesion molecules and Hh coreceptors Ihog and Boi. We describe that glypicans (Dally and Dally-like protein) are required to maintain Ihog, but not Boi, protein levels. We also show that ectopic Ihog stabilizes cytonemes through its interaction with glypicans, and we determine that two Ihog fibronectin III domains are essential for this interaction. Our data suggest that this interaction with Ihog in cytonemes confers the specificity of glypicans for Hh signaling.

developmental biology↗