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Animals defend a target level for their fundamental needs, including food, water, and sleep. Deviation from the target range, or "setpoint," triggers motivated behaviors to eliminate that difference. Whether and how the setpoint itself is encoded remains enigmatic for all motivated behaviors. Employing a high-throughput feeding assay in Drosophila, we demonstrate that the protein intake setpoint is set to different values in male, virgin female, and mated female flies to meet their varying protein demands. Leveraging this setpoint variability, we found, remarkably, that the information on the intake setpoint is stored within the protein hunger neurons as the resting membrane potential. Two RFamide G protein-coupled receptor (GPCR) pathways, by tuning the resting membrane potential in opposite directions, coordinately program and adjust the protein intake setpoint. Together, our studies map the protein intake setpoint to a single trackable physiological parameter and elucidate the cellular and molecular mechanisms underlying setpoint determination and modulation.
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Endoplasmic reticulum (ER) membrane junctions are formed by the dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long unbranched ER tubules in cells, and mutation of human ATL1 is linked to hereditary spastic paraplegia. Here, we demonstrate that COPII formation is drastically decreased in the periphery of ATL-deleted cells. ER export of cargo proteins becomes defective; ER exit site initiation is not affected, but many of the sites fail to recruit COPII subunits. The efficiency of cargo packaging into COPII vesicles is significantly reduced in cells lacking ATLs, or when the ER is transiently fragmented. Cargo is less mobile in the ER in the absence of ATL, but the cargo mobility and COPII formation can be restored by ATL R77A, which is capable of tethering, but not fusing, ER tubules. These findings suggest that the generation of ER junctions by ATL plays a critical role in maintaining the necessary mobility of ER contents to allow efficient packaging of cargo proteins into COPII vesicles.
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Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Retículo Endoplasmático/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mutantes/genética , Deleção de Sequência/genética , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologiaRESUMO
Planar cell polarity (PCP) is a process during which cells are polarized along the plane of the epithelium and is regulated by several transmembrane signaling proteins. After their synthesis, these PCP proteins are delivered along the secretory transport pathway to the plasma membrane, where they perform their physiological functions. However, the molecular mechanisms that regulate PCP protein transport remain largely unclear. Here, we found that the delivery of a PCP protein, Frizzled-6, to the cell surface is regulated by two conserved polybasic motifs: one located in its first intracellular loop and the other in its C-terminal cytosolic domain. We observed that the polybasic motif of Frizzled is also important for its surface localization in the Drosophila wing. Results from a mechanistic analysis indicated that Frizzled-6 packaging into vesicles at the endoplasmic reticulum (ER) is regulated by a direct interaction between the polybasic motif and the Glu-62 and Glu-63 residues on the secretion-associated Ras-related GTPase 1A (SAR1A) subunit of coat protein complex II (COPII). Moreover, we found that newly synthesized Frizzled-6 is associated with another PCP protein, cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), in the secretory transport pathway, and that this association regulates their surface delivery. Our results reveal insights into the molecular machinery that regulates the ER export of Frizzled-6. They also suggest that the association of CELSR1 with Frizzled-6 is important, enabling efficient Frizzled-6 delivery to the cell surface, providing a quality control mechanism that ensures the appropriate stoichiometry of these two PCP proteins at cell boundaries.
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Polaridade Celular/fisiologia , Receptores Frizzled/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Receptores Frizzled/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Domínios Proteicos/fisiologia , Transporte Proteico/fisiologia , Via SecretóriaRESUMO
In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.
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Membrana Celular/metabolismo , Polaridade Celular , Receptores Frizzled/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Sequência de Aminoácidos , Receptores Frizzled/genética , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Ligação Proteica , Transporte Proteico , Homologia de SequênciaRESUMO
Amyloid fibrils are involved in several neurodegenerative diseases. However, because of their polymorphism and low concentration, they are challenging to assess in real-time with conventional techniques. Here, we present a new approach for the characterization of the intermediates: protofibrils and "end-off" aggregates which are produced during the amyloid formation. To do so, we have fashioned conical track-etched nanopores that are functionalized to prevent the fouling. Using these nanopores, we have followed the kinetic of amyloid growth to discriminate the different intermediates protofibrils and "end-off. Then, the nanopore was used to characterize the effect of promoter and inhibitor of the fibrillation process. Finally, we have followed in real-time the degradation of amyloid with peptase. Compare with the SiN nanopore, the track-etched one features exceptionally high success rate via functionalization and detection in "one-pot". Our results demonstrate the potential for a conical nanopore to be used as a routine technique for the characterization of the amyloid growth and/or degradation.
