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A pair of designed transmembrane proteins form a dimer at the cell surface, as seen by single molecule imaging. Changes in the linker length or binding domain modulate the 2-dimensional binding affinity.

Characterizing interactions of proteins is pivotal for understanding their function. Recently, single-molecule imaging-based methods have proven useful for directly testing the stoichiometry of multi-subunit protein complexes. A limiting factor is the labeling of proteins with multiple spectrally discernible tags and low background. Here, we describe the use of zinc-finger (ZF)-mediated protein labeling for single-molecule imaging studies in living cells. A DNA-binding ZF is fused to the protein of interest and labeled by a DNA probe carrying the specific ZF binding sequence and an organic dye. Nonspecific binding is minimized by injecting the DNA/dye conjugate into the cell. With a reproducible labeling efficiency of 20%, we developed an approach to deduce the multiplicity of the subunits in a protein complex from the overall degree of labeling. We were able to confirm the fixed 2:2 assembly of the NMDA receptor in a three-color single-molecule imaging setup and reject alternative stoichiometries. Due to the modular design and small size of ZF proteins, this approach will allow the analysis of more complicated protein interaction patterns to understand the assembly rules for large protein complexes.

Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries., Small membrane proteins represent a largely unexplored yet abundant class of proteins, but how they are inserted into the bacterial membrane is unknown. This study identifies a novel posttranslational protein transport pathway that relies on the signal recognition particle and the SecYEG translocon/YidC insertase.

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane. ispartof: ELIFE vol:9 ispartof: location:England status: published

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10–100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities 2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities 2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

The detection of antibodies from blood sera is crucial for diagnostic purposes. Miniaturized protein assays in combination with microfluidic setups hold great potential by enabling automated handling and multiplexed analyses. Yet, the separate expression, purification, and storage of many individual proteins are time consuming and limit applicability. In vitro cell-free expression has been proposed as an alternative procedure for the generation of protein assays. We report the successful in vitro expression of different model proteins from DNA templates with an optimized expression mix. His10-tagged proteins were specifically captured and immobilized on a Ni-NTA coated sensor surface directly from the in vitro expression mix. Finally, the specific binding of antibodies from rabbit-derived blood sera to the immobilized proteins was monitored by imaging reflectometric interferometry (iRIf). Antibodies in the blood sera could be identified by binding to the respective epitopes with minimal cross reactivity. The results show the potential of in vitro expression and label-free detection for binding assays in general and diagnostic purposes in specific.

Phytochrome B (phyB) is the primary red light photoreceptor in plants, and regulates both growth and development. The relative levels of phyB in the active state are determined by the light conditions, such as direct sunlight or shade, but are also affected by light-independent dark reversion. Dark reversion is a temperature-dependent thermal relaxation process, by which phyB reverts from the active to the inactive state. Here, we show that the homologous phyB-binding proteins PCH1 and PCHL suppress phyB dark reversion, resulting in plants with dramatically enhanced light sensitivity. Moreover, far-red and blue light upregulate the expression of PCH1 and PCHL in a phyB independent manner, thereby increasing the response to red light perceived by phyB. PCH1 and PCHL therefore provide a node for the molecular integration of different light qualities by regulation of phyB dark reversion, allowing plants to adapt growth and development to the ambient environment., Dark reversion of the red-light receptor phytochrome B (phyB) converts the receptor to the inactive state. Here, Enderle et al. show that PCH1 and PCHL bind phyB and suppress dark reversion, and that far-red and blue-light induced upregulation of PCH1 and PCHL increases red light sensitivity in Arabidopsis.

The limit of subdiffraction imaging with fluorescent proteins currently lies at 20 nm, and therefore most protein complexes are too small (2-5 nm) to spatially resolve their individual subunits by optical means. However, the number and stoichiometry of subunits within an immobilized protein complex can be resolved by the observation of photobleaching steps of individual fluorophores or co-localization of single-molecule fluorescence emission in multiple colors. We give an overview of the proteins that have been investigated by this approach and the different techniques that can be used to immobilize and label the proteins. This minireview should serve as a guideline for scientists who want to employ single-molecule subunit counting for their research.

