RESUMEN
BACKGROUND: ICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Previous studies implied its involvement in generation, cargo storage, traffic, exocytosis and recycling of insulin secretory granules, as well as in ß-cell proliferation. While several ICA512 domains have been characterized, the function and structure of a large portion of its N-terminal extracellular (or lumenal) region are unknown. Here, we report a biophysical, biochemical, and functional characterization of ICA512-RESP18HD, a domain comprising residues 35 to 131 and homologous to regulated endocrine-specific protein 18 (RESP18). METHODS: Pure recombinant ICA512-RESP18HD was characterized by CD and fluorescence. Its binding to insulin and proinsulin was characterized by ELISA, surface plasmon resonance, and fluorescence anisotropy. Thiol reactivity was measured kinetically. Targeting of ΔRESP18HD ICA512-GFP to the membrane of insulinoma cells was monitored by immunofluorescence. RESULTS: ICA512-RESP18HD possesses a strong tendency to aggregate and polymerize via intermolecular disulfide formation, particularly at pH>4.5. Its cysteine residues are highly susceptible to oxidation forming an intramolecular disulfide between cysteine 53 and 62 and intermolecular disulfides via cysteine 40 and cysteine 47. The regulated sorting of ICA512 to secretory granules in INS-1 cells was impaired by deletion of RESP18HD. ICA512-RESP18HD binds with high-affinity to insulin and proinsulin. CONCLUSIONS: RESP18HD is required for efficient sorting of ICA512 to secretory granules. GENERAL SIGNIFICANCE: RESP18HD is a key determinant for ICA512 granule targeting.
Asunto(s)
Insulina/metabolismo , Proteínas del Tejido Nervioso/química , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/química , Secuencia de Aminoácidos/genética , Biofisica , Proliferación Celular/genética , Humanos , Insulina/química , Islotes Pancreáticos/química , Islotes Pancreáticos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Neuroendocrinas/química , Células Neuroendocrinas/metabolismo , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Secretoras/química , Vesículas Secretoras/metabolismoRESUMEN
This Commentary describes a call for contributions to an upcoming Special Issue (SI) of Biophysical Reviews on the Latin American Federation of Biophysical Societies (LAFeBS). It details the reason for the SI, the SI Editors' contact information, and the relevant submission details for those wishing to contribute a review manuscript.
RESUMEN
Lateral organization of biological membranes is frequently studied using fluorescence microscopy. One of the most widely used probes for these studies is 2-dimethylamino-6-lauroylnaphthalene (laurdan). The fluorescence of this probe is sensitive to the environment polarity, and thus laurdan reports the local penetration of water when inserted in membranes. Unfortunately, this probe can only be used under two-photon excitation due to its low photostability. This is a very important limitation, because there are not too many laboratories with capability for two-photon microscopy. In this work, we explored the performance of 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), a carboxyl-modified version of laurdan, for imaging biological membranes using a conventional confocal microscopy setup. We acquired generalized polarization (GP) images of C-laurdan inserted in giant unillamelar vesicles composed of binary mixtures of lipids and verified that the probe allows observing the coexistence of different phases. We also tested the performance of the probe for measurement with living cells and registered GP images of melanophore cells labeled with C-laurdan in which we could observe highly ordered regions such as filopodia. These findings show that C-laurdan can be successfully employed for studies of membrane lateral organization using a conventional confocal microscope and can open the possibility of studying a wide variety of membrane-related processes.
Asunto(s)
2-Naftilamina/análogos & derivados , Membrana Celular/química , Lauratos/química , Microscopía Confocal/métodos , 2-Naftilamina/química , Animales , Microscopía Fluorescente , Liposomas Unilamelares/químicaRESUMEN
This Commentary describes a call for contributions to an upcoming Special Issue (SI) of Biophysical Reviews on the Latin American Federation of Biophysical Societies (LAFeBS). It details the reason for the SI, the SI Editors contact information and the relevant submission details for those wishing to contribute.