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Amiloide/química , Lactoglobulinas/química , Nanoporos , Multimerização Proteica , Proteólise , Amiloide/antagonistas & inibidores , Curcumina/química , Sulfato de Dextrana/química , Etanol/química , Concentração de Íons de Hidrogênio , Cinética , Nanoporos/ultraestrutura , Pepsina A/química , Polietilenoglicóis/química , Multimerização Proteica/efeitos dos fármacos , Quercetina/químicaRESUMO
Single conical nanopores were functionalised layer by layer with weak polyelectrolytes. We studied their influence on the ionic diode properties We have considered different couples of polyelectrolytes: poly-l-lysine/poly(acrylic acid) and poly(ethyleneimine)/poly(acrylic acid) as well as the influence of cross-linking. The results show that the nanopores decorated with poly(ethyleneimine)/poly(acrylic acid) exhibit an interesting behavior. Indeed, at pH 3, the nanopore is open only at the low salt concentration, while at pH 7, it is already open. The nanopores functionalized with poly-l-lysine/poly(acrylic acid) do not show an inversion of ionic transport properties with the pH as expected. After cross-linked to prevent large conformational changes, the ionic diode properties are dependent on the pH.
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Multi-configurational Ehrenfest (MCE) approaches, which are intended to remedy the lack of correlations in the standard mean-field Ehrenfest method, have been proposed as coherent-state based ansätze for quantum propagation [D. V. Shalashilin, J. Chem. Phys. 130, 244101 (2009)] and also as the classical limit of the variational Gaussian-based multiconfiguration time dependent Hartree (G-MCTDH) method [S. Römer and I. Burghardt, Mol. Phys. 111, 3618 (2013)]. In the present paper, we establish the formal connection between these schemes and assess the performance of MCE for a coherent-state representation of the classical-limit subsystem. As a representative model system, we address the ultrafast, coherent charge transfer dynamics in an oligothiophene-fullerene donor-acceptor complex described by a two-state linear vibronic coupling model. MCE calculations are compared with reference calculations performed with the MCTDH method, for 10-40 vibrational modes. Beyond a dimensionality of 10 modes, it is shown that the correct representation of electronic coherence depends crucially on the sampling of initially unoccupied Gaussians.
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Nanopore sensors provide a unique platform to detect individual nucleic acids, proteins, and other biomolecules without the need for fluorescent labeling or chemical modifications. Solid-state nanopores offer the potential to integrate nanopore sensing with other technologies such as field-effect transistors (FETs), optics, plasmonics, and microfluidics, thereby attracting attention to the development of commercial instruments for diagnostics and healthcare applications. Stable nanopores with ideal dimensions are particularly critical for nanopore sensors to be integrated into other sensing devices and provide a high signal-to-noise ratio. Nanopore fabrication, although having benefited largely from the development of sophisticated nanofabrication techniques, remains a challenge in terms of cost, time consumption and accessibility. One of the latest developed methods-controlled breakdown (CBD)-has made the nanopore technique broadly accessible, boosting the use of nanopore sensing in both fundamental research and biomedical applications. Many works have been developed to improve the efficiency and robustness of pore formation by CBD. However, nanopores formed by traditional CBD are randomly positioned in the membrane. To expand nanopore sensing to a wider biomedical application, controlling the localization of nanopores formed by CBD is essential. This article reviews the recent strategies to control the location of nanopores formed by CBD. We discuss the fundamental mechanism and the efforts of different approaches to confine the region of nanopore formation.
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The modification of the inner nanopore wall by polymers is currently used to change the specific properties of the nanosystem. Among them, the polyethylene glycol (PEG) is the most used to prevent the fouling and ensure the wettability. However, its properties depend mainly on the chain structure that is very difficult to estimate inside this confined space. Combining experimental and simulation approaches, we provide an insight to the consequence of the PEG presence inside the nanopore on the nanopore properties. We show, in particular, that the cation type in the electrolyte, together with the type of electrolyte (water or urea), is at the origin of the ion transport modification in the nanopore.
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The switching of "ON" and "OFF" states of an ionic diode is investigated by considering a conical nanopore partially functionalized two polyelectrolyte (PE) layers via layer-by-layer deposition. Through observing the inversion of its rectification behavior, we demonstrate the function of the PE bilayers in ionic transport regulation. The ionic diode exhibits an ultrahigh ion rectification at a low level of pH. In an aqueous NaCl solution at pH 2, for example, the ratio of the current at "ON" state and that at "OFF" state can be about 800 and 200 for 1 and 100 mM, respectively. This remarkable gating behavior can be explained by the anion-pump-induced ion accumulation in the neutral region as well as the depletion zone at the interface. Our results further demonstrate the possibility of achieving an ultrahigh rectification in an ionic diode having a unipolar-like configuration.
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This research reports the transport behaviors of long flexible polymers (hyaluronic acid) through long conical track-etched nanochannels with and without grafted enzymes. The impacts of the channel diameter and the polymer regimes in solution (dilute and semi-dilute) have been investigated. Without enzymes, the experimental results can be well explained by the analytical models of the scaling law of de Gennes. Then, the corresponding enzymes (hyaluronidase) were grafted inside the channel. When enzymes are located at the base side, polymers get degraded at the entrance and the degraded products are detected. When enzymes are grafted at the tip side, the extension of translocation duration due to the binding of substrate-enzyme is observed. This is for the first time that the enzymatic degradation reactions are characterized in situ at the single molecule level by nanopore technology.
