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Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues., Cells in many tissues fuse into syncytia acquiring new functions. By investigating whether physical remodelling promotes differentiation, here, the authors show that plasma membrane diminution post-fusion causes transient nutrient stress that inhibits YAP1 activity and may reduce proliferation-promoting transcription.

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.One-Sentence SummaryGaining sequential genetic access to vertebrate cell lineages.

SummaryDeveloping cells divide and differentiate, and in many tissues, such as bone, muscle, and placenta, cells fuse acquiring specialized functions. While it is known that fused-cells are differentiated, it is unclear what mechanisms trigger the programmatic-change, and whether cell-fusion alone drives differentiation. To address this, we employed a fusogen-mediated cell-fusion system involving undifferentiated cells in tissue culture. RNA-seq analysis revealed cell-fusion initiates a dramatic transcriptional change towards differentiation. Dissecting the mechanisms causing this reprogramming, we observed that after cell-fusion plasma-membrane surface area decreases through increased endocytosis. Consequently, glucose-transporters are internalized, and cytoplasmic-glucose and ATP transiently decrease. This low-energetic state activates AMPK, which inhibits YAP1, causing cell-cycle arrest. Impairing either endocytosis or AMPK prevents YAP1 inhibition and cell-cycle arrest after fusion. Together these data suggest that cell-fusion-induced differentiation does not need to rely on extrinsic-cues; rather the plasma-membrane diminishment forced by the geometric-transformations of cell-fusion cause transient cell-starvation that induces differentiation.

Introdução: O voleibol é um esporte coletivo, que atualmente demonstra notoriedade na mídia, e como consequência dessa exposição houve grande aumento do número de adeptos desta modalidade esportiva. As metodologias de ensino para o esporte denotam papel importante no processo de aprendizagem. A partir desse pressuposto, novas aplicações pedagógicas foram evidenciadas na literatura, que retratam a pedagogia no esporte como um complemento à compreensão das atividades no âmbito da prática principalmente na educação física, pois além de realizarem os movimentos esportivos, a pedagogia agrega ao aluno valores em relação às atitudes que circundam a pratica em si, como análise dos porquês da realização de tais movimentos. Diante do atual quadro e pelo crescente apelo por atividades voltadas para a pedagogia do esporte aplicada no voleibol. Objetivo: Faz-se necessário a pesquisa integrativa na literatura expondo estudos que fazem parte da literatura específica para demonstrar as discussões sobre a aprendizagem do Voleibol. Método: Esse trabalho foi desenvolvido por meio de uma revisão integrativa, que tem como proposta revisar a literatura e identificar os estudos relacionados a este tema, aplicando métodos de buscas nas bases de dados nacionais e internacionais, sendo elas Scientific Electronic Library Online (SCIELO), Caribe em Ciências da Saúde (LILACS) e Centro Latino-Americano e do Caribe de informações em Ciências da Saúde (BIREME). Discussão: O voleibol tem sido pensado como esporte coletivo sob uma perspectiva de aprendizagem e treinamento não somente no método tradicional. Conclusão: Concluímos que o emprego da pedagogia do esporte favorece ao ensino da modalidade de voleibol, na fase de iniciação e ao alto rendimento, utilizando dos métodos pré-descritos no trabalho, sendo eles o global funcional e analítico sintético. Compreendendo todos os aspectos que envolvem uma prática esportiva, como desenvolvimento motor, integração social, modulação de caráter pessoal e habilidades técnicas e táticas pertinentes ao voleibol.Palavras chaves: Metodologia, Revisão Integrativa, Aprendizagem, Esporte Coletivo, Educação Física.

Yes-associated Protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a selective set of enhancers for potent target gene activation, but how YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus in response to macromolecular crowding. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized YAP’s DNA binding cofactor TEAD1 along with other YAP-related transcription co-activators, including TAZ, and subsequently induced transcription of YAP-specific proliferation genes. Super-resolution imaging using Assay for Transposase Accessible Chromatin with photoactivated localization microscopy (ATAC-PALM) revealed that YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA Polymerase II (Pol II), the accessible chromatin domains later acquired Pol II, producing newly transcribed RNA. Removal of YAP’s intrinsically-disordered transcription activation domain (TAD) prevented YAP condensate formation and diminished downstream YAP signaling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.

Objective The aim of this cross-sectional correlational study was to determine the association of pain with morphologic and inflammatory sonographic findings in patients with knee osteoarthritis. Methods A total of 113 participants with knee osteoarthritis were assessed using visual analog scale pain score and sonography. Ultrasound evaluation included morphologic changes (ie, articular cartilage degeneration, medial and lateral meniscal protrusion, and presence of osteophytes on the joint margins) and inflammatory changes (ie suprapatellar effusion and/or synovitis, Baker cyst, superficial and deep infrapatellar effusion, pes anserine tendinopathy, and Hoffa panniculitis). Results Cluster analysis via Ward's method grouped patients with minimal pain (visual analog scale score, 0-4) and with substantial pain (visual analog scale score, 5-10). Stepwise logistic regression yielded 5 variables that significantly explained the variation in the probability of perceived substantial pain at 10% level of significance: lateral cartilage clarity (LCC; P = .025), medial cartilage clarity (MCC; P = .20), medial cartilage thickness (MCT; P = .041), medial meniscus protrusion (MMP) (P = .029), and osteophytes at medial femoral margin (P = .082), with 63% overall prediction accuracy. When age and sex were added, 4 variables remained significant at a 10% level of significance: LCC, MCC, MCT, and MMP, with 65% overall prediction accuracy. The receiver operating characteristic curve of this model was 0.667. Conclusion The study was able to demonstrate that morphologic abnormalities in the ultrasound parameters for LCC, MCC, MCT, and MMP were able to predict significant joint pain in knee osteoarthritis. There were no inflammatory changes that contributed to significant joint pain in this study.

