Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway

Mecanismos moleculares de respuesta al estrés oxidativo mediados por la ruta de integridad celular (vía PKC1-MAP quinasa)de Saccharonmyces cerevisiae

La via d'integritat cel·lular o via Pkc1-MAP quinasa de Saccharomyces cerevisiae té un paper central en els mecanismes de resposta cel·lular enfront de diferents estressos ambientals, com són: l'estrès tèrmic, l'estrès hipoosmòtic o qualsevol estrès que afecti a la paret cel·lular. En aquest treball de tesi doctoral es demostra que aquesta via de transducció de senyal també està implicada en la supervivència iadaptació enfront els efectes de l'estrès oxidatiu.Hem observat que les proteïnes Pkc1 i Rom2 són essencials per a la supervivència cel·lular enfront de l'estrès oxidatiu generat per dos agents (peròxid d'hidrogen i diamida). A més, el citoesquelet d'actina és una de les dianes d'acció del peròxid d'hidrogen i de la diamida; aquests dos agents provoquen una despolarització del citoesquelet d'actina amb el conseqüent efecte en morfogènesi i viabilitat. Aquest fenomen succeeix de forma independent de les proteïnes de la via d'integritat cel·lular.Per altra banda, perquè el citoesquelet d'actina es repolaritzi com a resposta d'agents oxidants, són necessàries les proteïnes Mtl1, Rom2 i Pkc1, totes elles són elements que constitueixen la via d'integritat cel·lular.La diamida indueix la formació de ponts disulfur a les proteïnes de la superfície cel·lular. Aquest fet provoca canvis estructurals en la cèl·lula que, en darrer terme,causen l'activació de la via d'integritat cel·lular. Els nostres estudis han permès assignar una funció al receptor de la paret cel·lular Mtl1, com un sensor d'estrès oxidatiu en superfície i part integrant de la via Pkc1-MAP quinasa.El peròxid d'hidrogen afecta essencialment una funció cel·lular relacionada amb els últims estadis de secreció i la morfogènesi cel·lular. A més, observem dos funcions essencials de la proteïna Pkc1 com a resposta al tractament amb peròxid d'hidrogen: i)Pkc1 regula la biogènesi de ribosomes com a resposta als problemes en secreció que provoca l'agent oxidant. Així mateix, la inhibició de la transcripció de gens ribosomals es du a terme depenent d'un citoesquelet en polimerització activa, ii) Pkc1 indueix la restauració dels cables d'actina i també de la morfogènesi i polaritat cel·lular a través de l'activació de la profilina Pfy1.La vía de integridad celular o vía Pkc1-MAP quinasa de Saccharomyces cerevisiae posee un papel central en los mecanismos de respuesta celular frente a diferentes estreses ambientales, como son: el estrés térmico, el estrés hipoosmótico o cualquier estrés que afecte a la pared celular. En esta tesis doctoral se demuestra que esta vía de transducción de señal también está implicada en la supervivencia y adaptación frente a los efectos del estrés oxidativo.Hemos observado que las proteínas Pkc1 y Rom2 son esenciales para la supervivencia celular frente al estrés oxidativo mediado por dos agentes (peróxido de hidrógeno y diamida). Además, el citoesqueleto de actina es una de las dianas de acción del peróxido de hidrógeno y de la diamida, ambos agentes median la despolarización del citoesqueleto de actina con el consecuente efecto en morfogénesis y viabilidad. Este fenómeno ocurre de manera independiente de las proteínas de la vía de integridad celular. Sin embargo, para que el citoesqueleto de actina repolarice en respuesta a agentes oxidantes se requieren las proteínas Mtl1, Rom2 y Pkc1, todas ellas elementosconstituyentes de la vía de integridad celular.La diamida induce la formación de puentes disulfuro en las proteínas de la superficie celular, lo que provoca cambios estructurales en la misma, que en última instancia, causan la activación de la vía de integridad celular. Nuestros estudios han permitido asignar una función al receptor de pared celular Mtl1, como sensor de estrés oxidativo en superficie, y parte integrante de la vía Pkc1-MAP quinasa.El peróxido de hidrógeno afecta esencialmente a una función celular relacionada con los últimos estadios de secreción y con la morfogénesis celular. Además observamos dos funciones esenciales de la proteína Pkc1 en respuesta al tratamiento con peróxido de hidrógeno: i) Pkc1 regula la biogénesis de ribosomas en respuesta a los problemas en secreción provocado por el agente oxidante. Además, la inhibición de latrascripción de genes ribosomales se lleva a cabo dependiendo de un citoesqueleto en polimerización activa, ii) Pkc1 induce la restauración de los cables de actina y con ello de la morfogénesis y polaridad celular a través de la activación de la profilina Pfy1.The cell integrity pathway or Pkc1-MAP kinase pathway of Saccharomyces cerevisiae plays a central role in the cellular responses against different environmental stresses, such as: heat shock, hipoosmotic shock, starvation stresses, all of them affect the cell wall. This study shows that this transductional pathway is also involved in the survival and the adaptation of the cell against the affects of oxidative stress.We have observed that the proteins Pkc1 and Rom2 are essential for cell survival against oxidative stress caused by two agents: hydrogen peroxide and diamide. In addition, the actin cytoskeleton is one of the targets of both hydrogen peroxide and diamide, these agents cause depolarization of the actin cytoskeleton with a detrimental effect in cell morphogenesis and viability. This occurs independently on the cell integrity pathway proteins. However, for the actin cytoskeleton to repolarize in response to oxidant agents, the cells need the proteins Mtl1, Rom2 and Pkc1, all of them constitute the cell integrity pathway.Diamide causes the formation of disulphide bridges in the cell surface proteins, which provokes structural changes in the cell surface, and which finally induces the activation of the cell integrity pathway. In our investigation we have been able to assign a function to the cell wall receptor Mtl1, as a sensor of surface oxidative stress and an integral part of the Pkc1-MAP kinase pathway.Hydrogen peroxide affects primarily a cellular function related to the last stages of vesicular secretion. We also observe two essential functions of the protein Pkc1 in response to the treatment with hydrogen peroxide: i) Pkc1 regulates ribosomal biogenesis in response to problems in secretion, caused by the oxidant agent. The occurrence of the inhibition of ribosomal genes transcription is dependent on the active polymerization of the actin cytoskeleton, ii) Pkc1 induces the restoration of actin cables then promoting the restauration of cellular polarity and morphogenetic mechanisms through the activation of Pfy1 profilin