SMC and MukB complexes consist of a central SMC dimer and two essential binding partners, ScpA and ScpB (MukE and MukF), and are crucial for correct chromosome compaction and segregation. The complexes form two bipolar assemblies on the chromosome, one in each cell half. Using fluorescence recovery after photobleaching (FRAP), we provide evidence that the SMC complex has high exchange rates. This depends to a considerable degree on de novo protein synthesis, revealing that the bacterial SMC complex has high on and off rates for binding to the chromosome. A mutation in SMC that affects ATPase activity and results in exaggerated DNA binding in vitro causes a strong segregation defect in vivo and affects the localization of the entire SMC complex, which localizes to many more sites in the cell than under normal conditions. These data indicate that ATP turnover is important for the function of Bacillus subtilis SMC. In contrast, the centromere protein Spo0J and DNA gyrase showed much less exchange between distinct binding sites on the chromosome than that seen with SMC. Binding of Spo0J to the origin regions was rather static and remained partially conserved until the next cell cycle. Our experiments reveal that the SMC complex has a high, condensin-like turnover rate and that an alteration of the ATPase cycle affects SMC function in vivo , while several nucleoid-associated proteins feature limited or slow exchange between different sites on the nucleoid, which may be the basis for epigenetic-like phenomena observed in bacteria.

Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R–mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R–mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.

It has long been known that electrical fields (EFs) are able to influence the direction of migrating cells, a process commonly referred to as electrotaxis or galvanotaxis. Most studies have focused on migrating cells equipped with an existing polarity before EF application, making it difficult to delineate EF-specific pathways. Here we study the initial events in front–rear organization of spreading keratinocytes to dissect the molecular requirements for random and EF-controlled polarization. We find that Arp2/3-dependent protrusive forces and Rac1/Cdc42 activity were generally required for both forms of polarization but were dispensable for controlling the direction of EF-controlled polarization. By contrast, we found a crucial role for extracellular pH as well as G protein coupled–receptor (GPCR) or purinergic signaling in the control of directionality. The normal direction of polarization toward the cathode was reverted by lowering extracellular pH. Polarization toward the anode was also seen at neutral pH when GPCR or purinergic signaling was inhibited. However, the stepwise increase of extracellular pH in this scenario led to restoration of cathodal polarization. Overall our work puts forward a model in which the EF uses distinct polarization pathways. The cathodal pathway involves GPCR/purinergic signaling and is dominant over the anodal pathway at neutral pH., Molecular Biology of the Cell, 26 (24), ISSN:1939-4586, ISSN:1059-1524

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in TRPP2 and PKD1, which form an ion channel/receptor complex containing three TRPP2 and one PKD1. A TRPP2 C-terminal coiled-coil trimer, critical for the assembly of this complex, associates with a single PKD1 C-terminal coiled-coil. Many ADPKD pathogenic mutations result in the abolishment of the TRPP2/PKD1 coiled-coil complex. To gain molecular and functional insights into this heterotetrameric complex, we computationally constructed a structural model by using a two-step docking strategy, based on a known crystal structure of the TRPP2 coiled-coil trimer. The model shows that this tetrameric complex has a novel di-trimer configuration: An upstream trimer made of three TRPP2 helices and a downstream trimer made of two TRPP2 helices and one PKD1 helix. Mutagenesis and biochemical analysis identified critical TRPP2/PKD1 interface contacts essential for the heteromeric coiled-coil complex. Mutation of these interface positions in the full-length proteins showed that these interactions were critical for the assembly of the full-length complex in cells. Our results provide a means to specifically weaken the TRPP2 and PKD1 association, thus facilitating future in vitro and in vivo studies on the functional importance of this association.

