Subunit Counting Research Articles

Quantitative Single-Molecule Analysis of Ryanodine Receptor 2 Subunit Assembly in Cardiac and Neuronal Tissues.

We developed a method for ex vivo receptor encapsulation and single-molecule imaging techniques from neuronal and cardiac tissues, illustrating the method's broad applicability for measuring membrane receptor assembly. Ryanodine receptor 2 (RyR2) is a tetrameric Ca2+ channel governing intracellular Ca2+ dynamics, which is critical for muscle contraction. Employing GFP-RyR2 knock-in mice, we isolated individual receptor proteins in tissue-specific nanovesicles and performed subunit counting analyses to yield quantitative assessment of stoichiometric distributions across different organs. With this method, we explored the potential heterogeneity of brain-derived RyR2, which has been reported to form heteromeric assemblies with other ryanodine receptor isoforms.

Single-molecule Diffusion and Assembly on Polymer-crowded Lipid Membranes.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.

Analysis of single-molecule brightness allows subunit counting of high-order oligomeric biomolecular complexes. Although the theory behind the method has been extensively assessed, systematic analysis of the experimental conditions required to accurately quantify the stoichiometry of biological complexes remains challenging. In this work, we develop a high-throughput, automated computational pipeline for single-molecule brightness analysis that requires minimal human input. We use this strategy to systematically quantify the accuracy of counting under a wide range of experimental conditions in simulated ground-truth data and then validate its use on experimentally obtained data. Our approach defines a set of conditions under which subunit counting by brightness analysis is designed to work optimally and helps in establishing the experimental limits in quantifying the number of subunits in a complex of interest. Finally, we combine these features into a powerful, yet simple, software that can be easily used for the analysis of the stoichiometry of such complexes.

Stoichiometry of GluA1/GluA2 Heteromer of AMPA Receptor

AMPA receptors are one of the ionotropic glutamate receptors in neurons which play an important role in synaptic plasticity. Out of four main subunits of the AMPA receptor, GluA1 and GluA2 heteromers are most abundant in brain. Crystallography and single molecule studies show that GluA1 and GluA2 prefers to assemble in 2:2 stoichiometry while forming an AMPA receptor tetramer. The reason behind this preference is not yet understood. One possibility is that the Q/R editing site in the pore forming loop of GluA2's trans-membrane domain, which renders the subunit calcium impermeable, drives the preference for 2:2 stoichiometry. To test the hypothesis, two different chimera structures GluA1-2-1 and GluA2-1-2 are cloned by swapping the trans-membrane domain (containing the RNA editing site) of either subunit. The assembly of chimeras are observed in Xenopus laevis oocytes by single-molecule microscopy. A subunit counting method based on the bleaching steps of chimeras tagged with fluorescent proteins tagged is used to understand the stoichiometry of the assembled chimeras. Thus it is aimed to decipher the molecular determinants behind the 2:2 stoichiometry preference of AMPA receptor subunits.

Markov State Model Analysis of Dynamic Dimerization Interfaces between Transmembrane Domains of Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluRs) are an important family of class C G protein-coupled receptors (GPCRs) characterized by a large extracellular ligand-binding domain (LBD) and a constitutive dimeric arrangement in the cell membrane. While the role of inter-LBD conformational dynamics in mGluR dimerization and activation has been extensively studied, the contribution of the transmembrane domains (TMDs) has only recently been explored using single molecule subunit counting experiments and inter-TMD fluorescence resonance energy transfer (FRET) measurements in living cells.

Determining stoichiometry of ion channel complexes using single subunit counting.

Most membrane proteins, and ion channels in particular, assemble to multimeric biological complexes. This starts with the quarternary structure and continues with the recruitment of auxiliary subunits and oligomerization or clustering of the complexes. While the quarternary structure is best determined by atomic-scale structures, stoichiometry of heteromers and dynamic changes in the assembly cannot necessarily be investigated with structural methods. Here, single subunit counting has proven a powerful method to study the composition of these complexes. Single subunit counting uses the irreversible photodestruction of fluorescent tags as means to directly count a labeled subunit and thereby derive the composition of the assemblies. In this chapter, we discuss single subunit counting and its limitations. We present alternative methods and provide a detailed protocol for recording and analysis of single subunit counting data.

