Cadherin-mediated adhesion takes control.

The formation of a centralised apical membrane initiation site (AMIS) is a key event in epithelial cell polarisation. A recent study by Liang et al demonstrates that AMIS localisation relies on cadherin-mediated cell adhesion.

Multiple ciliary localization signals control INPP5E ciliary targeting.

Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoes.

Deciphering the interplay between autophagy and polarity in epithelial tubulogenesis.

The Metazoan complexity arises from a primary building block, the epithelium, which comprises a layer of polarized cells that divide the organism into compartments. Most of these body compartments are organs formed by epithelial tubes that enclose an internal hollow space or lumen. Over the last decades, multiple studies have unmasked the paramount events required to form this lumen de novo. In epithelial cells, these events mainly involve recognizing external clues, establishing and maintaining apicobasal polarity, endo-lysosomal trafficking, and expanding the created lumen. Although canonical autophagy has been classically considered a catabolic process needed for cell survival, multiple studies have also emphasized its crucial role in epithelial polarity, morphogenesis and cellular homeostasis. Furthermore, non-canonical autophagy pathways have been recently discovered as atypical secretory routes. Both canonical and non-canonical pathways play essential roles in epithelial polarity and lumen formation. This review addresses how the molecular machinery for epithelial polarity and autophagy interplay in different processes and how autophagy functions influence lumenogenesis, emphasizing its role in the lumen formation key events.

Intercalate or invaginate: PI(3,4,5)P3 governs a membrane constriction switch in cell shaping.

Although contractile processes, from tissue invagination to cell intercalation, utilize diverse ratcheting mechanisms, little is known about how ratcheting becomes engaged at specific cell surfaces. In this issue of Developmental Cell, Maio et al. demonstrate that PI(3,4,5)P3 is a paramount regulator of the Sbf/RabGEF-Rab35 ratchet mechanism.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis.

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.

Conocimientos y percepciones de profesionales de Atención Primaria sobre el acceso y la atención sanitaria de la población inmigrante / Knowledge and perceptions of Primary Care professionals regarding access and healthcare assistance provided to the immigrant population

OBJETIVOS: describir conocimientos y percepciones de profesionales de Atención Primaria sobre el acceso al Sistema Nacional de Salud (SNS) y la atención sanitaria de la población inmigrante, así como analizar diferencias entre categorías profesionales. MÉTODOS: se realizó un estudio observacional descriptivo transversal mediante una encuesta a 352 profesionales sanitarios y no sanitarios en centros de salud de la Comunidad de Madrid, y residentes de Medicina y Enfermería de la Unidad Docente Multiprofesional de Atención Familiar y Comunitaria Sur de Madrid (UDMAFyC Sur) entre febrero y marzo de 2019. RESULTADOS: respondieron 179 profesionales (50,9%). El 50,3% opinó que los inmigrantes tienen frecuentes dificultades de acceso y el 65,9% que el estatus migratorio condiciona su salud, señalando el idioma como barrera relevante el 80,4%. El 72,7% de los sanitarios afirmó que no consumen más recursos. Expresaron nulo/escaso conocimiento del Real Decreto-Ley (RDL) 16/2012 el 72%, y del RDL 7/2018, un 66,7%. Un 30,7% consideró que el RDL 16/2012 tuvo efectos negativos en salud, y el 28,5%, que el RDL 7/2018 recuperó la universalidad. Un 54,9% de sanitarios apoyó la universalidad del SNS, contrastando con el 19,1% de personal no sanitario. CONCLUSIONES: la mitad de profesionales percibe frecuentes dificultades de la población inmigrante en su acceso al SNS, aunque el conocimiento de las normativas es limitado. Una ajustada mayoría respalda la universalidad, existiendo posicionamientos restrictivos con relativa frecuencia, especialmente entre el personal no sanitario. Se reproducen ciertos mitos sobre la atención a población inmigrante, no así acerca del consumo de recursos sanitarios

OBJECTIVES: To report the knowledge and perceptions of Primary Care professionals regarding access to the Spanish National Healthcare System and healthcare assistance provided to the immigrant population, as well as to analyse differences by professional categories. METHODS: A cross-sectional study was conducted by sending a questionnaire to 352 professionals: healthcare workers (HCWs) and non-healthcare workers (non-HCWs) from primary health care centres in the southern region of the Community of Madrid and resident doctors and nurses assigned to this primary healthcare area, from February to March 2019. RESULTS: Overall, 179 professionals (50.9%) replied. Of these, 50.3% thought that migrants face frequent difficulties in access, whereas 65.9% considered that migrants' health is conditioned by their migratory status. Moreover, 80.4% identified language as a relevant barrier and 72.7% of HCWs believed that migrants do not consume more resources. Regarding the Spanish Royal Decree-laws (RDL), 72% admitted having no or little knowledge of Spanish RDL 16/2012, and 66.7% of Spanish RDL 7/2018. However, 30.7% considered that Spanish RDL 16/2012 had negative effects on health, 28.5% agreed that Spanish RDL 7/2018 recovered universal healthcare coverage (UHC). Overall, 54.9% of HCWs support UHC in comparison to 19.1% of non-HCW. CONCLUSIONS: Half the professionals perceive that immigrants encounter frequent difficulties when accessing the Spanish Healthcare System, although their knowledge of legislation is limited. A slight majority supports UHC and finds a large number of restrictive stances, especially among non-HCWs. Certain misconceptions regarding care provided to immigrants persist, but not those related to health resources consumption

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes.

T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule-organizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1 ß phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1 ß phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1 ß re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1 ß regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization. ABBREVIATIONS: Ab, antibody; AICD, activation-induced cell death; AIP, average intensity projection; APC, antigen-presenting cell; BCR, B-cell receptor for antigen; C, centre of mass; cent2, centrin 2; cIS, central region of the immune synapse; CMAC, CellTracker™ Blue (7-amino-4-chloromethylcoumarin); cSMAC, central supramolecular activation cluster; CTL, cytotoxic T lymphocytes; DAG, diacylglycerol; DGKα, diacylglycerol kinase α; Dia1, Diaphanous-1; dSMAC, distal supramolecular activation cluster; ECL, enhanced chemiluminescence; ESCRT, endosomal sorting complex required for traffic; F-actin, filamentous actin; Fact-low cIS, F-actin-low region at the centre of the immune synapse; FasL, Fas ligand; FMNL1, formin-like 1; fps, frames per second; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; HRP, horseradish peroxidase; ILV, intraluminal vesicles; IS, immune synapse; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MIP, maximal intensity projection; MVB, multivesicular bodies; MTOC, microtubule-organizing centre; NS, not significant; PBL, peripheral blood lymphocytes; PKC, protein kinase C; PKCδ, protein kinase C δ isoform; PLC, phospholipase C; PMA, phorbol myristate acetate; Pol. Index, polarization index; pSMAC, peripheral supramolecular activation cluster; PSF, point spread function; ROI, region of interest; SD, standard deviation; shRNA, short hairpin RNA; SEE, Staphylococcus enterotoxin E; SMAC, supramolecular activation cluster; TCR, T-cell receptor for antigen; T-helper (Th); TRANS, transmittance; WB, Western blot.

Hypoxia compensates cell cycle arrest with progenitor differentiation during angiogenesis.

Angiogenesis, the main mechanism that allows vascular expansion for tissue regeneration or disease progression, is often triggered by an imbalance between oxygen consumption and demand. Here, by analyzing changes in the transcriptomic profile of endothelial cells (ECs) under hypoxia we uncovered that the repression of cell cycle entry and DNA replication stand as central responses in the early adaptation of ECs to low oxygen tension. Accordingly, hypoxia imposed a restriction in S-phase in ECs that is mediated by Hypoxia-Inducible Factors. Our results indicate that the induction of angiogenesis by hypoxia in Embryoid Bodies generated from murine Stem Cells is accomplished by the compensation of decreased S-phase entry in mature ECs and differentiation of progenitor cells. This conditioning most likely allows an optimum remodeling of the vascular network. Identification of the molecular underpinnings of cell cycle arrest by hypoxia would be relevant for the design of improved strategies aimed to suppress angiogenesis in pathological contexts where hypoxia is a driver of neovascularization.

Micropattern-based platform as a physiologically relevant model to study epithelial morphogenesis and nephrotoxicity.

Tubulogenesis in epithelial organs often initiates with the acquisition of apicobasal polarity, giving rise to the formation of small lumens that expand and fuse to generate a single opened cavity. In this study, we present a micropattern-based device engineered to generate epithelial tubes through a process that recapitulates in vivo tubule morphogenesis. Interestingly, tubulogenesis in this device is dependent on microenvironmental cues such as cell confinement, extracellular matrix composition, and substrate stiffness, and our set-up specifically allows the control of these extracellular conditions. Additionally, proximal tubule cell lines growing on micropatterns express higher levels of drug transporters and are more sensitive to nephrotoxicity. These tubes display specific morphological defects that can be linked to nephrotoxicity, which would be helpful to predict potential toxicity when developing new compounds. This device, with the ability to recapitulate tube formation in vitro, has emerged as a powerful tool to study the molecular mechanisms involved in organogenesis and, by being more physiologically relevant than existing cellular models, becomes an innovative platform to conduct drug discovery assays.