The KCNQ1 voltage-gated potassium channel and its auxiliary subunit KCNE1 play a crucial role in the regulation of the heartbeat. The stoichiometry of KCNQ1 and KCNE1 complex has been debated, with some results suggesting that the four KCNQ1 subunits that form the channel associate with two KCNE1 subunits (a 4∶2 stoichiometry), while others have suggested that the stoichiometry may not be fixed. We applied a single molecule fluorescence bleaching method to count subunits in many individual complexes and found that the stoichiometry of the KCNQ1 - KCNE1 complex is flexible, with up to four KCNE1 subunits associating with the four KCNQ1 subunits of the channel (a 4∶4 stoichiometry). The proportion of the various stoichiometries was found to depend on the relative expression densities of KCNQ1 and KCNE1. Strikingly, both the voltage-dependence and kinetics of gating were found to depend on the relative densities of KCNQ1 and KCNE1, suggesting the heart rhythm may be regulated by the relative expression of the auxiliary subunit and the resulting stoichiometry of the channel complex.

Voltage-gated proton channels have been described in different cells and organisms since the early ’80s, but the first member of the family, Hv1, was cloned only recently. The Hv1 channel was found to contain a voltage-sensing domain (VSD), similar to those of voltage-gated sodium, potassium and calcium channels. All these other channels also contain a pore domain, which forms a central pore at the interface of the four subunits. The pore domain is missing in Hv1. This raised several questions on the location of the proton pore and on the mechanism of gating. Here, we briefly review our effort to understand the structural organization of Hv1 channels and discuss the relationship between the gating of Hv1 and the gating of ion-conducting pores recently discovered in the VSDs of mutant voltage-gated potassium and sodium channels.

SummaryIn voltage-gated channels, ions flow through a single pore located at the interface between membrane-spanning pore domains from each of four subunits, and the gates of the pore are controlled by four peripheral voltage-sensing domains. In a striking exception, the newly discovered voltage-gated Hv1 proton channels lack a homologous pore domain, leaving the location of the pore unknown. Also unknown are the number of subunits and the mechanism of gating. We find that Hv1 is a dimer and that each subunit contains its own pore and gate, which is controlled by its own voltage sensor. Our experiments show that the cytosolic domain of the channel is necessary and sufficient for dimerization and that the transmembrane part of the channel is functional also when monomerized. The results suggest a mechanism of gating whereby the voltage sensor and gate are one and the same.

Significance Our work concerns the stoichiometry of acid-sensing ion channels (ASICs), proton-gated Na + channels. In the central nervous system ASICs are composed of either only ASIC1a subunits or ASIC1a and ASIC2a subunits. Blockade of the ASIC1a/2a heteromer causes strong analgesia. Because ligands and drugs bind at subunit interfaces of ASICs, knowing the exact stoichiometry of heteromeric ASICs is pivotal for an informed drug design. We used a single-molecule method where we image individual ion channels that we fused to fluorescent proteins to clearly demonstrate that ASICs assemble into trimers and that coexpression of ASIC1a and ASIC2a leads to a mix of ASIC1a and ASIC2a homomers and ASIC1a:ASIC2a heteromers. Heteromers assemble randomly in both 1:2 and 2:1 compositions.

Voltage-sensing domains (VSDs) confer voltage dependence on effector domains of membrane proteins. Ion channels use four VSDs to control a gate in the pore domain, but in the recently discovered phosphatase Ci-VSP, the number of subunits has been unknown. Using single-molecule microscopy to count subunits and voltage clamp fluorometry to detect structural dynamics, we found Ci-VSP to be a monomer, which operates independently, but nevertheless undergoes multiple voltage-dependent conformational transitions.

The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.

