Ca2+-activated K+ channels modulate membrane potential in the human parathyroid cell: Possible role in exocytosis.

The relationships between parathyroid hormone (PTH) secretion and parathyroid cell membrane potential, including the identities and roles of K+ channels that regulate and/or modulate membrane potential are not well defined. Here we have used Western blot/immunohistochemistry as well as patch-clamp and perifusion techniques to identify and localize specific K+ channels in parathyroid cells and to investigate their roles in the control of membrane potential and PTH secretion. We also re-investigated the relationship between membrane potential and exocytosis. We showed that in single human parathyroid cells K+ current is dependent on at least two types of Ca2+-activated K+ channels: a small-conductance Ca2+-activated K+ channel (KSK) and a large-conductance voltage and Ca2+-activated K+ channel (KBK). These channels were sensitive to specific peptide blocking toxins including apamin, charybdotoxin, and iberiotoxin. These channels confer sensitivity of the membrane potential in single cells to high extracellular K+, TEA, and peptide toxins. Blocking of KBK potently inhibited K+ channel current, and KBK was shown to be expressed in the plasma membrane of parathyroid cells. In addition, when using the capacitance technique as an indicator of exocytosis, clamping the parathyroid cell at -60 mV prevented exocytosis, whereas holding the membrane potential at 0 mV facilitated it. Taken together, the results show that human parathyroid cells have functional KBK and KSK channels but the data presented herein suggest that KBK/KSK channels likely contribute to the maintenance of the membrane potential, and that membrane potential, per se, modulates exocytosis independently of [Ca2+]i.

Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS).

Nucleocytoplasmic transport of transcription factors is vital for normal cellular function, and its breakdown is a major contributing factor in many diseases. The glucocorticoid receptor (GR) is an evolutionarily conserved, ligand-dependent transcription factor that regulates homeostasis and response to stress and is an important target for therapeutics in inflammation and cancer. In unstimulated cells, the GR resides in the cytoplasm bound to other molecules in a large multiprotein complex. Upon stimulation with endogenous or synthetic ligands, GR translocation to the cell nucleus occurs, where the GR regulates the transcription of numerous genes by direct binding to glucocorticoid response elements or by physically associating with other transcription factors. While much is known about molecular mechanisms underlying GR function, the spatial organization of directionality of GR nucleocytoplasmic transport remains less well characterized, and it is not well understood how the bidirectional nucleocytoplasmic flow of GR is coordinated in stimulated cells. Here, we use two-foci cross-correlation in a massively parallel fluorescence correlation spectroscopy (mpFCS) system to map in live cells the directionality of GR translocation at different positions along the nuclear envelope. We show theoretically and experimentally that cross-correlation of signals from two nearby observation volume elements (OVEs) in an mpFCS setup presents a sharp peak when the OVEs are positioned along the trajectory of molecular motion and that the time position of the peak corresponds to the average time of flight of the molecule between the two OVEs. Hence, the direction and velocity of nucleocytoplasmic transport can be determined simultaneously at several locations along the nuclear envelope. We reveal that under ligand-induced GR translocation, nucleocytoplasmic import/export of GR proceeds simultaneously but at different locations in the cell nucleus. Our data show that mpFCS can characterize in detail the heterogeneity of directional nucleocytoplasmic transport in a live cell and may be invaluable for studies aiming to understand how the bidirectional flow of macromolecules through the nuclear pore complex (NPC) is coordinated to avoid intranuclear transcription factor accretion/abatement.

Plaque Evaluation by Ultrasound and Transcriptomics Reveals BCLAF1 as a Regulator of Smooth Muscle Cell Lipid Transdifferentiation in Atherosclerosis.

BACKGROUND: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. METHODS: Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). RESULTS: Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. CONCLUSIONS: Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

A strategy for designing allosteric modulators of transcription factor dimerization.

