Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors

2008 ◽  
Vol 366 (1880) ◽  
pp. 3445-3462 ◽  
Author(s):  
Marat Mukhtarov ◽  
Olga Markova ◽  
Eleonore Real ◽  
Yves Jacob ◽  
Svetlana Buldakova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Glycine Receptor ◽  
Yellow Fluorescent Protein ◽  
High Sensitivity ◽  
Functional Expression ◽  
Molecular Probes ◽  
Molecular Probe ◽  
Non Invasive ◽  
Spectroscopic Monitoring

Genetically encoded probes have become powerful tools for non-invasive monitoring of ions, distributions of proteins and the migration and formation of cellular components. We describe the functional expression of two molecular probes for non-invasive fluorescent monitoring of intracellular Cl ([Cl] i ) and the functioning of glycine receptor (GlyR) channels. The first probe is a recently developed cyan fluorescent protein–yellow fluorescent protein-based construct, termed Cl-Sensor, with relatively high sensitivity to Cl ( K app ∼30 mM). In this study, we describe its expression in retina cells using in vivo electroporation and analyse changes in [Cl] i at depolarization and during the first three weeks of post-natal development. An application of 40 mM K + causes an elevation in [Cl] i of approximately 40 mM. In photoreceptors from retina slices of a 6-day-old rat (P6 rat), the mean [Cl] i is approximately 50 mM, and for P16 and P21 rats it is approximately 30–35 mM. The second construct, termed BioSensor-GlyR, is a GlyR channel with Cl-Sensor incorporated into the cytoplasmic domain. This is the first molecular probe for spectroscopic monitoring of the functioning of receptor-operated channels. These types of probes offer a means of screening pharmacological agents and monitoring Cl under different physiological and pathological conditions and permit spectroscopic monitoring of the activity of GlyRs expressed in heterologous systems and neurons.

scholarly journals Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

2010 ◽  
Vol 298 (4) ◽  
pp. E807-E814 ◽  
Author(s):  
Lara R. Nyman ◽  
Eric Ford ◽  
Alvin C. Powers ◽  
David W. Piston
Keyword(s):  
Blood Flow ◽  
Blood Glucose ◽  
Pancreatic Islets ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Types ◽  
Β Cells ◽  
Islet Blood Flow ◽  
Significant Difference

Pancreatic islets are highly vascularized and arranged so that regions containing β-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic (∼300 mg/dl or 16.8 mM) and hypoglycemic (∼50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the β-cells or yellow fluorescent protein in the α-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of β-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

2008 ◽  
Vol 294 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Ellen Steward Pentz ◽  
Maria Luisa S. Sequeira Lopez ◽  
Magali Cordaillat ◽  
R. Ariel Gomez
Keyword(s):  
Chromatin Remodeling ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Histone H4 Acetylation ◽  
Renin Gene ◽  
Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

scholarly journals A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

2021 ◽  
Author(s):  
Giovanni Gallo ◽  
Ioannis Mougiakos ◽  
Mauricio Bianco ◽  
Miriam Carbonaro ◽  
Andrea Carpentieri ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Thermus Thermophilus ◽  
Yellow Fluorescent Protein ◽  
Arsenic Resistance ◽  
Wide Range ◽  
Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

LDL Receptor Mediates Effective Endocytosis of Activated Factor V.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1766-1766 ◽  
Author(s):  
Alexander B. Meijer ◽  
Sigrid D. Roosendaal ◽  
Vincent Limburg ◽  
Carmen van der Zwaan ◽  
Kees W. Rodenburg ◽  
...  
Keyword(s):  
Cho Cells ◽  
Fluorescent Protein ◽  
Low Density Lipoprotein ◽  
Yellow Fluorescent Protein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Functional Expression ◽  
Factor V ◽  
Early Endosomes ◽  
Density Lipoprotein Receptor

Abstract The platelet α-granules contain a unique pool of partially activated factor V, which has been suggested to originate form megakaryocyte endocytosis of factor V (FV) from plasma. The presence of activated FV (FVa) in plasma itself should be tightly controlled as dysfunction therein may predispose to thrombotic disorders. Previously, we have reported that FVa but not FV can bind the low-density lipoprotein receptor related protein (LRP). This multifunctional receptor can bind a multitude of ligands including FV’s homologue factor VIII (FVIII). We now investigated whether FV or FVa can, like FVIII, also bind the more restricted LDL receptor (LDLR). To this end, the endocytosis of FV and FVa by CHO cells expressing LDLR (CHO-LDLR+ cells) was assessed utilizing confocal microscopy. In the experimental setup, FV and FVa were visualized employing immuno-fluorescence staining techniques. The results showed that within 10 minutes after addition of FVa, fluorescent spots appeared inside the CHO-LDLR+ cells. In contrast, no fluorescent spots were observed after 10 minutes of incubation with FV. These observations suggest that FVa but not FV effectively interacts with the LDLR expressing CHO cells. We then assessed whether FVa can compete with FVIII for endocytosis by CHO-LDLR+ cells. In the presence of an equimolar amount of FVa and a FVIII derivative (FVIIIYFP) containing yellow fluorescent protein, both proteins were detected within the same vesicles inside the CHO-LDLR+ cells. Employing co-localization studies, we established that these vesicles represented early endosomes. In the presence of an access of FVa, however, the yellow fluorescence of FVIIIYFP was no longer observed. These results demonstrate that FVa can block the FVIII endocytosis by CHO-LDLR+ cells. The observations together suggest that LDLR can not only bind FVIII but also FVa. In line with this notion, we established that CHO-ldlA cells, which lack functional expression of LDLR, were unable to internalize neither FVa nor FVIIIYFP. In addition, the ligand-binding clusters II and cluster IV of LRP effectively inhibited the endocytic uptake of FVa by CHO-LDLR+ cells. This implies that structural elements of FVa involved in LRP binding may overlap with those required for LDLR dependent internalization. Our results suggest a so far unidentified role for members of the LDL receptor family in the regulation of FVa in plasma.

