RESUMO
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
Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one-third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging.
Assuntos
Envelhecimento/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Doenças Neurodegenerativas/prevenção & controle , Substâncias Protetoras/farmacologia , Deficiências na Proteostase/prevenção & controle , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Estresse do Retículo Endoplasmático/genética , Guanabenzo/farmacologia , Humanos , Indóis/farmacologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Proteoma/genética , Proteoma/metabolismo , Proteostase/efeitos dos fármacos , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
Sensory integration is vital for motile organisms constantly exposed to changing surroundings. Chlamydomonas reinhardtii is a single-celled green alga found swimming in freshwater. In this type of alga, sensory input is first detected by membrane receptors located in the cell body, and then transduced to the beating cilia by membrane depolarization. Many components of the machinery associated with sensory integration in C. reinhardtii, such as chemoreceptors and repolarization-associated channels, are yet uncharacterized. TRP channels are known mediators for cellular sensing in animal cells and it has been suggested that the C. reinhardtii genome encodes for a set of TRP proteins. Here, by combining behavioral studies with electrophysiological experiments conducted on both population and single alga, we test whether TRP channel blockers affect algal swimming behavior. Our results suggest that a TRP conductance is associated to the repolarization that follows a depolarizing receptor potential, highlighting a primitive function of TRP proteins.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Cílios/fisiologia , Potenciais da Membrana , Canais de Potencial de Receptor Transitório/metabolismo , Fenômenos Biológicos , Chlamydomonas reinhardtii/genética , Genoma , Dados de Sequência Molecular , Transdução de SinaisRESUMO
Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.
Assuntos
Membrana Celular/química , Proteínas de Fluorescência Verde/análise , Proteínas de Membrana/análise , Microscopia Confocal/métodos , Células HEK293 , Humanos , Canais Iônicos/análise , FotodegradaçãoRESUMO
BACKGROUND: The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane. METHODOLOGY/PRINCIPAL FINDINGS: We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2-8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MßCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability. CONCLUSIONS/SIGNIFICANCE: These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons.