RESUMO
L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.
Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/metabolismo , Ácido Láctico/análise , Proteínas Periplásmicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cristalografia por Raios X , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Ácido Láctico/metabolismo , Microscopia de Fluorescência , Proteínas Periplásmicas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos TestesRESUMO
Human mesenchymal stem cells (hMSCs), which have the ability to differentiate into osteoblasts, show promise for bone tissue engineering and bone defect treatment. While there are a number of approaches currently available to accomplish this, e.g., utilizing biodegradable materials loaded with the synthetic glucocorticoid osteogenic inducer dexamethasone (DEX), there are still many disadvantages with the current technologies. Here, we generated light-responsive microgels that we showed are capable of loading and releasing DEX in a light-triggered fashion, with the released DEX being able to induce hMSC differentiation into osteoblasts. Specifically, light-responsive poly(N-isopropylacrylamide-co-nitrobenzyl methacrylate) (pNIPAm-co-NBMA) microgels were synthesized via free radical precipitation polymerization and their size, morphology, and chemical composition were characterized. We then went on to show that the microgels could be loaded with DEX (via what we think are hydrophobic interactions) and released upon exposure to UV light. We went on to show that the DEX released from the microgels was still capable of inducing osteogenic differentiation of hMSCs using an alamarBlue assay and normalized alkaline phosphatase (ALP) activity assay. We also investigated how hMSC differentiation was impacted by intermittent DEX released from UV-exposed microgels. Finally, we confirmed that the microgels themselves were not cytotoxic to hMSCs. Taken together, the DEX-loaded light-responsive microgels reported here may find a use for niche clinical applications, e.g., bone tissue repair.
Assuntos
Dexametasona/farmacologia , Luz , Células-Tronco Mesenquimais/efeitos dos fármacos , Microgéis/química , Antraquinonas/química , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dexametasona/química , Humanos , Estrutura Molecular , Osteogênese/efeitos dos fármacos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor's functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.
Assuntos
Cálcio/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Fluorescência , Células HeLa , Humanos , Ligação de Hidrogênio , Prótons , Espectrometria de Fluorescência/métodos , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Store-operated calcium entry (SOCE), primarily mediated by Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that has been linked to human diseases including myopathy, epilepsy, immunodeficiency, and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms underlying SOCE activation, the development of therapies against dysfunctional SOCE significantly lags behind, partly due to the lack of more specific pharmacological tools and poor understanding of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin. OBJECTIVE AND METHODS: Capitalizing on Ca2+ imaging and pharmacological tools, we aimed to systemically delineate the mechanism of action of digitoxin by defining how it impinges on Orai1 to exert its suppressive effect on SOCE. RESULTS: The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27-S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1. CONCLUSIONS: Our study identified two regions that are critical for the inhibition on Orai1 channels, providing valuable hotspots for future design of SOCE inhibitors.