Transcription factors (TFs) are fundamental in the regulation of gene expression in the development and differentiation of cells. They may act as oncogenes and when overexpressed in tumors become plausible targets for the design of antitumor agents. Homodimerization or heterodimerization of TFs are required for DNA binding and the association interface between subunits, for the design of allosteric modulators, appears as a privileged structure for the pharmacophore-based computational strategy. Based on this strategy, a set of compounds were earlier identified as potential suppressors of OLIG2 dimerization and found to inhibit tumor growth in a mouse glioblastoma cell line and in a whole-animal study. To investigate whether the antitumor activity is due to the predicted mechanism of action, we undertook a study of OLIG2 dimerization using fluorescence cross-correlation spectroscopy (FCCS) of live HEK cells transfected with 2 spectrally different OLIG2 clones. The selected compounds showed an effect with potency, which correlated with the earlier observed antitumor activity. The OLIG2 proteins showed change in diffusion time under compound treatment in line with dissociation from DNA. The data suggest a general approach of drug discovery based on the design of allosteric modulators of protein-protein interaction.

Control of Hox transcription factor concentration and cell-to-cell variability by an auto-regulatory switch.

The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, as this requires the use of quantitative methods with single-molecule sensitivity. Using Fluorescence Correlation Spectroscopy (FCS), we have characterized the concentration and variability of 14 endogenously tagged TFs in live Drosophila imaginal discs. For the Hox TF Antennapedia, we investigated whether protein variability results from random stochastic events or is developmentally regulated. We found that Antennapedia transitioned from low concentration/high variability early, to high concentration/low variability later, in development. FCS and temporally resolved genetic studies uncovered that Antennapedia itself is necessary and sufficient to drive a developmental regulatory switch from auto-activation to auto-repression, thereby reducing variability. This switch is controlled by progressive changes in relative concentrations of preferentially activating and repressing Antennapedia isoforms, which bind chromatin with different affinities. Mathematical modeling demonstrated that the experimentally supported auto-regulatory circuit can explain the increase of Antennapedia concentration and suppression of variability over time.

Cytosolic GPR37, but not GPR37L1, multimerization and its reversal by Parkin: A live cell imaging study.

Biochemical data have shown aggregated G protein-coupled receptor 37 (GPR37) in the cytoplasm and Lewy bodies in Parkinson's disease (PD). Properly folded GPR37 at the plasma membrane appears to be neuroprotective. GPR37, and its homologue GPR37L1, are orphan G protein-coupled receptors and their homo- and hetero-dimers have not been established. We therefore examined GPR37 and GPR37L1 dimerization and extended studies of multimerization of GPR37 to live cells. In this study, we investigated GPR37 and GPR37L1 dimerization and multimerization in live cells using three quantitative imaging methods: Fluorescence Cross-Correlation Spectroscopy, Förster Resonance Energy Transfer, and Fluorescence Lifetime Imaging Microscopy. Our data show that GPR37 and GPR37L1 form homo- and heterodimers in live N2a cells. Importantly, aggregation of GPR37, but not GPR37L1, was identified in the cytoplasm, which could be counteracted by Parkin overexpression. These data provide further evidence that GPR37 participate in cytosolic aggregation processes implicated in PD pathology.

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation.

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP−5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain.

Smallest Secondary Nucleation Competent Aß Aggregates Probed by an ATP-Independent Molecular Chaperone Domain.