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

Dietary restriction increases the longevity of many organisms, but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy (µ-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger µ-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interacted with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating µ-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate µ-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms. DOI: http://dx.doi.org/10.7554/eLife.21690.001

Different multicellular organisms undergo cell-cell fusion to form functional syncytia that support specialized functions necessary for proper development and survival. For years, monitoring the structural consequences of this process using live-cell imaging has been challenging due to the unpredictable timing of cell fusion events in tissue systems. Here we present a triggered vesicular stomatitis virus G-protein (VSV-G)-mediated cell-cell fusion assay that can be used to synchronize fusion between cells. This allows the study of cellular changes that occur during cell fusion. The process is induced using a fast wash of low pH isotonic buffer, promoting the fusion of plasma membranes of two or more adjacent cells within seconds. This approach is suitable for studying mixing of small cytoplasmic molecules between fusing cells as well as changes in organelle distribution and dynamics. © 2018 by John Wiley & Sons, Inc.

Consumption of ready-to-eat meals in Spain is limited, although an increasing trend is observed. This paper examines consumers’ reactions to the possible launching of a new ready-to-eat roast lamb dish, which could be applied for home consumption or foodservice/catering. This dish is cooked following traditional methods, is appreciated by most consumers and is consumed quite often. A survey has been undertaken to gain information about consumers’ opinions, purchasing habits and consumption of ready-to-eat dishes. The consumption of this type of product is strongly related to consumers’ age, education and income. Spanish consumers do not trust ready-to-eat dishes because they perceive these dishes as incorrectly processed and containing preservatives. In this study a hypothetical ready-to-eat roast lamb dish was also presented to consumers with two non-traditional side dishes. Consumers tasted the dish, and their preferences were analysed using a conjoint analysis. Results show that some preferences are linked with tradition, whereas others are more innovative and typical of modern-time behaviour. The right combination of features could be critical for a proper launching process.

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

During endocytosis, actin polymerization nucleated by the Arp2/3 complex provides force needed to drive internalization. Las17 is the strongest activator of the Arp2/3 complex in yeast cells. This study shows that Las17 is associated into a stable complex with Sla1, an adaptor that inhibits Las17 activity both in vitro and in vivo., During clathrin-mediated endocytosis, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (yeast WASp) is the strongest activator of the Arp2/3 complex in yeast cells; it is not autoinhibited and arrives to endocytic sites 20 s before actin polymerization begins. It is unclear how Las17 is kept inactive for 20 s at endocytic sites, thus restricting actin polymerization to late stages of endocytosis. In this paper, we demonstrate that Las17 is part of a large and biochemically stable complex with Sla1, a clathrin adaptor that inhibits Las17 activity. The interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I and class II. In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on the class I/II Las17 polyproline motifs and is based on competition between Sla1 and monomeric actin for binding to Las17. Furthermore, live-cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, inhibition during the initial 20 s, and efficient endocytosis. These results advance our understanding of the regulation of actin polymerization in endocytosis.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery. Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E] but variants have been subsequently discovered. The W-box conforms to the sequence PWxxW (where x is any residue). Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery. Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E] but variants have been subsequently discovered. The W-box conforms to the sequence PWxxW (where x is any residue). Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

During clathrin-mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin-binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile alpha-motif (SAM) domain and shows a propensity to oligomerize. By co-immunoprecipitation, Sla1p binds to clathrin and self-associates in vivo. Mutations in the clathrin-binding motif that abolish clathrin binding and structure-based mutations in SHD2 that impede self-association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin-mediated endocytosis.

To understand the early hebdomadal complications in newborns from mothers with both mild and severe preeclampsia.A transversal, observational trial was conducted in HGR No 1 IMSS, Morelia, Michoacán, México. The clinical records of gestating women were reviewed to detect those who had preeclampsia (PE) or eclampsia in the tokosurgical ward of the same hospital. Newborns were sent up to neonatology room, where their perinatal clinical history was done, and were also observed to detect complications.During the trial 1,644 gestating women were attended: 49 (3%) had PE, 25 (51%) of which had mild PE, 24 (49%) severe PE. Gestational age in 12 (25%) was less at the 37 weeks. With regard to delivery resolution, cesarian section was performed in 28 (57%). As to neonates, one or more early hebdomadale complications were observed in 32 (65%), in which asphyxia, hypoglucemia, and hypocalcemia as well as necrotizing enterocolitis predominated. Multivariate and univariate analysis showed severe preeclampsia to be associated as a significant and independent risk factor for asphyxia, hypocalcemia and necrotizing enterocolitis (RR 7.2, CI 95% 1.8-2.8; RR 1.3 CI 95% 1.0-1.7; and RR 1.4 CI 95% 1.0-1.9 respectively) (p0.05).Disseminating the prenatal control significance to detect PE is important because of the repercussions over gestating women and fetus, which increases perinatal morbidity and mortality.