Diseño de un nuevo sensor de ca2+ y su aplicación a los orgánulos intracelulares ex vivo e in vivo
- Autores: Paloma Navas Navarro
- Directores de la Tesis: María Teresa Alonso Alonso (dir. tes.), Javier García Sancho (codir. tes.)
- Lectura: En la Universidad de Valladolid ( España ) en 2016
- Idioma: español
- Tribunal Calificador de la Tesis: Javier Álvarez Martín (presid.), Pablo Chamero Benito (secret.), Juan Llopis Borrás (voc.), Miguel Angel Valdeolmillos López (voc.), M. José Sánchez Barrena (voc.)
- Programa de doctorado: Programa de Doctorado en Investigación Biomédica por la Universidad de Valladolid
- Materias:
- Enlaces
- Tesis en acceso abierto en: UVADOC
- Resumen
Ca2+ is a ubiquitous messenger involved in a plethora of cellular processes, including muscle contraction, secretion, gene expression or differentiation. Ca2+ signals occur when its cytosolic concentration ([Ca2+]C) increases. However, maintained high [Ca2+]C is toxic and it must be extruded to the extracellular space or sequestered into the endoplasmic reticulum (ER).
The ER is the main cellular Ca2+ store and it contains high Ca2+ concentration in its lumen, neccesary for the correct function of this organelle, involved in protein folding and transport. Chronic decrease of the concentration of Ca2+ in the ER ([Ca2+]ER) triggers the unfolded protein response, and eventually, cell death. The ER actively contributes to cytosolic Ca2+ signalling, releasing Ca2+ through the channels present in its membrane to the cytosol, following a favorable electrochemical gradient. Once the stimulus ceases, the ER uptakes Ca2+ back to its lumen. For all these reasons, monitoring the Ca2+ homeostasis directly in the ER lumen is essential to fully understand the cellular physiology and pathology.
At present, a broad variety of genetically encoded Ca2+ sensors exist, either bioluminescent or fluorescent, whose light emission changes depending on [Ca2+]. However, most of the available sensors are of high affinity for Ca2+, not suitable to measure the high [Ca2+] existing inside the ER. The photoprotein aequorin, originally isolated from the jellyfish Aequorea victoria, was the first Ca2+ sensor used to monitor this ion in the ER. Aequorin has provided important insights in the Ca2+ signalling field, but it has two caveats: first, its poor spatial resolution seriously limits its use to do cell imaging; second, it needs to be reconstituted with its cofactor coelenterazine, which complicates in vivo measurements in the ER. By contrast, low affinity fluorescent Ca2+ sensors (composed of one or two fluorescent proteins and one Ca2+ binding protein) elicit imaging measurements with good spatial and temporal resolution.
Our group has recently developed a new class of fluorescent Ca2+ sensors based on the fusion of two proteins from the jellyfish Aequorea, green fluorescent protein (GFP) and aequorin, called GAP (GFP Aequorin Protein). The use of aequorin instead of the most widely used calmodulin as the Ca2+ binding protein makes it bio-orthogonal, that is, not perturbed by undesired interactions with endogenous effectors. GAP exhibits an excitation spectrum with two maximums, at 405 and 470 nm; in the presence of Ca2+, the first peak decreases and the second one increases, allowing ratiometric measurements. GAP affinity is tunable, with a high affinity variant (GAP, Kd = 660 nM) and an intermediate affinity variant (GAP1, Kd = 12 microM).
The aim of this project was to engineer a low affinity variant of GAP, which enables to measure the Ca2+ concentration in high Ca2+ content organelles, such as the ER or the Golgi apparatus. For this purpose, aequorin was first mutated in 22 residues of the three EF hands. The resulting proteins were produced and purified in E. coli, and screened in vitro in a plate reader fluorescent assay. One of the variants, the double mutant D24N/D119A, named GAP2, displayed an ideal affinity (Kd = 410 microM) to measure [Ca2+]ER. GAP2 was somewhat dimmer than its parental GAP or GAP1. In order to compensate this loss of fluorescence, additional substitutions within the GFP moiety were introduced, generating GAP3, which displayed a 2-fold increased fluorescence with respect to GAP2. The new sensors were fully characterized in vitro and in the ER of HeLa cells. They were ratiometric, displayed a dynamic range of 3-4 fold, a Hill coefficient of 1 and they were quite insensitive to pH or Mg2+.
GAP2 and GAP3 are targetable to the ER, where they successfully recorded stimuli-induced Ca2+ releases from the ER in HeLa cells, with IP3-coupled stimuli and with SERCA pump inhibitors. Moreover, simultaneous imaging of [Ca2+]ER and [Ca2+]C were recorded by combining GAP with spectrally compatible synthetic sensors, such as Rhod-3.
The ability of new Ca2+ sensors to record [Ca2+]ER in more intact tissues was assessed by expressing ER-targeted GAP in transgenic mice, under the control of a ubiquitous promoter. Expression of the erGAP transgene was found in virtually all tissues tested. We measured ER Ca2+ dynamics in the following cell systems: monocytes, hippocampal neurons (in dissociated cultures and acute slices), pancreatic islets and hypophysis. The ability of GAP to measure [Ca2+]ER in vivo was addressed by creating transgenic flies for erGAP3 specifically expressed in the sarcoplasmic reticulum of skeletal muscles. In vivo recordings were performed in thoracic fly muscles electrically stimulated through the motoneurons.
The use of the new Ca2+ sensors in combination with new disease models, such as those provided by iPS cell technology or aging models, promises novel approaches to investigate the pathophysiological relevance of organellar Ca2+ dyshomeostasis.
