RESUMO
The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.
Assuntos
Lisossomos , Organelas , Potenciais da Membrana , Organelas/metabolismo , Lisossomos/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismoRESUMO
Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.
Assuntos
Técnicas Biossensoriais , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Potenciais da Membrana , Microscopia de Fluorescência , Imagem Óptica , Animais , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Optogenética , Células PC12 , RatosRESUMO
Acute serum amyloid A (A-SAA) has been considered a major acute-phase reactant and an effector of innate immunity in all vertebrates. The work presented here shows that the expression of A-SAA is strongly induced in a wide variety of immune-relevant tissues in rainbow trout, either naturally infected with Flavobacterium psychrophilum or challenged with lipopolysaccharide (LPS) or CpG oligonucleotides (CpG ODN). Nevertheless, A-SAA was undetectable by Western blot either in the plasma or in high-density lipoprotein (HDL) of infected or challenged fish, using either an anti-mouse SAA1 IgG or an anti-trout A-SAA peptide serum, which recognise both the intact recombinant trout A-SAA and fragments derived from it. However, the anti-peptide serum was the immunoreactive in all primary defence barriers and in mononuclear cells of head kidney, spleen and liver. These findings reveal that, unlike mammalian SAA, trout A-SAA does not increase significantly in the plasma of diseased fish, suggesting it is more likely to be involved in local defence.