Protein Kinase C δ Regulates the Depletion of Actin at the Immunological Synapse Required for Polarized Exosome Secretion by T Cells.

Multivesicular bodies (MVB) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, ILVs are secreted as Fas ligand-bearing, pro-apoptotic exosomes following T cell receptor (TCR)-induced fusion of MVB with the plasma membrane at the immune synapse (IS). In this study we show that protein kinase C δ (PKCδ), a novel PKC isotype activated by diacylglycerol (DAG), regulates TCR-controlled MVB polarization toward the IS and exosome secretion. Concomitantly, we demonstrate that PKCδ-interfered T lymphocytes are defective in activation-induced cell death. Using a DAG sensor based on the C1 DAG-binding domain of PKCδ and a GFP-PKCδ chimera, we reveal that T lymphocyte activation enhances DAG levels at the MVB endomembranes which mediates the association of PKCδ to MVB. Spatiotemporal reorganization of F-actin at the IS is inhibited in PKCδ-interfered T lymphocytes. Therefore, we propose PKCδ as a DAG effector that regulates the actin reorganization necessary for MVB traffic and exosome secretion.

Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

Chromatin conformation regulates the coordination between DNA replication and transcription.

Chromatin is the template for the basic processes of replication and transcription, making the maintenance of chromosomal integrity critical for cell viability. To elucidate how dividing cells respond to alterations in chromatin structure, here we analyse the replication programme of primary cells with altered chromatin configuration caused by the genetic ablation of the HMGB1 gene, or three histone H1 genes. We find that loss of chromatin compaction in H1-depleted cells triggers the accumulation of stalled forks and DNA damage as a consequence of transcription-replication conflicts. In contrast, reductions in nucleosome occupancy due to the lack of HMGB1 cause faster fork progression without impacting the initiation landscape or fork stability. Thus, perturbations in chromatin integrity elicit a range of responses in the dynamics of DNA replication and transcription, with different consequences on replicative stress. These findings have broad implications for our understanding of how defects in chromatin structure contribute to genomic instability.

The timing of monozygotic twinning: a criticism of the common model.

In the dominant model, monozygotic (MZ) twinning is universally accepted as a post-fertilization event resulting from splitting of the embryo along its first 2 weeks of development. The stage at which splitting occurs determines chorionicity and amnionicity. A short history on how the model was built is presented, stressing the role played by some embryologists, in particular George Corner, in its completion and final success. Strikingly, for more than 60 years no deep criticisms have been raised against the model, which, in virtue of its rational and plausible character, enjoys the status of undisputed truth. At close examination, the embryological support of the model shows some important weak points, particularly when dealing with late splitting. In the author's view, the model not only has contributed to 'suspend' our knowledge on the timing of MZ twinning, but seems indefensible and claims to be substituted. That factor could imply relevant consequences for embryology and bioethics. As an alternative to the model, a new theory to explain the timing of MZ twinning is proposed. It is based on two premises. First, MZ twinning would be a fertilization event. In that case, due to an alteration of the zygote-blastomere transition, the first zygotic division, instead of producing two blastomeres, generates twin zygotes. Second, monochorionicity and monoamnionicity would not depend on embryo splitting, but on fusion of membranes. Some support for this theory can be found in recent embryological advances and also in some explanations of old.

[The fictitious embryo: a critical history of a biological myth. The author explains his book]. / El Embrión Ficticio: Historia de un Mito Biológico. El Autor Explica su Libro.

Basically, the commented upon book is the result of the writer's reaction to the absence of serious biological science that for years is reigning in the bioethics of the human embryo. The embryology used by bioethicists, both in their theoretical studies and in their interventions before public policy drafting committees, has consisted essentially not in primary research materials, but in text-book descriptions, with which resulted easier to support the theory of the pre-embryo. In this way, biologists were able to provide philosophers and jurists with a number of apparently scientific ″arguments″ so convincing that no one felt the need to review them critically. The lack or inferior condition of the biological status of the human supported by the arguments on the irrelevance of fertilization; on the numerical predominance of the extraembryonic cell population over the proper embryonic one; on the formation of monozygotic twins along the first two weeks of development; on the formation of tetragametic chimeras by fusion in one of two previously independent dizygotic embryos; on the totipotency of the cells of the young embryo; and, finally, on the massive spontaneous wastage of early embryos. Those arguments sought to consolidate the thesis that the biological entities exhibiting those behaviours were so precarious biologically that they couldn't claim a full ontological status of humanhood and, therefore, they cannot demand from us the ethical full respect due to human beings. Throughout the book I try to refute, in a reasonable and convincing way, and, more importantly in my view, with biological data, the above arguments. The author warns that the book can not be read without effort, but he considers that to get rid of the prevailing prejudices in embryoethics is a worthwhile goal.