Determining the correct stoichiometry of Kv2.1/Kv6.4 heterotetramers, functional in multiple stoichiometrical configurations

The electrically silent (KvS) members of the voltage-gated potassium (Kv) subfamilies Kv5, Kv6, Kv8, and Kv9 selectively modulate Kv2 subunits by forming heterotetrameric Kv2/KvS channels. Based on the reported 3:1 stoichiometry of Kv2.1/Kv9.3 channels, we tested the hypothesis that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. We investigate the Kv2.1/Kv6.4 stoichiometry using single subunit counting and functional characterization of tetrameric concatemers. For selecting the most probable stoichiometry, we introduce a model-selection method that is applicable for any multimeric complex by investigating the stoichiometry of Kv2.1/Kv6.4 channels. Weighted likelihood calculations bring rigor to a powerful technique. Using the weighted-likelihood model-selection method and analysis of electrophysiological data, we show that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. Within this stoichiometry, the Kv6.4 subunits have to be positioned alternating with Kv2.1 to express functional channels. The variability in Kv2/KvS assembly increases the diversity of heterotetrameric configurations and extends the regulatory possibilities of KvS by allowing the presence of more than one silent subunit.

Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes

The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown. Using single-molecule subunit counting, we determined that the number of KCNE2 subunits in complex with the pore-forming subunits of human HCN channels differs with each HCN isoform and is dynamic with respect to concentration. These interactions can be altered by KCNE2 gene-variants with functional implications. The results provide an additional consideration necessary to understand heart rhythm, pain, and epileptic disorders.

Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors (mGluRs) are class C, synaptic G-protein-coupled receptors (GPCRs) that contain large extracellular ligand binding domains (LBDs) and form constitutive dimers. Despite the existence of a detailed picture of inter-LBD conformational dynamics and structural snapshots of both isolated domains and full-length receptors, it remains unclear how mGluR activation proceeds at the level of the transmembrane domains (TMDs) and how TMD-targeting allosteric drugs exert their effects. Here, we use time-resolved functional and conformational assays to dissect the mechanisms by which allosteric drugs activate and modulate mGluR2. Single-molecule subunit counting and inter-TMD fluorescence resonance energy transfer measurements in living cells reveal LBD-independent conformational rearrangements between TMD dimers during receptor modulation. Using these assays along with functional readouts, we uncover heterogeneity in the magnitude, direction, and the timing of the action of both positive and negative allosteric drugs. Together our experiments lead to a three-state model of TMD activation, which provides a framework for understanding how inter-subunit rearrangements drive class C GPCR activation.

Inter‐Subunit Conformational Dynamics and Allosteric Modulation of Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluRs) are class C G protein‐coupled receptors (GPCRs) which function in synapses throughout the central nervous system where they respond to the excitatory neurotransmitter glutamate to initiate intracellular signaling cascades. A comprehensive understanding of mGluR activation requires a mechanistic description of the effects of both extracellular ligand binding domain(LBD)‐targeting “orthosteric” and transmembrane domain (TMD)‐targeting “allosteric” compounds, both of which have therapeutic potential. Here we use a combination of high resolution, time‐resolved functional and conformational assays to dissect the mechanisms by which allosteric compounds activate and modulate mGluRs. First, using an electrophysiological assay we find that mGluR2 can be activated by both orthosteric or allosteric ligands and that allosteric activation does not require LBD closure. We then use a single molecule fluorescence‐based subunit counting assay to find that mGluR transmembrane domains efficiently dimerize with variable efficiency to a degree that is consistently higher than that observed for class A GPCRs and is not sensitive to allosteric drugs. Next, we develop an inter‐TMD FRET assay that reveals that activation‐associated inter‐TMD rearrangement occurs independently of allosteric input from the LBDs. Using this assay, we test a panel of allosteric compounds to uncover diversity in apparent affinity, efficacy and kinetics between these compounds. Strikingly, we find that negative allosteric modulators are either neutral or induce TMD rearrangement which correlates with degree of inverse agonism. Together this data is consistent with a model where multiple (>2) intersubunit reorientations are part of the activation process and motivates further work on the role of multimeric assembly and inter‐domain coupling in the activation of class C GPCRs.Support or Funding InformationThis work is supported by an NIGMS R35 (R35 GM124731) grant to JL. JKT is supported by an NSF GRFP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Super-Resolution Microscopy as a Tool for Counting Proteins in a Sub-Cellular Environment

