Representative publications

Theresa S P Rothenbücher, Hakan Gürbüz, Marta P Pereira, Arto Heiskanen, Jenny Emneus and Alberto Martinez-Serrano

Brain organoids are considered to be a highly promising in vitro model for the study of the human brain and, despite their various shortcomings, have already been used widely in neurobiological studies. Especially for drug screening applications, a highly reproducible protocol with simple tissue culture steps and consistent output, is required. Here we present an engineering approach that addresses several existing shortcomings of brain organoids. By culturing brain organoids with a polycaprolactone scaffold, we were able to modify their shape into a flat morphology. Engineered flat brain organoids (efBOs) possess advantageous diffusion conditions and thus their tissue is better supplied with oxygen and nutrients, preventing the formation of a necrotic tissue core. Moreover, the efBO protocol is highly simplified and allows to customize the organoid size directly from the start. By seeding cells onto 12 by 12 mm scaffolds, the brain organoid size can be significantly increased. In addition, we were able to observe folding reminiscent of gyrification around day 20, which was self-generated by the tissue. To our knowledge, this is the first study that reports intrinsically caused gyrification of neuronal tissue in vitro. We consider our efBO protocol as a next step towards the generation of a stable and reliable human brain model for drug screening applications and spatial patterning experiments.

Mariam Hachimi, Catalina Grabowski, Silvia Campanario, Gonzalo Herranz, Gabriel Baonza, Juan M.Serrador, Sergio Gomez-Lopez, Maria D.Barea, Minerva Bosch-Fortea, Darren Gilmour, Michel Bagnat, Alejo E.Rodriguez-Fraticelli, Fernando Martin-Belmonte

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

Leticia Labat-de-Hoz, Armando Rubio-Ramos, Javier Casares-Arias, Miguel Bernabé-Rubio, Isabel Correas, and Miguel A. Alonso

Primary cilia are solitary, microtubule-based protrusions surrounded by a ciliary membrane equipped with selected receptors that orchestrate important signaling pathways that control cell growth, differentiation, development and homeostasis. Depending on the cell type, primary cilium assembly takes place intracellularly or at the cell surface. The intracellular route has been the focus of research on primary cilium biogenesis, whereas the route that occurs at the cell surface, which we call the “alternative” route, has been much less thoroughly characterized. In this review, based on recent experimental evidence, we present a model of primary ciliogenesis by the alternative route in which the remnant of the midbody generated upon cytokinesis acquires compact membranes, that are involved in compartmentalization of biological membranes. The midbody remnant delivers part of those membranes to the centrosome in order to assemble the ciliary membrane, thereby licensing primary cilium formation. The midbody remnant's involvement in primary cilium formation, the regulation of its inheritance by the ESCRT machinery, and the assembly of the ciliary membrane from the membranes originally associated with the remnant are discussed in the context of the literature concerning the ciliary membrane, the emerging roles of the midbody remnant, the regulation of cytokinesis, and the role of membrane compartmentalization. We also present a model of cilium emergence during evolution, and summarize the directions for future research.

Pilar López‐Nieva Laura González‐Sánchez María Ángeles Cobos‐Fernández Raúl Córdoba Javier Santos José Fernández‐Piqueras

The NOTCH1 gene encodes a transmembrane receptor protein with activating mutations observed in many T‐cell acute lymphoblastic leukemias (T‐ALLs) and lymphomas, as well as in other tumor types, which has led to interest in inhibiting NOTCH1 signaling as a therapeutic target in cancer. Several classes of Notch inhibitors have been developed, including monoclonal antibodies against NOTCH receptors or ligands, decoys, blocking peptides, and γ‐secretase inhibitors (GSIs). GSIs block a critical proteolytic step in NOTCH activation and are the most widely studied. Current treatments with GSIs have not successfully passed clinical trials because of side effects that limit the maximum tolerable dose. Multiple γ‐secretase–cleavage substrates may be involved in carcinogenesis, indicating that there may be other targets for GSIs. Resistance mechanisms may include PTEN inactivation, mutations involving FBXW7, or constitutive MYC expression conferring independence from NOTCH1 inactivation. Recent studies have suggested that selective targeting γ‐secretase may offer an improved efficacy and toxicity profile over the effects caused by broad‐spectrum GSIs. Understanding the mechanism of GSI‐induced cell death and the ability to accurately identify patients based on the activity of the pathway will improve the response to GSI and support further investigation of such compounds for the rational design of anti‐NOTCH1 therapies for the treatment of T‐ALL.

