Biology subjects

Lopez-Iglesias, C.

Publications and source records attributed to Lopez-Iglesias, C..

7 recordsLinked to original sources

bioRxiv2025

A human organoid model of alveolar regeneration reveals distinct epithelial responses to interferon-gamma

Chronic obstructive pulmonary disease is characterized by inflammation and emphysema, leading to progressive alveolar destruction. Currently, no therapies effectively regenerate the alveolar epithelium. Here, we describe a feeder- and serum-free primary adult human organoid model to investigate how inflammation influences alveolar regeneration. We achieve long-term expansion of multipotent progenitor-like cells, while an alveolar type 2 (AT2) maturation protocol enhances surfactant production and supports tubular myelin formation. Introducing a LATS inhibitor to the expansion condition induces alveolar type 1 (AT1) differentiation while maintaining AT2 cells. Using this platform, we find that interferon-gamma exerts cytotoxic effects on AT1 cells while promoting the growth of regenerating AT2 cells, illustrating how a single inflammatory stimulus can have divergent effects on alveolar epithelial cell types. These findings underscore the nuanced influence of pro-inflammatory cytokines on alveolar regeneration. Our organoid model provides a reductionist platform for mechanistic studies, aimed to identify strategies to enhance alveolar regeneration.

cell biology↗

bioRxiv2024

The curse of the red pearl: a fibroblast specific pearl-necklace mitochondrial phenotype caused by phototoxicity

The dynamic nature of mitochondria makes live cell imaging an important tool in mitochondrial research. Although imaging using fluorescent probes is the golden standard in studying mitochondrial morphology, these probes might introduce a-specific features. In this study, live cell fluorescent imaging was applied to investigate a pearl-necklace shaped mitochondrial phenotype that arises when mitochondrial fission is restricted. In this fibroblast specific pearl-necklace phenotype, constricted and expanded mitochondrial regions alternate. Imaging studies revealed that the formation time of this pearl-necklace phenotype differs between laser scanning confocal, widefield and spinning disk confocal microscopy. We found that the phenotype formation correlates with the excitation of the fluorescent probe and is the result of phototoxicity. Interestingly, the phenotype only arises in cells stained with red mitochondrial dyes. Serial section electron tomography pearl-necklace mitochondria revealed that the mitochondrial membranes remained intact, while the cristae structure was altered. Furthermore, filaments and ER were present at the constricted sites. This study illustrates the importance of considering experimental conditions for live cell imaging to prevent imaging artefacts that can have a major impact on the obtained results.

molecular biology↗

bioRxiv2024

Tuft cells act as regenerative stem cells in the human intestine

In mice, intestinal tuft cells have been described as a long-lived, post-mitotic cell type of which two distinct subsets have been identified, named tuft-1 and tuft-21. By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase upon interleukin (IL)-4 and IL-13 exposure, as reported previously in mouse2-4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem- and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

cell biology↗

bioRxiv2021

Priming mycobacterial ESX-secreted protein B to form a channel-like structure

ESX-1 is a major virulence factor of Mycobacterium tuberculosis, a secretion machinery directly involved in the survival of the microorganism from the immune system defence. It disrupts the phagosome membrane of the host cell through a contact-dependent mechanism. Recently, the structure of the inner-membrane core complex of the homologous ESX-3 and ESX-5 was resolved; however, the elements involved in the secretion through the outer membrane or those acting on the host cell membrane are unknown. Protein substrates might form this missing element. Here, we describe the oligomerisation process of the ESX-1 substrate EspB, which occurs upon cleavage of its C-terminal region and is favoured by an acidic environment. Cryo-electron microscopy data are presented which show that EspB from different mycobacterial species have a conserved quaternary structure, except for the non-pathogenic species M. smegmatis. EspB assembles into a channel with dimensions and characteristics suitable for the transit of ESX-1 substrates, as shown by the presence of another EspB trapped within. Our results provide insight into the structure and assembly of EspB, and suggests a possible function as a structural element of ESX-1.

biochemistry↗

bioRxiv2020

Mycobacteria-host interactions in human bronchiolar airway organoids

Tuberculosis, one of the oldest human pathogens remains a major global health threat. Recent advances in organoid technology offer a unique opportunity to grow different human "organs" in vitro, including the human airway, that faithfully recapitulate tissue architecture and function. We have explored the potential of human airway organoids (AOs) as a novel system in which to model tuberculosis infection. To this end, we adapted biosafety containment level 3-approved procedures to allow successful microinjection of Mycobacterium tuberculosis, the causative agent of tuberculosis, into AOs. We reveal that mycobacteria infected epithelial cells with low efficiency, and that the organoid microenvironment was able to control, but not eliminate the pathogen. We demonstrate that AOs responded to infection by inducing cytokine and antimicrobial peptide production, and inhibiting mucins. Given the importance of myeloid cells in tuberculosis infection, we co-cultured mycobacteria-infected organoids with human monocyte-derived macrophages, and found that these cells were recruited to the organoid epithelium. We conclude that adult stem cell-derived airway organoids can be used to model early events of tuberculosis infection and offer new avenues for fundamental and therapeutic research.

systems biology↗

bioRxiv2020

Exostosin-1 Glycosyltransferase Regulates Endoplasmic Reticulum Architecture and Dynamics

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including ER shape-dynamics-function relationships in mammalian cells. We have observed that, inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated to ER network extension. Our findings suggest that EXT1 drives glycosylation reactions involving ER structural proteins and high-energy nucleotide sugars, which might also apply to other organelles.

cell biology↗

bioRxiv2019

Automated cryo-EM sample preparation by pin-printing and jet vitrification

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide controlled and efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The devices performance was validated by resolving 4 standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 [A] resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

biophysics↗