Protein oligomerization is a commonly encountered strategy by which the functional repertoire of proteins is increased. This, however, is a double-edged sword strategy because protein oligomerization is notoriously difficult to control. Living organisms have therefore developed a number of chaperones that prevent protein aggregation. The small ATP-independent molecular chaperone domain proSP-C BRICHOS, which is mainly trimeric, specifically inhibits fibril surface-catalyzed nucleation reactions that give rise to toxic oligomers during the aggregation of the Alzheimer's disease-related amyloid-ß peptide (Aß42). Here, we have created a stable proSP-C BRICHOS monomer mutant and show that it does not bind to monomeric Aß42 but has a high affinity for Aß42 fibrils, using surface plasmon resonance. Kinetic analysis of Aß42 aggregation profiles, measured by thioflavin T fluorescence, reveals that the proSP-C BRICHOS monomer mutant strongly inhibits secondary nucleation reactions and thereby reduces the level of catalytic formation of toxic Aß42 oligomers. To study binding between the proSP-C BRICHOS monomer mutant and small soluble Aß42 aggregates, we analyzed fluorescence cross-correlation spectroscopy measurements with the maximum entropy method for fluorescence correlation spectroscopy. We found that the proSP-C BRICHOS monomer mutant binds to the smallest emerging Aß42 aggregates that are comprised of eight or fewer Aß42 molecules, which are already secondary nucleation competent. Our approach can be used to provide molecular-level insights into the mechanisms of action of substances that interfere with protein aggregation.

Dynamic Cellular Cartography: Mapping the Local Determinants of Oligodendrocyte Transcription Factor 2 (OLIG2) Function in Live Cells Using Massively Parallel Fluorescence Correlation Spectroscopy Integrated with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM).

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with single-molecule sensitivity, high temporal resolution (∼10 µs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function. In particular, we show that cytoplasmic OLIG2-eGFP is largely monomeric and freely diffusing, with the fraction of freely diffusing OLIG2-eGFP molecules being fD,freecyt = (0.75 ± 0.10) and the diffusion time τD,freecyt = (0.5 ± 0.3) ms. In contrast, OLIG2-eGFP homodimers are abundant in the cell nucleus, constituting ∼25% of the nuclear pool, some fD,boundnuc = (0.65 ± 0.10) of nuclear OLIG2-eGFP is bound to chromatin DNA, whereas freely moving OLIG2-eGFP molecules diffuse at the same rate as those in the cytoplasm, as evident from the lateral diffusion times τD,freenuc = τD,freecyt = (0.5 ± 0.3) ms. OLIG2-eGFP interactions with chromatin DNA, revealed through their influence on the apparent diffusion behavior of OLIG2-eGFP, τD,boundnuc (850 ± 500) ms, are characterized by an apparent dissociation constant Kd,appOLIG2-DNA = (45 ± 30) nM. The apparent dissociation constant of OLIG2-eGFP homodimers was estimated to be Kd,app(OLIG2-eGFP)2 ≈ 560 nM. The allosteric inhibitor of OLIG2 dimerization, compound NSC 50467, neither affects OLIG2-eGFP properties in the cytoplasm nor does it alter the overall cytoplasmic environment. In contrast, it significantly impedes OLIG2-eGFP homodimerization in the cell nucleus, increasing five-fold the apparent dissociation constant, Kd,app,NSC50467(OLIG2-eGFP)2 ≈ 3 µM, thus reducing homodimer levels to below 7% and effectively abolishing OLIG2-eGFP specific binding to chromatin DNA. The mpFCS/FLIM methodology has a myriad of applications in biomedical research and pharmaceutical industry. For example, it is indispensable for understanding how biological functions emerge through the dynamic integration of location-specific molecular processes and invaluable for drug development, as it allows us to quantitatively characterize the interactions of drugs with drug targets in live cells.

Dynamic lateral organization of opioid receptors (kappa, muwt and muN40D ) in the plasma membrane at the nanoscale level.

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt ), and its naturally occurring isoform (MOPN40D ) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble-averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside-enriched domains and partial association with cholesterol-enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor-specific. KOP-containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D . Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt , whereas this effect was not observed for MOPN40D .