The bacterial SMC (structural maintenance of chromosomes) complex binds nonspecifically to DNA in vitro and forms two discrete subcellular centers in vivo, one in each cell half. How this distribution is maintained is unclear. We show by time-lapse imaging of single molecules that the localization is achieved through limited, yet rapid movement of the SMC subunits through the nucleoid. Accessory ScpAB subunits mediate the arrest of 20% of SMC molecules at the center of a cell half and do not move together with the 80% mobile SMC molecules. Only free SMC, but not the preformed SMC/ScpAB complex, was able to bind to DNA in vitro, revealing distinct functions of SMC fractions. Thus, whereas SMC alone dynamically interacts with many sites on the chromosome, it forms static assemblies together with ScpAB complex partners. Our findings reveal two distinct modes of interaction of SMC with the chromosome and indicate that limited diffusion within a confined space and transient arrest may be a general mechanism for positioning proteins within a chromosome and within a noncompartmentalized cell., Cell Reports, 3 (5), ISSN:2666-3864, ISSN:2211-1247

Summary Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here, we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed coreceptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into the use of IRs as genetically encoded chemical sensors. Video Abstract

In this chapter, we describe how single-molecule fluorescence can be used to analyze protein–protein interactions by enabling the direct visualization of protein complexes and the number and species of their constituent subunits. The prerequisites for this visualization are a low protein density, facilitating the discrimination of individual fluorescent complexes, and high labeling efficiency and specificity to allow for quantitative measurements of protein interactions. We describe three experimental realizations of quantitative imaging techniques made possible by the single-molecule approach: counting the photobleaching steps of fluorescent complexes, the analysis of a histogram of measured spot intensities, and the colocalization of fluorescent tags of multiple colors. Applications of this approach range from the determination of a protein’s oligomerization state to the analysis of the subunit content of large protein assemblies containing multiple subunit types. We describe the minimal setup required for these experiments and present several examples of this approach applied to current biological questions.

Mutations in PKD1 and TRPP2 account for nearly all cases of autosomal dominant polycystic kidney disease (ADPKD). These 2 proteins form a receptor/ion channel complex on the cell surface. Using a combination of biochemistry, crystallography, and a single-molecule method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that this complex contains 3 TRPP2 and 1 PKD1. A newly identified coiled-coil domain in the C terminus of TRPP2 is critical for the formation of this complex. This coiled-coil domain forms a homotrimer, in both solution and crystal structure, and binds to a single coiled-coil domain in the C terminus of PKD1. Mutations that disrupt the TRPP2 coiled-coil domain trimer abolish the assembly of both the full-length TRPP2 trimer and the TRPP2/PKD1 complex and diminish the surface expression of both proteins. These results have significant implications for the assembly, regulation, and function of the TRPP2/PKD1 complex and the pathogenic mechanism of some ADPKD-producing mutations.

Voltage-gated proton channels have been described in different cells and organisms since the early ’80s, but the first member of the family, Hv1, was cloned only recently. The Hv1 channel was found to contain a voltage-sensing domain (VSD), similar to those of voltage-gated sodium, potassium and calcium channels. All these other channels also contain a pore domain, which forms a central pore at the interface of the four subunits. The pore domain is missing in Hv1. This raised several questions on the location of the proton pore and on the mechanism of gating. Here, we briefly review our effort to understand the structural organization of Hv1 channels and discuss the relationship between the gating of Hv1 and the gating of ion-conducting pores recently discovered in the VSDs of mutant voltage-gated potassium and sodium channels.

SUMMARY In voltage-gated sodium, potassium, and calcium channels the functions of ion conduction and voltage sensing are performed by two distinct structural units: the pore domain and the voltage-sensing domain (VSD). In the Hv1 voltage-gated proton channel, the VSD has the remarkable property of performing both functions. Hv1 was recently found to dimerize and to form channels made of two pores. However, the channels were also found to function when dimerization was prevented, raising a question about the functional role of dimerization. Here we show that the two subunits of the Hv1 dimer influence one another during gating, with positive cooperativity shaping the response to voltage of the two pores. We also find that the two voltage sensors undergo conformational changes that precede pore opening and that these conformational changes are allosterically coupled between the two subunits. Our results point to a major role of dimerization in the modulation of Hv1 activity.