El embrión ficticio: historia de un mito biológico. El autor explica su libro / The fictitious embryo: A critical history of a biological myth. The authro explains his book

En el fondo, el libro comentado es fruto de la reacción del autor ante la ausencia de ciencia biológica seria que domina desde hace años la bioética del embrión humano. La embriología usada por los bioéticos, tanto en sus estudios teóricos como en sus intervenciones en los comités que han elaborado la normativa legal, ha sido en esencia una embriología secundaria, divulgativa, que sirvió para sustentar la teoría del preembrión. Los biólogos proporcionaron a filósofos y juristas una serie de "argumentos" aparentemente científicos y tan convincentes que nadie sintió la necesidad de revisarlos críticamente. Son los argumentos de la irrelevancia de la fecundación; del predominio numérico de las células de destino extraembrionario sobre las propiamente embrionarias; de la formación de gemelos monocigóticos a lo largo de las dos primeras semanas del desarrollo; de la formación de quimeras tetragaméticas mediante fusión de dos embrionesdizigóticos en los primeros catorce días; de la totipotencialidad de las células del embrión joven; y, finalmente, de la masiva pérdida espontánea de embriones. Se ha pretendido consolidar la tesis de que las entidades biológicas que expresan esos comportamientos son biológicamente tan precarias que no pueden reclamar un estatus ontológico de plena humanidad, no pueden exigir de nosotros el respeto que se debe a los seres propiamente humanos. A lo largo del libro trato de refutar -me parece que razonable y convincentemente, y lo que me parece más importante, con datos biológicos- los argumentos arriba citados. El autor advierte lealmente que el libro no puede leerse sin esfuerzo, pero considera que liberarse de los prejuicios reinantes en la embrioética es un objetivo que merece la pena

Basically, the commented upon book is the result of the writer’s reaction to the absence of serious biological science that for years is reigning in the bioethics of the human embryo. The embryology used by bioethicists, both in their theoretical studies and in their interventions before public policy drafting committees, has consisted essentially not in primary research materials, but in text-book descriptions, with which resulted easier to support the theory of the pre-embryo. In this way, biologists were able to provide philosophers and jurists with a number of apparently scientific "arguments" so convincing that no one felt the need to review them critically. The lack or inferior condition of the biological status of the human supported by the arguments on the irrelevance of fertilization; on the numerical predominance of the extraembryonic cell population over the proper embryonic one; on the formation of monozygotic twins along the first two weeks of development; on the formation of tetragametic chimeras by fusion in one of two previously independent dizygotic embryos; on the totipotency of the cells of the young embryo; and, finally, on the massive spontaneous wastage of early embryos. Those arguments sought to consolidate the thesis that the biological entities exhibiting those behaviours were so precarious biologically that they couldn’t claim a full ontological status of humanhood and, therefore, they cannot demand from us the ethical full respect due to human beings. Throughout the book I try to refute -in a reasonable and convincing way, and, more importantly in my view, with biological data- the above arguments. The author warns that the book can not be read without effort, but he considers that to get rid of the prevailing prejudices in embryoethics is a worthwhile goal

High-resolution analysis of DNA synthesis start sites and nucleosome architecture at efficient mammalian replication origins.

DNA replication origins are poorly characterized genomic regions that are essential to recruit and position the initiation complex to start DNA synthesis. Despite the lack of specific replicator sequences, initiation of replication does not occur at random sites in the mammalian genome. This has lead to the view that DNA accessibility could be a major determinant of mammalian origins. Here, we performed a high-resolution analysis of nucleosome architecture and initiation sites along several origins of different genomic location and firing efficiencies. We found that mammalian origins are highly variable in nucleosome conformation and initiation patterns. Strikingly, initiation sites at efficient CpG island-associated origins always occur at positions of high-nucleosome occupancy. Origin recognition complex (ORC) binding sites, however, occur at adjacent but distinct positions marked by labile nucleosomes. We also found that initiation profiles mirror nucleosome architecture, both at endogenous origins and at a transgene in a heterologous system. Our studies provide a unique insight into the relationship between chromatin structure and initiation sites in the mammalian genome that has direct implications for how the replication programme can be accommodated to diverse epigenetic scenarios.