Rga6, una GAP de Cdc42, implicada en el control de la morfología celular de Schizosaccharomyces pombe

Memoria presentada por Mª Teresa Revilla Guarinos para optar al Grado de Doctor en Biología Funcional y Genómica, y que ha sido realizada en el Instituto de Biología Funcional y Genómica, centro mixto de la Universidad de Salamanca (Departamento de Microbiología y Genética) y el Consejo Superior de Investigaciones Científicas.La morfogénesis constituye junto con el crecimiento y la diferenciación celular, uno de los aspectos más importantes del desarrollo de un organismo. Muchas de las principales moléculas implicadas en el control del crecimiento polarizado y la morfogénesis, como las GTPasas Rho, están conservadas desde levaduras a mamíferos. En nuestro grupo estudiamos el papel de las GTPasas Rho en el crecimiento polarizado empleando como organismo modelo la levadura de fisión Schizosaccharomyces pombe. Esta levadura posee una morfología cilíndrica y crece por los polos. Su forma, tamaño y división a lo largo del ciclo son muy reproducibles en el laboratorio. Gracias a ello, es un buen organismo modelo para estudiar el control de la morfología celular. S. pombe contiene 6 genes que codifican GTPasas de la familia Rho, Rho1-Rho5 y Cdc42. Cdc42 es esencial para la viabilidad celular. Se localiza tanto en la membrana plasmática, como en las membranas internas y es la GTPasa implicada en el control del crecimiento celular polarizado. Como todas las GTPasas de la familia Rho, la señalización de Cdc42 está regulada por proteínas activadoras denominadas GEFs (del inglés “Guanine nucleotide Exchange Factors”) e inhibidoras denominadas GAPs (“GTPases Activating Proteins”). En la levadura de fisión, el control espacial de la activación de Cdc42 determina su morfología celular. Los GEFs, Scd1 y GEF1, se localizan en las zonas de crecimiento, polos y septo, donde Cdc42 está activa y la única proteína GAP descrita hasta la fecha, Rga4, se localiza en la membrana lateral de la célula, en las zonas sin crecimiento, donde Cdc42 está inactiva. Scd1 y Rga4 tienen papeles aditivos controlando la morfología celular. Gef1 por su parte, parece estar más implicado en los procesos de citocinesis y en la transición del crecimiento monopolar a bipolar denominada NETO (New End Take Off). En los polos, la dinámica de activación de Cdc42 presenta un comportamiento oscilatorio. Cdc42 controla el crecimiento celular polarizado mediante su proteína efectora For3. Esta formina promueve la formación de cables de actina en interfase, implicados, junto con las miosinas de clase V, en el movimiento de las vesículas secretoras, que contienen el nuevo material sintetizado, hacia las zonas de crecimiento activo. Además existe relación entre Cdc42 y el complejo proteico denominado exocisto, implicado en la fusión de las vesículas procedentes del Aparato de Golgi con la membrana plasmática. Aunque hasta la fecha no está claro como Cdc42 regula el exocisto, sí se sabe que la secreción polarizada llevada a cabo por For3 y el correcto funcionamiento del exocisto, son 2 procesos fundamentales para la correcta morfología de la célula.Esta tesis doctoral ha sido financiada por una beca predoctoral de Formación de Personal Investigador (FPI) del Ministerio de Economía y Competitividad del Gobierno de España con referencia BES-2011-045514 y asociada al proyecto de investigación BFU2010-15641 en el Organismo Consejo Superior de Investigaciones Científicas (CSIC).Peer Reviewe

Characterization of a Regulatory Network of Peptide Antibiotic Detoxification Modules in <em>Lactobacillus casei</em> BL23

Two-component systems (TCS) are major signal transduction pathways that allow bacteria to detect and respond to environmental and intracellular changes. A group of TCS has been shown to be involved in the response against antimicrobial peptides (AMPs). These TCS are characterized by the possession of intramembrane-sensing histidine kinases, and they are usually associated with ABC transporters of the peptide-7 exporter family (Pep7E). Lactobacillus casei BL23 encodes two TCS belonging to this group (TCS09 and TCS12) that are located next to two ABC transporters (ABC09 and ABC12), as well as a third Pep7E ABC transporter not genetically associated with any TCS (orphan ABC). This study addressed the involvement of modules TCS09/ABC09 and TCS12/ABC12 in AMP resistance. Results showed that both systems contribute to L. casei resistance to AMPs, and that each TCS constitutes a functional unit with its corresponding ABC transporter. Analysis of transcriptional levels showed that module 09 is required for the induction of ABC09 expression in response to nisin. In contrast, module 12 controls a wider regulon that encompasses the orphan ABC, the dlt operon (d-alanylation of teichoid acids), and the mprF gene (l-lysinylation of phospholipids), thereby controlling properties of the cell envelope. Furthermore, the characterization of a dltA mutant showed that Dlt plays a major role in AMP resistance in L. casei. This is the first report on the regulation of the response of L. casei to AMPs, giving insight into its ability to adapt to the challenging environments that it encounters as a probiotic microorganism

Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions.This work was supported by Grants BFU2013-43439-P and BIO2015-69958-P from the Ministry of Economy and Competitiveness, Spain, and Grant CSI037U14 from Junta de Castilla y León, Spain.Peer Reviewe

Rga6 is a Rho GAP involved in fission yeast polarized growth

Resumen del póster presentado al American Society for Cell Biology (ASCB) Annual Meeting: "Cell Biology", celebrado en San Diego, California (US) del 12 al 16 de diciembre de 2015.Fission yeast Cdc42 is essential for polarized growth and the maintenance of cell dimensions and cylindrical morphology. This GTPase is positively regulated by the GEFs Scd1 and Gef1 and negatively regulated by the GAP Rga4. This study establishes that the protein Rga6 is also a Cdc42 GAP that collaborates with Rga4 in the maintenance of fission yeast shape and the restriction of growth to the tip area. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking simultaneously both GAPs are rounded. In these cells, active Cdc42 is observed all along the membrane. Overexpression of rga6+ for brief periods of time (12-20h) causes cells to elongate, and grow in a monopolar way, as does overexpression of Rga4, and for longer times is deleterious. Rga6 localizes to the plasma membrane forming clusters which are different from those formed by Rga4. Rga6 clusters are very dynamic and move by lateral diffusion. A polybasic region at the Rga6 C-terminus is responsable for its membrane localization. Rga6-GFP fluorescence is considerably reduced at the growing tips and this reduction is dependent on the N-terminal region of Rga6 and on the actin cables made by the formin For3. We propose here that Rga6 participates in the regulation of active Cdc42 localization and in the amplitude of active Cdc42 oscillations at the cell tips.Peer Reviewe