Canonical NMDA receptors assemble from two glycine-binding NR1 subunits with two glutamate-binding NR2 subunits to form glutamate-gated excitatory receptors that mediate synaptic transmission and plasticity. The role of glycine-binding NR3 subunits is less clear. Whereas in Xenopus laevis oocytes, two NR3 subunits coassemble with two NR1 subunits to form a glycine-gated receptor, such a receptor has yet to be found in mammalian cells. Meanwhile, NR1, NR2, and NR3 appear to coassemble into triheteromeric receptors in neurons, but it is not clear whether this occurs in oocytes. To test the rules that govern subunit assembly in NMDA receptors, we developed a single-molecule fluorescence colocalization method. The method focuses selectively on the plasma membrane and simultaneously determines the subunit composition of hundreds of individual protein complexes within an optical patch on a live cell. We find that NR1, NR2, and NR3 follow an exclusion rule that yields separate populations of NR1/NR2 and NR1/NR3 receptors on the surface of oocytes. In contrast, coexpression of NR1, NR3A, and NR3B yields triheteromeric receptors with a fixed stoichiometry of two NR1 subunits with one NR3A and one NR3B. At least part of this regulation of subunit stoichiometry appears to be caused by internal retention. Thus, depending on the mixture of subunits, functional receptors on the cell surface may follow either an exclusion rule or a stoichiometric combination rule, providing an important constraint on functional diversity. Cell-to-cell differences in the rules may help sculpt distinct physiological properties.

Many membrane proteins including ion channels form multi-molecular complexes. Because the composition of a molecular complex may define its functional properties, it is important to know its stoichiometry. KCNQ1 encodes a voltage-gated potassium channel alpha subunit, and four KCNQ1 subunits form one ion channel. KCNQ1 channel forms a molecular complex with auxiliary subunit KCNE proteins. In the heart the KCNQ1-KCNE1 complex underlies slowly-activating IKs current, which plays a significant role in regulation of the cardiac action potential. Assuming a fixed KCNQ1-KCNE1 stoichiometry macroscopic current measurements led earlier investigators to the conclusion that each 4-subunit channel is associated with two KCNE1 subunits (4:2 subunit stoichiometry). We asked whether the KCNQ1-KCNE1 stoichiometry is indeed fixed by counting subunits in many individual complexes using TIRF microscopy (Ulbrich and Isacoff, 2007, 2008). We expressed GFP-tagged KCNQ1 or KCNE1 in Xenopus oocytes at low density and counted bleaching steps in many fluorescent spots corresponding to single channel complexes. First, we confirmed that KCNQ1 forms a tetramer. Next we counted GFP-tagged KCNE1 subunits co-expressed with mCherry-tagged KCNQ1. We observed up to four bleaching steps from GFP-KCNE1 co-localized with mCherry, indicating that up to four KCNE1 subunits can bind to one KCNQ1 tetrameric channel. We find that the number of KCNE1 subunits per complex increases as the expression of KCNE1 is raised relative to that of KCNQ1. Our results suggest that modulation of KCNQ channels may be regulated by the level of expression of KCNE subunits.

