Application of an improvement cycle in classroom in the subject Fundamentals of life science in the degree of primary education

En los tiempos actuales necesitamos de ciudadanos capaces de lidiar con la incertidumbre. En un ambiente tecnológico y social en continuo cambio nuestros estudiantes necesitan poseer una actitud proactiva ante el aprendizaje si quieren mantener una mentalidad crítica. La universidad tiene la responsabilidad de ayudar a los estudiantes a encontrar su propia estrategia de aprendizaje. Para alcanzar este objetivo necesitamos que el estudiante desarrolle un aprendizaje consciente. En este artículo se presenta una experiencia de innovación docente que se ha llevado a cabo en la asignatura de Fundamentos de la vida en el grado de educación primaria. El ciclo de mejora (CIMA) se ha realizado en torno a la temática del cuerpo humano. Para abordar este tema se ha planteado un modelo didáctico centrado en el alumno que parte de sus ideas previas y trabaja en base a preguntas motivantes y actividades de contraste. Una vez implementado el CIMA se ha procedido a realizar una evaluación del aprendizaje mediante las escaleras de aprendizaje, la reflexión sobre el diario de las sesiones y la opinión del alumno. La experiencia docente ha sido muy constructiva y en general el alumno se siente satisfecho.The current times demand citizens able to deal with uncertainty. Society and technology are changing fast and our students will need to pursue constant learning if they want to keep a critical mind. The university has the responsibility of helping them to become independent learners. To achieve this, we need teaching that promotes the learning by the students. In this article we present an innovation experience about biology in the primary education grade. We made an improvement cycle in classroom (ICC) within the theme of the human body. The didactic model focused on the student, uses exciting questions and activities that challenge their mental models. Once the ICC was finished, we proceeded to evaluate the experience through learning stairs, the classroom diary and the feedback of the students. The experience was, in general, constructive for both the teacher and the students

A Role for Lipids in Protein Sorting?

Lipid and protein diversity provides structural and functional identity to the membrane compartments that define the eukaryotic cell. This compositional heterogeneity is maintained by the secretory pathway, which feeds newly synthesized proteins and lipids to the endomembrane systems. The precise sorting of lipids and proteins through the pathway guarantees the achievement of their correct delivery. Although proteins have been shown to be key for sorting mechanisms, whether and how lipids contribute to this process is still an open discussion. Our laboratory, in collaboration with other groups, has recently addressed the long-postulated role of membrane lipids in protein sorting in the secretory pathway, by investigating in yeast how a special class of lipid-linked cell surface proteins are differentially exported from the endoplasmic reticulum. Here we comment on this interdisciplinary study that highlights the role of lipid diversity and the importance of protein-lipid interactions in sorting processes at the cell membrane.FEDER/Ministerio de Ciencia, Innovación y Universidades PID2020-119505GB-I00 and BFU2017-89700-

Limited ER quality control for GPI-anchored proteins

Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast. We could efficiently route Gas1* to Hrd1-dependent ERAD and provide evidence that it contains a GPI anchor, ruling out that a GPI anchor obstructs ERAD. Instead, we show that the normally decreased susceptibility of Gas1* to ERAD is caused by canonical remodeling of its GPI anchor, which occurs in all GPI-APs and provides a protein-independent ER export signal. Thus, GPI anchor remodeling is independent of protein folding and leads to efficient ER export of even misfolded species. Our data imply that ER quality control is limited for the entire class of GPI-APs, many of them being clinically relevantEspaña, Ministerio de Ciencia e Innovación BFU2014-59309-PEspaña, Ministerio de Ciencia e Innovación BFU2009-07290España, Junta de Andalucía P09-CVI-450

Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast

Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.Ministerio de Ciencia e Innovación BFU2011-24513Junta de Andalucía P09-CVI-450

The ceramide synthase subunit lac1 regulates cell growth and size in fission yeast

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.Junta de Andalucía P18-FRJ1132Universidad de Sevilla VIPPIT-2020-I.5Japan Society for the Promotion of Science JP19H02922, JP21K19088Ministerio de Ciencia, Innovación y Universidades BFU2017-89700-

The yeast p24 complex regulates gpi-anchored protein Transport and quality control by monitoring anchor remodeling

Glycosylphosphatidylinositol (GPI)-anchored proteins are secretory proteins that are attached to the cell surface of eukaryotic cells by a glycolipid moiety. Once GPI anchoring has occurred in the lumen of the endoplasmic reticulum (ER), the structure of the lipid part on the GPI anchor undergoes a remodeling process prior to ER exit. In this study, we provide evidence suggesting that the yeast p24 complex, through binding specifically to GPI- anchored proteins in an anchor-dependent manner, plays a dual role in their selective traffick - ing. First, the p24 complex promotes efficient ER exit of remodeled GPI-anchored proteins after concentration by connecting them with the COPII coat and thus facilitates their incorpo - ration into vesicles. Second, it retrieves escaped, unremodeled GPI-anchored proteins from the Golgi to the ER in COPI vesicles. Therefore the p24 complex, by sensing the status of the GPI anchor, regulates GPI-anchored protein intracellular transport and coordinates this with correct anchor remodelin

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein crosslinking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system

Dual Independent Roles of the p24 Complex in Selectivity of Secretory Cargo Export from the Endoplasmic Reticulum

The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors. Here, we show biochemically that the p24 complex associates at the ER with other cargo receptors in a COPII-dependent manner, forming high-molecular weight multireceptor complexes. Furthermore, live cell imaging analysis reveals that the p24 complex is required to retain in the ER secretory cargos when their specific receptors are absent. This requirement does not involve neither the unfolded protein response nor the retrograde transport from the Golgi. Our results suggest that, in addition to its role as a cargo receptor in the specialized GPI-anchored protein pathway, the p24 complex also plays an independent role in secretory cargo selectivity during its exit through the general ER export pathway, preventing the non-selective bulk flow of native secretory cargos. This mechanism would ensure receptor-regulated cargo transport, providing an additional layer of regulation of secretory cargo selectivity during ER export.Ministerio de Economía y Competitividad BFU2017-89700-P, BFU2016-78265-

Ceramide chain length–dependent protein sorting into selective endoplasmic reticulum exit sites

Protein sorting in the secretory pathway is crucial to maintain cellular compartmentalization and homeostasis. In addition to coat-mediated sorting, the role of lipids in driving protein sorting during secretory transport is a longstanding fundamental question that still remains unanswered. Here, we conduct 3D simultaneous multicolor high-resolution live imaging to demonstrate in vivo that newly synthesized glycosylphosphatidylinositol-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized endoplasmic reticulum exit sites that are distinct from those used by transmembrane proteins. Furthermore, we show that the chain length of ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity. Our study provides the first direct in vivo evidence for lipid chain length–based protein cargo sorting into selective export sites of the secretory pathway.Spanish Ministry of Economy and Competitiveness (MINECO; grant number BFU2017-89700-P)University of Seville (VIPPIT-2020-I.5)Japan Society for the Promotion of Science (JP25221103, JP17H06420, and JP18H05275)Swiss National Science Foundation (grant no. 163966)Swiss National Supercomputing Centre (CSCS) under project IDs s726 and s84

Structural analysis of the GPI glycan

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.FEDER/Ministerio de Ciencia, Innovación y Universidades BFU2017-89700-PJapan Society for the Promotion of Science JP19H0292