scholarly journals Conventional Kinesin Mediates Microtubule-Microtubule Interactions In Vivo

2006 ◽  
Vol 17 (2) ◽  
pp. 907-916 ◽  
Author(s):  
Anne Straube ◽  
Gerd Hause ◽  
Gero Fink ◽  
Gero Steinberg
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Activity ◽  
C Terminus ◽  
Cross Bridges ◽  
Conventional Kinesin ◽  
Motor Head

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.

In vivo associations of Escherichia coli NarJ with a peptide of the first 50 residues of nitrate reductase catalytic subunit NarG

2009 ◽  
Vol 55 (2) ◽  
pp. 179-188 ◽  
Author(s):  
Haiming Li ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Catalytic Subunit ◽  
Bimolecular Fluorescence Complementation ◽  
E Coli ◽  
N Terminus ◽  
Tat System

The catalytic subunit of many Escherichia coli redox enzymes bares a twin-arginine translocation (Tat)-dependent signal peptide in its precursor, which directs the redox enzyme complex to this Sec-independent pathway. NarG of the E. coli nitrate reductase NarGHI complex possesses a vestige twin-arginine motif at its N terminus. During the cofactor insertion, and assembly and folding of the NarG–NarH complex, a chaperone protein, NarJ, is thought to interact with the N terminus and an unknown second site of NarG. Our previous in vitro study provided evidence that NarJ’s role shows some Tat system dependence. In this work, we investigated the associations of NarJ with a peptide of the first 50 residues of NarG (NarG50) in living cells. Two approaches were used: the Förster resonance energy transfer (FRET) based on yellow fluorescent protein – cyan fluorescent protein (YFP–CFP) and the bimolecular fluorescence complementation (BiFC). Compared with the wild-type (WT) E. coli cotransformants expressing both NarJ–YFP and NarG50–CFP, tat gene mutants gave an apparent FRET efficiency (Eapp) that was on the order of 25%–40% lower. These experiments implied a Tat system dependency of the in vivo associations between NarJ and the NarG50 peptide. In the BiFC assay, a 4-fold lower specific fluorescence intensity was observed for the E. coli WT cotransformants expressing both NarJ–Yc and NarG50–Yn than for its tat mutants, again suggesting a Tat dependence of the interactions. Fluorescence microscopy showed a “dot”/unipolar distribution of the reassembled YFP–NarJ:NarG50 both in WT and tat mutants, demonstrating a distinct localization of the interaction. Thus, although the degree of the interaction shows Tat dependence, the cell localization is less so. Taken together, these data further support that NarJ’s activity on NarG may be assisted by the Tat system.

Accelerated axon outgrowth, guidance, and target reinnervation across nerve transection gaps following a brief electrical stimulation paradigm

2012 ◽  
Vol 116 (3) ◽  
pp. 498-512 ◽  
Author(s):  
Bhagat Singh ◽  
Qing-Gui Xu ◽  
Colin K. Franz ◽  
Rumi Zhang ◽  
Colin Dalton ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Microelectrode Array ◽  
Fluorescent Protein ◽  
Recovery Of Function ◽  
Yellow Fluorescent Protein ◽  
Sensory Axons ◽  
Regenerated Fibers ◽  
Stimulation Paradigm

Object Regeneration of peripheral nerves is remarkably restrained across transection injuries, limiting recovery of function. Strategies to reverse this common and unfortunate outcome are limited. Remarkably, however, new evidence suggests that a brief extracellular electrical stimulation (ES), delivered at the time of injury, improves the regrowth of motor and sensory axons. Methods In this work, the authors explored and tested this ES paradigm, which was applied proximal to transected sciatic nerves in mice, and identified several novel and compelling impacts of the approach. Using thy-1 yellow fluorescent protein mice with fluorescent axons that allow serial in vivo tracking of regeneration, the morphological, electrophysiological, and behavioral indices of nerve regrowth were measured. Results The authors show that ES is associated with a 30%–50% improvement in several indices of regeneration: regrowth of axons and their partnered Schwann cells across transection sites, maturation of regenerated fibers in gaps spanning transection zones, and entry of axons into their muscle and cutaneous target zones. In parallel studies, the authors analyzed adult sensory neurons and their response to extracellular ES while plated on a novel microelectrode array construct designed to deliver the identical ES paradigm used in vivo. The ES accelerated neurite outgrowth, supporting the concept of a neuron-autonomous mechanism of action. Conclusions Taken together, these results support a robust role for brief ES following peripheral nerve injuries in promoting regeneration. Electrical stimulation has a wider repertoire of impact than previously recognized, and its impact in vitro supports the hypothesis that a neuron-specific reprogrammed injury response is recruited by the ES protocol.

scholarly journals Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision

2011 ◽  
Vol 192 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Wanda Kukulski ◽  
Martin Schorb ◽  
Sonja Welsch ◽  
Andrea Picco ◽  
Marko Kaksonen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Cellular Processes ◽  
Cellular Events ◽  
Time Period ◽  
Fluorescence And Electron Microscopy ◽  
3D Electron Microscopy ◽  
3D Electron

Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.

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