RESUMEN
Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)-mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.
Asunto(s)
Técnicas Biosensibles , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Ratones , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Proteínas de Unión al ARN/química , Reproducibilidad de los ResultadosRESUMEN
Calcineurin is responsible for mediating a wide variety of cellular processes in response to dynamic calcium (Ca(2+)) signals, yet the precise mechanisms involved in the spatiotemporal control of calcineurin signaling are poorly understood. Here, we use genetically encoded fluorescent biosensors to directly probe the role of cytosolic Ca(2+) oscillations in modulating calcineurin activity dynamics in insulin-secreting MIN6 ß-cells. We show that Ca(2+) oscillations induce distinct temporal patterns of calcineurin activity in the cytosol and plasma membrane vs at the ER and mitochondria in these cells. Furthermore, we found that these differential calcineurin activity patterns are determined by variations in the subcellular distribution of calmodulin (CaM), indicating that CaM plays an active role in shaping both the spatial and temporal aspects of calcineurin signaling. Together, our findings provide new insights into the mechanisms by which oscillatory signals are decoded to generate specific functional outputs within different cellular compartments.
Asunto(s)
Calcineurina/metabolismo , Señalización del Calcio/genética , Calcio/metabolismo , Calmodulina/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Calcineurina/genética , Calmodulina/genética , Compartimento Celular , Línea Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Secretoras de Insulina/citología , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
Fluorescence-based, genetically encodable biosensors are widely used tools for real-time analysis of biological processes. Over the last few decades, the number of available genetically encodable biosensors and the types of processes they can monitor have increased rapidly. Here, we aim to introduce the reader to general principles and practices in biosensor development and highlight ways in which biosensors can be used to illuminate outstanding questions of biological function. Specifically, we focus on sensors developed for monitoring kinase activity and use them to illustrate some common considerations for biosensor design. We describe several uses to which kinase and second-messenger biosensors have been put, and conclude with considerations for the use of biosensors once they are developed. Overall, as fluorescence-based biosensors continue to diversify and improve, we expect them to continue to be widely used as reliable and fruitful tools for gaining deeper insights into cellular and organismal function.
Asunto(s)
Técnicas Biosensibles , Proteínas Quinasas/metabolismo , Animales , Animales Modificados Genéticamente , Transferencia Resonante de Energía de Fluorescencia , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.