Margarita Saiz, Encarnacion Martinez‐Salas

RNA viruses have developed specialized mechanisms to subvert host RNA‐binding proteins (RBPs) favoring their own gene expression. The Leader (L) protein of foot‐and‐mouth disease virus, a member of the Picornaviridae family, is a papain‐like cysteine protease that self‐cleaves from the polyprotein. Early in infection, the L protease cleaves the translation initiation factors eIF4GI and eIF4GII, inducing the shutdown of cap‐dependent translation. However, the cleavage sites on the viral polyprotein, eIF4GI, and eIF4GII differ in sequence, challenging the definition of a consensus site for L targets. Identification of Gemin5 and Daxx proteolytic products in infected cells unveiled a motif centered on the RKAR sequence. The RBP Gemin5 is a member of the survival of motor neurons complex, a ribosome interacting protein, and a translation downregulator. Likewise, the Fas‐ligand Daxx is a multifunctional adaptor that plays key roles in transcription control, apoptosis, and innate immune antiviral response. Remarkably, the cleavage site on the RNA helicases MDA5 and LGP2, two relevant immune sensors of the retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors family, resembles the L target site of Gemin5 and Daxx, and similar cleavage sites have been reported in ISG15 and TBK1, two proteins involved in type I interferon response and signaling pathway, respectively. In this review we dissect the features of the L cleavage sites in essential RBPs, eventually helping in the discovery of novel L targets.

Carl P. Lehmann, Irene Saugar, José Antonio Tercero

The analysis of protein relocalization by fluorescence microscopy has been important for studying processes involved in genome integrity maintenance at the cellular level. Structure-specific endonucleases are required for genome stability, and work in budding yeast has revealed that these proteins accumulate and colocalize at discrete subnuclear foci following DNA damage. Here we describe protocols for fluorescence microscopy analysis of live budding-yeast cells containing fluorescent-tagged proteins that have been useful for the study of endonuclease relocalization during the cell cycle and under DNA-damaging conditions, all of which can be extended to the analysis of other proteins.

Cesar Cobaleda, Carolina Vicente-Dueñas & Isidro Sanchez-Garcia

B cell acute lymphoblastic leukaemia (B-ALL) is the most common form of childhood cancer. Although treatment has advanced remarkably in the past 50 years, it still fails in ~20% of patients. Recent studies revealed that more than 5% of healthy newborns carry preleukaemic clones that originate in utero, but only a small percentage of these carriers will progress to overt B-ALL. The drivers of progression are unclear, but B-ALL incidence seems to be increasing in parallel with the adoption of modern lifestyles. Emerging evidence shows that a major driver for the conversion from the preleukaemic state to the B-ALL state is exposure to immune stressors, such as infection. Here, we discuss our current understanding of the environmental triggers and genetic predispositions that may lead to B-ALL, highlighting lessons from epidemiology, the clinic and animal models, and identifying priority areas for future research.

Lydia Horndler, Pilar Delgado, David Abia, Ivaylo Balabanov, Pedro Martínez-Fleta, Georgina Cornish, Miguel A Llamas, Sergio Serrano-Villar, Francisco Sánchez-Madrid, Manuel Fresno, Hisse M van Santen, Balbino Alarcón

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infection.

César Gago-Córdoba, Jorge Val-Calvo, David Abia, Alberto Díaz-Talavera, Andrés Miguel-Arribas, Rocío Aguilar Suárez, Jan Maarten van Dijl, Ling Juan Wu, Wilfried J. J. Meijer

Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation.

IMPORTANCE Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of Bacillus subtilis encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation.

Berta Alcover-Sanchez, Gonzalo Garcia-Martin, Juan Escudero-Ramirez, Carolina Gonzalez-Riano, Paz Lorenzo, Alfredo Gimenez-Cassina, Laura Formentini, Pedro de la Villa-Polo, Marta P Pereira, Francisco Wandosell, Beatriz Cubelos