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

Transepithelial transport of proteins is an important step in the immune response to food allergens. Mammalian meat allergy is characterized by an IgE response against the carbohydrate moiety galactosyl-α-1,3-galactose (α-Gal) present on mammalian glycoproteins and glycolipids, which causes severe allergic reactions several hours after red meat consumption. The delayed reaction may be related to the processing of α-Gal carrying proteins in the gastrointestinal tract. The aim of this study was to investigate how protein glycosylation by α-Gal affects the susceptibility to gastric digestion and transport through the Caco-2 cell monolayer. We found that α-Gal glycosylation altered protein susceptibility to gastric digestion, where large protein fragments bearing the α-Gal epitope remained for up to 2 h of digestion. Furthermore, α-Gal glycosylation of the protein hampered transcytosis of the protein through the Caco-2 monolayer. α-Gal epitope on the intact protein could be detected in the endosomal fraction obtained by differential centrifugation of Caco-2 cell lysates. Furthermore, the level of galectin-3 in Caco-2 cells was not affected by the presence of α-Gal glycosylated BSA (bovine serum albumin) (BSA-α-Gal). Taken together, our data add new knowledge and shed light on the digestion and transport of α-Gal glycosylated proteins.

Conformation-specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen.

We engineered and employed a chaperone-like amyloid-binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross-reacted with amyloid beta-peptide (Aß42) protofibrils, but not with Aß40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1-hIAPP complex cross-react with Aß42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation-specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation-sensitive and sequence-independent and can target more than one type of protofibril species.

Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells.

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 µs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy.

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

Erratum to: Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.

Disruption of Ankyrin B and Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Membrane Diffusion.

The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes.

WNT3 involvement in human bladder exstrophy and cloaca development in zebrafish.

Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30,000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT-pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3, WNT6, WNT7A, WNT8B, WNT10A, WNT11, WNT16, FZD5, LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild-type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G>A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT-pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC.

Phenotypic Modulation of Smooth Muscle Cells in Atherosclerosis Is Associated With Downregulation of LMOD1, SYNPO2, PDLIM7, PLN, and SYNM.

OBJECTIVE: Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. APPROACH AND RESULTS: Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7, and PLN expression positively correlated to typical SMC markers in plaques (Pearson r>0.6, P<0.0001) and in rat intimal hyperplasia (r>0.8, P<0.0001). By immunohistochemistry, the proteins were expressed in SMCs in normal vessels, but largely absent in human plaques and intimal hyperplasia. Subcellularly, most proteins localized to the cytoskeleton in cultured SMCs and were regulated by active enhancer histone modification H3K27ac by chromatin immunoprecipitation-sequencing. Functionally, the genes were downregulated by PDGFB (platelet-derived growth factor beta) and IFNg (interferron gamma), exposure to shear flow stress, and oxLDL (oxidized low-density lipoprotein) loading. Genetic variants in PDLIM7, PLN, and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. CONCLUSIONS: We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

The HPA axis and ethanol: a synthesis of mathematical modelling and experimental observations.

Stress and alcohol use are interrelated-stress contributes to the initiation and upholding of alcohol use and alcohol use alters the way we perceive and respond to stress. Intricate mechanisms through which ethanol alters the organism's response to stress remain elusive. We have developed a stoichiometric network model to succinctly describe neurochemical transformations underlying the stress response axis and use numerical simulations to model ethanol effects on complex daily changes of blood levels of cholesterol, 6 peptide and 8 steroid hormones. Modelling suggests that ethanol alters the dynamical regulation of hypothalamic-pituitary-adrenal (HPA) axis activity by affecting the amplitude of ultradian oscillations of HPA axis hormones, which defines the threshold with respect to which the response to stress is being set. These effects are complex-low/moderate acute ethanol challenge (<8 mM) may reduce, leave unaltered or increase the amplitude of ultradian cortisol (CORT) oscillations, giving rise to an intricate response at the organism level, offering also a potential explanation as to why apparently discordant results were observed in experimental studies. In contrast, high-dose acute ethanol challenge (>8 mM) increases instantaneous CORT levels and the amplitude of ultradian CORT oscillations in a dose-dependent manner, affecting the HPA axis activity also during the following day(s). Chronic exposure to ethanol qualitatively changes the HPA axis dynamics, whereas ethanol at intoxicating levels shuts down this dynamic regulation mechanism. Mathematical modelling gives a quantitative biology-based framework that can be used for predicting how the integral HPA axis response is perturbed by alcohol.