32 research outputs found

    Plasma Membrane Localization of Signaling Proteins in Yeast: a Dissertation

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    In response to external stimuli, many intracellular signaling proteins undergo dynamic changes in localization to the plasma membrane. Using the Saccharomyces cerevisiaemating pathway as a model, I investigated the molecular interactions that govern plasma membrane localization of signaling proteins, and how the plasma membrane compartmentalization of a signaling complex influences the overall signaling behavior of the pathway. Signaling proteins often consist of multiple interaction domains that collectively dictate their localization and function. Ste20 is a p21-activated kinase (PAK) that functions downstream of the Rho-type GTPase Cdc42 to activate several mitogen-activated protein (MAP) kinase pathways in budding yeast, including the mating pathway. I identified a short domain in Ste20 that directly binds to membrane lipids via electrostatic interaction. A mutation in this domain abolishes both the localization and function of Ste20. Thus, the previously known Cdc42 binding is necessary but not sufficient; instead, direct membrane binding by Ste20 is also critical. By replacing this domain with heterologous membranebinding domains, I demonstrated that phospholipid specificity is not essential in vivo. Functionally important short membrane-binding domains were also found in the Cdc42 effectors Gic1 and Gic2, indicating that generic membrane binding can work in concert with the CRIB domain to regulate activation of Cdc42 targets. These results underscore the importance of cooperation between protein-protein and protein-membrane interaction in achieving proper localization of signaling proteins at the cell cortex. At the system level, MAP kinase cascades can be graded or switch-like. The budding yeast mating pathway exhibits a graded response to increasing levels of pheromone. Previously the scaffold protein Ste5 was hypothesized to contribute to this graded response. To test this idea, I activated the pathway in a variety of ways and measured the response at the single cell level. I found that the graded response is not perturbed by the deletion of negative regulators of the pathway whereas the response became switch-like when the pathway was activated by a crosstalk stimulus that bypasses the upstream components. Interestingly, activation of the pathway in the cytoplasm using the graded expression of MAPKKK resulted in an ultrasensitive response. In contrast, activation of the pathway at the plasma membrane using the graded expression of membranetargeted active pathway components remained graded. In these settings, the scaffold protein Ste5 increased ultrasensitivity when limited to the cytosol; however, if Ste5 was allowed to function at the plasma membrane, signaling was graded. The results suggest that, in the mating pathway, the inherently ultrasensitive MAPK cascade is converted to a graded system by the scaffoldmediated assembly of signaling complexes at the plasma membrane. Therefore, the plasma membrane localization of Ste5 helps shape the input-output properties of the mating MAPK pathway in a manner that is suitable for the biology of mating. Taken together, this thesis underscores the importance of plasma membrane localization during mating pathway signaling in yeast. The examples described here provide further appreciation of how multiple interaction domains can function together to achieve specific targeting of the signaling proteins, as well as advances in understanding the role of scaffold proteins in modulating signaling behavior to promote graded signaling at the plasma membrane

    Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

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    [Background:] Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. [Objective:] To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. [Methods:] We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. [Results:] The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. [Conclusion:] These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres

    Pensée et action socio-éducative dans des contextes de l’enseignement secondaire. Une étude descriptive et de corrélation