Fast synaptic transmission in vertebrates is critically dependent on myelin for insulation and metabolic support. Myelin is produced by oligodendrocytes (OLs) that maintain multilayered membrane compartments that wrap around axonal fibers. Alterations in myelination can therefore lead to severe pathologies such as multiple sclerosis. Given that hypomyelination disorders have complex etiologies, reproducing clinical symptoms of myelin diseases from a neurological perspective in animal models has been difficult. We recently reported that R-Ras1-/- and/or R-Ras2-/- mice, which lack GTPases essential for OL survival and differentiation processes, present different degrees of hypomyelination in the central nervous system with a compounded hypomyelination in double knockout (DKO) mice. Here, we discovered that the loss of R-Ras1 and/or R-Ras2 function is associated with aberrant myelinated axons with increased numbers of mitochondria, and a disrupted mitochondrial respiration that leads to increased reactive oxygen species levels. Consequently, aberrant myelinated axons are thinner with cytoskeletal phosphorylation patterns typical of axonal degeneration processes, characteristic of myelin diseases. Although we observed different levels of hypomyelination in a single mutant mouse, the combined loss of function in DKO mice lead to a compromised axonal integrity, triggering the loss of visual function. Our findings demonstrate that the loss of R-Ras function reproduces several characteristics of hypomyelinating diseases, and we therefore propose that R-Ras1-/- and R-Ras2-/- neurological models are valuable approaches for the study of these myelin pathologies.

Alba C Arcones, Rocío Vila-Bedmar, Mercedes Mirasierra, Marta Cruces-Sande, Mario Vallejo, Ben Jones, Alejandra Tomas, Federico Mayor Jr, Cristina Murga

Background: Insulin secretion from the pancreatic β-cell is finely modulated by different signals to allow an adequate control of glucose homeostasis. Incretin hormones such as glucagon-like peptide-1 (GLP-1) act as key physiological potentiators of insulin release through binding to the G protein-coupled receptor GLP-1R. Another key regulator of insulin signaling is the Ser/Thr kinase G protein-coupled receptor kinase 2 (GRK2). However, whether GRK2 affects insulin secretion or if GRK2 can control incretin actions in vivo remains to be analyzed.

Results: Using GRK2 hemizygous mice, isolated pancreatic islets, and model β-cell lines, we have uncovered a relevant physiological role for GRK2 as a regulator of incretin-mediated insulin secretion in vivo. Feeding, oral glucose gavage, or administration of GLP-1R agonists in animals with reduced GRK2 levels (GRK2+/- mice) resulted in enhanced early phase insulin release without affecting late phase secretion. In contrast, intraperitoneal glucose-induced insulin release was not affected. This effect was recapitulated in isolated islets and correlated with the increased size or priming efficacy of the readily releasable pool (RRP) of insulin granules that was observed in GRK2+/- mice. Using nanoBRET in β-cell lines, we found that stimulation of GLP-1R promoted GRK2 association to this receptor and that GRK2 protein and kinase activity were required for subsequent β-arrestin recruitment.

Conclusions: Overall, our data suggest that GRK2 is an important negative modulator of GLP-1R-mediated insulin secretion and that GRK2-interfering strategies may favor β-cell insulin secretion specifically during the early phase, an effect that may carry interesting therapeutic applications.

Verónica Miguel, Jessica Tituaña, J. Ignacio Herrero, Laura Herrero, Dolors Serra, Paula Cuevas, Coral Barbas, Diego Rodríguez Puyol, Laura Márquez-Expósito, Marta Ruiz-Ortega, Carolina Castillo, Xin Sheng, Katalin Susztak, Miguel Ruiz-Canela, Jordi Salas-Salvadó, Miguel A. Martínez González, Sagrario Ortega, Ricardo Ramos, and Santiago Lamas

Chronic kidney disease (CKD) remains a major epidemiological, clinical, and biomedical challenge. During CKD, renal tubular epithelial cells (TECs) present a persistent inflammatory and profibrotic response. Fatty acid oxidation (FAO), the main source of energy for TECs, is reduced in kidney fibrosis and contributes to its pathogenesis. To determine whether gain of function in FAO (FAO-GOF) could protect from fibrosis, we generated a conditional transgenic mouse model with overexpression of the fatty acid shuttling enzyme carnitine palmitoyl-transferase 1A (CPT1A) in TECs. Cpt1a-knockin (CPT1A-KI) mice subjected to 3 models of renal fibrosis (unilateral ureteral obstruction, folic acid nephropathy [FAN], and adenine-induced nephrotoxicity) exhibited decreased expression of fibrotic markers, a blunted proinflammatory response, and reduced epithelial cell damage and macrophage influx. Protection from fibrosis was also observed when Cpt1a overexpression was induced after FAN. FAO-GOF restored oxidative metabolism and mitochondrial number and enhanced bioenergetics, increasing palmitate oxidation and ATP levels, changes that were also recapitulated in TECs exposed to profibrotic stimuli. Studies in patients showed decreased CPT1 levels and increased accumulation of short- and middle-chain acylcarnitines, reflecting impaired FAO in human CKD. We propose that strategies based on FAO-GOF may constitute powerful alternatives to combat fibrosis inherent to CKD.

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