Super-resolution microscopy, and in particular single molecule localization microscopy (such as stochastic optical reconstruction microscopy, STORM), offers a unique opportunity for quantifying protein copy numbers with nanoscale resolution [1,2]. The development of methods able to access a precise molecular counting of protein copy numbers is essential, clearing the way to address several biological questions using super-resolution microscopy. The challenges of molecular counting using super-resolution [3,4] can be addressed using calibration standards such as DNA origami, since they provide direct characterization for quantitative imaging when immunofluorescence is used. Here we propose to use quantitative approaches based on DNA origami calibration to study the distribution of adhesion proteins and membrane proteins. First, we focus on quantitative studies of the stoichiometry of membrane proteins (i.e membrane channels and their accessory subunits). Furthermore, we apply this method for average protein copy number estimation of adhesion proteins (i.e. vinculin) in mammalian cells and neurons. 1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): p. 156-62. 2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound proteins. Nat Methods, 2007. 4(4): p. 319-21. 3. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Nature methods doi:10.1038/nmeth.3804 (2016) 4. Cella Zanacchi F. et al., DNA Origami: Versatile super-resolution calibration standard for quantifying protein copy-number, Nature Methods 14, 789–792 (2017) (2017)

Inter-Domain Interactions and Allosteric Modulation of Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluRs) are class C G protein-coupled receptors (GPCRs) which function in synapses throughout the central nervous system where they respond to the excitatory neurotransmitter glutamate to initiate intracellular signaling cascades. A comprehensive understanding of mGluR activation requires a mechanistic description of the effects of both extracellular ligand binding domain(LBD)-targeting “orthosteric” and transmembrane domain (TMD)-targeting “allosteric” compounds. Here we use a combination of high resolution, time-resolved functional and conformational assays to dissect the mechanisms by which allosteric compounds activate and modulate mGluRs. First, using an electrophysiological assay we find that mGluR2 can be activated by both orthosteric or allosteric ligands and that allosteric activation does not require LBD closure. We then use a single molecule fluorescence-based subunit counting assay to find that mGluR transmembrane domains efficiently dimerize with variable efficiency to a degree that is consistently higher than that observed for class A GPCRs and is not sensitive to allosteric drugs. Next, we develop an inter-TMD FRET assay that reveals that activation-associated inter-TMD rearrangement occurs independently of allosteric input from the LBDs. Using this assay, we test a panel of allosteric compounds to uncover diversity in apparent affinity, efficacy and kinetics between these compounds. Strikingly, we find that negative allosteric modulators are either neutral or induce TMD rearrangement which correlates with degree of inverse agonism. Together this data is consistent with a model where multiple (>2) intersubunit reorientations are part of the activation process and motivates further work on the role of multimeric assembly and inter-domain coupling in the activation of class C GPCRs.

Versatile Super-Resolution Calibration Standard for Quantifying Protein Copy Number

Single molecule based super-resolution microscopy offers a unique opportunity for quantifying protein copy numbers with nanoscale resolution [1,2]. While fluorescent proteins have been extensively characterized for quantitative imaging using calibration standards, similar calibration tools for small organic fluorophores used in conjunction with immunofluorescence are lacking.Within this framework, the development of methods able to access a precise molecular counting of protein copy numbers is essential, clearing the way to address several biological questions using super-resolution techniques such as stochastic optical reconstruction microscopy (STORM).The development of a suitable calibration method represents the best way to address the challenges of molecular counting using super-resolution [3,4]. Within this project, we demonstrate that DNA origami in combination with GFP antibodies is a versatile platform for quantifying protein copy number in immunofluorescence based super-resolution microscopy. We show that this calibration method, besides quantifying the average protein copy number in a cell, allows determining the abundance of various oligomeric states. Furthermore, we apply this calibration method to quantify nucleoporins (NUP107) [5] and molecular motors (dynein intermediate chain) [6] in vivo. Overall, we provide a versatile strategy for quantifying a large number of proteins of interest using various labeling approaches.1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): p. 156-62.2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound proteins. Nat Methods, 2007. 4(4): p. 319-21.3. Schmied, J.J., et al., DNA origami-based standards for quantitative fluorescence microscopy. Nat Protoc, 2014. 9(6): p. 1367-91.4. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Nature methods doi:10.1038/nmeth.3804 (2016)5. Szymborska, A., et al., Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging. Science, 2013. 341(6146): p. 655-658.6. Derr, N.D., et al., Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science, 2012. 338(6107): p. 662-5.

Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors

G protein-coupled receptors (GPCRs) mediate cellular responses to a wide variety of extracellular stimuli. GPCR dimerization may expand signaling diversity and tune functionality, but little is known about the mechanisms of subunit assembly and interaction or the signaling properties of heteromers. Using single-molecule subunit counting on class C metabotropic glutamate receptors (mGluRs), we map dimerization determinants and define a heterodimerization profile. Intersubunit fluorescence resonance energy transfer measurements reveal that interactions between ligand-binding domains control the conformational rearrangements underlying receptor activation. Selective liganding with photoswitchable tethered agonists conjugated to one or both subunits of covalently linked mGluR2 homodimers reveals that receptor activation is highly cooperative. Strikingly, this cooperativity is asymmetric in mGluR2/mGluR3 heterodimers. Our results lead to a model of cooperative activation of mGluRs that provides a framework for understanding how class C GPCRs couple extracellular binding to dimer reorganization and G protein activation.

Production of Heteromeric Transmembrane Receptors with Defined Subunit Stoichiometry

Signal transduction across cell membranes often requires assembly of heteromeric receptors with defined stoichiometry. In this issue of Structure, Morales-Perez etal. (2016) present elegant methods for the expression of heteromeric nicotinic acetylcholine receptors with a defined α4β2 stoichiometry involving controlled baculovirus-mediated transduction and subunit counting by measurement of two fluorescent signals.

Heterodimerization within the TREK Channel Subfamily

TREK1, TREK2, and TRAAK form the TREK subfamily of two-pore-domain K+ (K2P) channels. Despite sharing up to 78% sequence identity and showing overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli, such as phosopholipids or external pH. For instance, TREK1 is inhibited by external acidification while TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single molecule pull-down and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2 and TRAAK readily co-assemble to form heterodimers. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the different heterodimers and found that all combinations form outwardly-rectifying potassium-selective channels but with variable voltage-sensitivity and regulation. TREK1/TREK2 heterodimers show low levels of activity at physiological pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modelling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel can be regulated independently of each other via titratable histidines and their interaction with nearby charged residues. TREK1/TRAAK heterodimers is also functional and unlike the TRAAK homodimer is able to be regulated by PLD2 which binds the TREK1 moiety. Together, this study greatly expands the number of channels within the TREK subfamily and defines how heterodimerization can alter the physiological properties of K2P channels.

Mapping CFTR Clusters in 3D via Single Molecule Step Photobleaching Analysis in Epithelial Cells

Protein oligomerization is crucial for cellular regulation. Various methods have been used to study the oligomerization of membrane proteins and multimeric channels including single molecule step photobleaching subunit counting. However, the membrane proteins function in a quasi 2D environment within a larger 3D cellular context encompassing membrane delivery and recycling via exocytosis and endocytosis and scaffolding and functional interactions within multiprotein complexes. We present PISA (photobleaching intensity step analysis), a new single molecule approach to map oligomerization states of fluorescently tagged proteins in 3D in cells where the membrane interface is imaged via TIRF microscopy. PISA counts oligomer subunits via sequential photobleaching steps, and additionally maps these distributions with nm precision in z due to magnitude differences of the intensity steps within the TIRF evanescent field. We applied PISA to map distributions of clusters of cystic fibrosis transmembrane conductance regulator (CFTR) in primary human airway epithelial cells. CFTR is an anion channel which conducts chloride and bicarbonate ions through the apical plasma membrane of epithelial cells and is known to interact with other proteins which form a macromolecular complex that regulates its activity at the cell surface. We measured membrane distributions of CFTR-eGFP as well as intracellular clusters of CFTR that were distributed at distances over 100 nm from the membrane. Treatment of the cells with vasoactive intestinal peptide (VIP) and cholesterol oxidase (COase) both resulted in a shift of CFTR clusters towards the membrane. VIP treatment resulted in larger CFTR cluster size (mean 2.9 subunits/cluster) and population (mean 28 units/ROI) as compared to untreated cells (mean 2.1 subunits/cluster) and cluster population (mean 22 units/ROI). COase treatment resulted only in lower cluster population (mean 9 units/ROI). Results on untreated cells were corroborated by TEM.

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem–loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells

Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h.

Kv4.2 and Accessory Dipeptidyl Peptidase-like Protein 10 (DPP10) Subunit Preferentially Form a 4:2 (Kv4.2:DPP10) Channel Complex

Kv4 is a member of the voltage-gated K(+) channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2-DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of the DPP10 formed dimers in the plasma membrane, and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2-DPP10 complex; Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2-DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2-DPP10 complex was variable depending on the relative expression level of each subunit, with a preference for 4:2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10.