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    Este artículo refiere resultados de un proyecto de investigación más amplio que engloba diferentes unidades de análisis relacionadas con el educador social y su acción profesional. Pretendemos describir y justificar la intervención socioeducativa que desempeñan los educadores sociales en los centros de enseñanza, por este motivo planteamos en las comunidades de Castilla La Mancha y Extremadura un estudio descriptivo-correlacional, dentro del clásico diseño de encuesta, a través de un cuestionario. Los resultados corroboran la creciente irrupción de los educadores sociales en el sistema educativo y las funciones realizadas que nos permite revisar y debatir el papel de los educadores en los centros. Presentamos los resultados parciales que hacen referencia a una de las dimensiones estudiadas referidas, en líneas generales, al perfil del educador social y, más concretamente, a las funciones del mismo en los centros de secundaria colaborando en la respuesta que la sociedad actual demanda a la educación.Cet article présente les résultats d’un projet plus étendu de recherche qui comporte différentes unités d’analyse à propos de l’éducateur spécialisé et son action professionnelle. Pour décrire et justifier l’intervention socio-éducative menée par les éducateurs spécialisés dans les centres d’enseignement, une étude descriptive- corrélationnelle a été réalisée dans les Communautés Autonomes de Castilla la Mancha et de Extremadura au moyen d’un questionnaire crée dans le cadre classique des enquêtes. Les résultats corroborent l’irruption croissante des éducateurs spécialisés dans le système éducatif et les fonctions qui leur sont attachées, ce qui permet de réviser et de débattre le rôle des éducateurs dans les centres. On y présente des résultats partiels d’une des dimensions étudiées concernant, grosso modo, le profil de l’enseignant spécialisé et, plus en particulier, les fonctions qui lui sont attachées dans les centres d’enseignement secondaire pour répondre aux demandes que la société actuelle pose à l’éducation

    Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells

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    Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A, encoding Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium channel (mKir2.1) and HypoPP2-associated Nav1.4 variants. Whole cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane potential to comparable levels to myofibers, and that some Nav1.4 variants induced notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants using a ratiometric pH indicator, SNARF-4F. Our optical method provides a potential in vitro platform for high-throughput drug screen, not only for HypoPP but also for other channelopathies caused by VSD mutations.Kubota T., Takahashi S., Yamamoto R., et al. Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells. DMM Disease Models and Mechanisms 16, A18 (2023); https://doi.org/10.1242/dmm.049704

    Membrane localization of scaffold proteins promotes graded signaling in the yeast MAP kinase cascade

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    BACKGROUND: Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or whether it must be enforced by separate mechanisms. It has been hypothesized that scaffold proteins promote graded behavior. RESULTS: Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust in that it resists perturbation by loss of several negative-feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. CONCLUSIONS: The results suggest that the MAPK cascade module is inherently ultrasensitive but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context

    Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

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    The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP2-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase–dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein–protein and protein–membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex

    A cmap-enabled gene expression signature-matching approach identifies small-molecule inducers of accelerated cell senescence

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    Abstract Background Diverse stresses including genotoxic therapy can induce proliferating cancer cells to undergo cellular senescence and take on the characteristic phenotypes of replicative cellular aging. This accelerated or therapy-induced senescence has been alternatively proposed to contribute to therapeutic efficacy or resistance. Toward better understanding this cell state, we sought to define the core transcriptome of accelerated senescence in cancer cells. Results We examined senescence induced by ionizing irradiation or ectopic overexpression of the stoichiometric cyclin-dependent kinase (CDK) inhibitor p21CIP/WAF1/SDI1 in the human breast cancer cell line MCF7. While radiation produces a strong DNA damage response, ectopic expression of p21 arrests cell cycle progression independently of DNA damage. Both conditions promoted senescence within 5 days. Microarray analysis revealed 378 up- and 391 down-regulated genes that were shared between the two conditions, representing a candidate signature. Systems analysis of the shared differentially expressed genes (DEGs) revealed strong signals for cell cycle control and DNA damage response pathways and predicted multiple upstream regulators previously linked to senescence. Querying the shared DEGs against the Connectivity Map (cmap) database of transcriptional responses to small molecules yielded 20 compounds that induce a similar gene expression pattern in MCF7 cells. Of 16 agents evaluated, six induced senescence on their own. Of these, the selective estrogen receptor degrader fulvestrant and the histone acetyltransferase inhibitor vorinostat did so without causing chromosomal damage. Conclusions Using a systems biology approach with experimental validation, we have defined a core gene expression signature for therapy-induced senescence

    EU member states\u27 exit taxation on individuals in conflict with the EU law

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