Polycystic kidney disease (PKD) family proteins associate with transient receptor potential (TRP) channel family proteins to form functionally important complexes. PKD proteins differ from known ion channel-forming proteins and are generally thought to act as membrane receptors. Here we find that PKD1L3, a PKD protein, functions as a channel-forming subunit in an acid-sensing heteromeric complex formed by PKD1L3 and TRPP3, a TRP channel protein. Single amino acid mutations in the putative pore region of both proteins alter the channel's ion selectivity. The PKD1L3/TRPP3 complex in the plasma membrane of live cells contains one PKD1L3 and three TRPP3. A TRPP3 C-terminal coiled-coil domain forms a trimer in solution and in crystal and plays a crucial role in the assembly and surface expression of the PKD1L3/TRPP3 complex. These results demonstrate that PKD subunits constitute a new class of channel-forming proteins, enriching our understanding of the function of PKD proteins and PKD/TRPP complexes.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the genes encoding TRPP2 and PKD1, which form an ion channel/receptor complex containing three TRPP2 and one PKD1. PKD1 is a large integral membrane protein containing 11 putative transmembrane regions, while TRPP2 has 6 transmembrane regions and belongs to the transient receptor potential (TRP) channel family. TRPP2 and PKD1 associate through their C-termini, where a single PKD1 coiled coil binds to a TRPP2 coiled coil trimer. Many ADPKD pathogenic mutations result in the abolishment of the TRPP2/PKD1 coiled coil complex. Obtaining the structure of this complex would help us better understand its crucial role in the assembly and function of the full length TRPP2/PKD1 complex. By combing computational and experimental approaches, we generated a structural model for this coiled coil complex, based on a crystal structure of the TRPP2 coiled coil trimer. The structural model was constructed by a two-step docking strategy, which combines iterative rigid-body search and molecular dynamics (MD) simulations. In this structural model, the N-terminal region of the TRPP2 coiled coil remains as a trimer, but the C-terminal regions of two of the three TRPP2 helixes interact with a single PKD1 coiled coil to form a new trimer. Disruption of predicted critical TRPP2/PKD1 interface contacts abolished or greatly weakened the association between TRP2 and PKD1 coiled coils, supporting the accuracy of the structural model. Some mutations also greatly attenuated the assembly of the full-length TRPP2/PKD1 complex, providing the means to specifically disrupt this complex and study its functional importance in vitro and in vivo. The structural model also sheds light on the pathogenic mechanisms of some ADPKD-causing mutations.∗J.Z. and Y.Y. contributed equally to this work.

Obesity and type 2 diabetes are strongly associated with adipose tissue dysfunction and impaired adipogenesis. Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold‐free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi‐omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R 2 = 0.97). Integration of multi‐omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFβ signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis., A scaffold‐free high‐throughput compatible 3D human adipocyte culture platform in chemically defined media is presented. The model is long‐term stable and closely resembles human adipocytes in vivo on transcriptomic, lipidomic, and functional level. Furthermore, transcription factor activity analysis reveals a complex signaling network that links the organotypic microenvironment to improved adipogenesis.

Proteins in the polycystic kidney disease (PKD) family associate with subunits of the transient receptor potential polysystin (TRPP) subfamily to form functionally important complexes. For example, PKD1 and TRPP2 form a receptor/ion channel complex critical for kidney development and function, and mutations in both proteins cause human polycystic kidney disease. Another PKD member, PKD1L3, coassemble with TRPP3 to form a candidate sour taste receptor. The molecular mechanisms of the assembly of these complexes are largely unknown. Unlike TRP channel subunits, which contain six transmembrane segments, PKD proteins have 11 putative transmembrane segments, with a large extracellular N-terminus containing well-recognized protein-protein, protein-sugar, and protein-ligand interaction motifs. PKD proteins are therefore generally believed to function as cell surface receptors. In this study, we find that PKD1L3, when expressed in Xenopus oocytes, functions as a channel-forming subunit in an acid-sensing heteromeric channel complex formed by PKD1L3 and TRPP3. Single amino acid mutations in the putative pore region of both proteins alter the channel's permeability to monovalent and divalent cations. Single-molecule imaging analysis indicates that the PKD1L3/TRPP3 complex in the plasma membrane of live Xenopus oocytes contains one PKD1L3 and three TRPP3 subunits. We identify a C-terminal coiled-coil domain in TRPP3 and find it to be a trimer in solution and crystal structure. This coiled-coil domain trimer is critical for the assembly and surface expression of TRPP3 homomeric complexes and PKD1L3/TRPP3 heteromeric complexes. These results reinforce the notion that PKD and TRPP proteins associate with a 1:3 stoichiometry. They also provide evidence demonstrating that PKD proteins can function as channel-forming subunits.