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Ácido Hialurônico/química , Hialuronoglucosaminidase/química , Modelos Químicos , NanoporosRESUMO
The possibility of tuning the current rectification and selectivity in nanofluidic diodes is demonstrated both experimentally and theoretically through dynamically functionalizing a conical nanopore with poly-l-lysine. We identified an optimum functionalization time equivalent to optimum modification depth that assures the highest rectification degrees. Results showed that the functionalization time-dependent rectification behavior of nanofluidic diodes is dominated by the properties of current at positive voltages that in our electrode configuration indicate the "on" state of the diode and accumulation of ions in the nanopore. The functionalization time also tunes the ion selectivity of the diode. If the functionalization time is sufficiently short, an unusual depletion of counterions near the bipolar interface results in a cation-selective nanopore. However, a further increase in the duration of functionalization renders a nanopore that is an anion-selective nanopore. The dynamic functionalization presented in this Letter enables tuning ion selectivity of nanopores.
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The heparin contamination by oversulfated chondroitin (OSCS) was at the origin of one major sanitary problem of last decade. Here we propose a novel strategy to detect OSCS from heparin solution based on conical nanopore functionalized with poly-L-lysine deposition to ensure its re-usability. This sensor is an excellent to detect low heparin concentration (from 25â¯ng/ml to 3⯵g/ml) using the modification of ionic current rectification. It also allows following the kinetic of heparin degradation by heparinase with a good correlation with results obtained by classical methods. The sensor is sensitive to the inhibition of heparinase by OSCS until a concentration of 200â¯pg/ml representing 0.01% in weight in a heparin. This resolution is one order of magnitude lower than the one obtained by chromatography. For the first time, it was reached without fluorescence labeling.
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Técnicas Biossensoriais , Sulfatos de Condroitina/isolamento & purificação , Contaminação de Medicamentos , Heparina/química , Sulfatos de Condroitina/química , Heparina Liase/química , Humanos , NanoporosRESUMO
The salinity gradient between brine and fresh water is an abundant source of power which can be harvested by two major membrane methods: pressure-retarded osmosis and reversed electrodialysis. Nowadays, the latter technology is close to real application, but it still suffers from low power yield. Low membrane selectivity and complex membrane fabrication are the main limiting factors. To improve that, we design a couple of ion-selective membranes based on the track-etched polymer nanopore functionalized by highly charged hydrogels. Two nanopore geometries are compared (cylindrical and conical shape) to generate osmotic energy with gel functions and more importantly can be scaled up. Experiments from the single nanopore and multipore membrane to stacked membranes show complete characterization from ionic transportation to energy generation and a clear relationship from the single pore to stacked membranes. In the actual experiment conditions, a power density of 0.37 W m-2 at pH 7 was achieved. By improving ionic tracks and reducing intermembrane distances, it can be a good candidate for industrial applications.
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The trans-Golgi network (TGN) and endosomes are essential protein sorting stations in the secretory transport pathway. Protein sorting is fundamentally a process of spatial segregation, but the spatial relationships among the proteins that constitute the sorting machinery have not been systematically analyzed at high resolution in mammalian cells. Here, using two-color STORM imaging, we show that the TGN/endosome-localized cargo adaptors, AP-1, GGA2 and epsinR, form elongated structures of over 250 nm in length at the juxta-nuclear Golgi area. Many of these structures are associated with clathrin. We found that AP-1 is spatially segregated from AP-3 and GGA2, whereas a fraction of AP-1 and GGA2 punctae are associated with epsinR. Moreover, we observed that the planar cell polarity cargo proteins, Vangl2 and Frizzled6 associate with different cargo adaptors-AP-1 and GGA2 or epsinR, respectively-when exiting the TGN. Knockdown analysis confirms the functional significance of this segregation. Our data indicates that TGN/endosome-localized cargo adaptors have distinct spatial relationships. The spatially segregated cargo adaptors GGA2 and AP-1 regulate sorting of Frizzled6 and Vangl2, respectively and spatially associated cargo adaptors can cooperatively regulate a specific sorting process.
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In nature, ion channels are highly selective pores and act as gate to ensure selective ion transport, allowing ions to cross the membrane. By mimicking them, single solid state nanopore devices emerge as a new, powerful class of molecule sensors that allow for the label-free detection of biomolecules (DNA, RNA, and proteins), non-biological polymers, as well as small molecules. In this review, we exhaustively describe the fabrication and functionalization techniques to design highly robust and selective solid state nanopores. First we outline the different materials and methods to design nanopores, we explain the ionic conduction in nanopores, and finally we summarize some techniques to modify and functionalize the surface in order to obtain biomimetic nanopores, responding to different external stimuli.