Pkc1 and the upstream elements of the cell integrity pathway in Saccharomyces cerevisiae, Rom2 and Mtl1, are required for cellular responses to oxidative stress

In this study we analyze the participation of the PKC1-MAPK cell integrity pathway in cellular re- sponses to oxidative stress in Saccharomyces cerevisiae. Evidence is presented demonstrating that only Pkc1 and the upstream elements of the cell integrity pathway are essential for cell survival upon treatment with two oxidizing agents, diamide and hydrogen peroxide. Mtl1 is characterized for the first time as a cell-wall sensor of oxidative stress. We also show that the actin cytoskele- ton is a cellular target for oxidative stress. Both diamide and hydrogen peroxide provoke a marked depolariza- tion of the actin cytoskeleton, being Mtl1, Rom2 and Pkc1 functions all required to restore the correct actin organization. Diamide induces the formation of disul- fide bonds in newly secreted cell-wall proteins. This mainly provokes structural changes in the cell outer layer, which activate the PKC1-MAPK pathway and hence the protein kinase Slt2. Our results led us to the conclusion that Pkc1 activity is required to overcome the effects of oxidative stress by: (i) enhancing the ma- chinery required to repair the altered cell wall and (ii) restoring actin cytoskeleton polarity by promoting actin cable formation

Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle

In a human menstrual cycle the endometrium undergoes remodeling, shedding and regeneration, all of which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, our understanding of this unique type of tissue homeostasis remains rudimentary. We characterized the transcriptomic transformation of human endometrium at single-cell resolution across the menstrual cycle, resolving cellular heterogeneity in multiple dimensions. We profiled the behavior of seven endometrial cell types, including a previously uncharacterized ciliated cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that the human window of implantation opens with an abrupt and discontinuous transcriptomic activation in the epithelia, accompanied with a widespread decidualization feature in the stromal fibroblasts. Our study provides a high-resolution molecular and cellular characterization of human endometrial transformation across the menstrual cycle, providing insights into this essential physiological process

The human periconceptional maternal-embryonic space in health and disease

Pregnancy is established during the periconceptional period as a continuum beginning with blastocyst attachment to the endometrial epithelial surface followed by embryo invasion and placenta formation. This period sets the foundation for the child and mother's health during pregnancy. Emerging evidence indicates that prevention of downstream pathologies in both the embryo/newborn and pregnant mother may be possible at this stage. In this review, we discuss current advances in the periconceptional space, including the preimplantation human embryo and maternal endometrium. We also discuss the role of the maternal decidua, the periconceptional maternal-embryonic interface, the dialogue between these elements, and the importance of the endometrial microbiome in the implantation process and pregnancy. Finally, we discuss the myometrium in the periconceptional space and review its role in determining pregnancy health