Dimerization mechanism of an inverted-topology ion channel in membranes.

Many ion channels are multisubunit complexes where oligomerization is an obligatory requirement for function as the binding axis forms the charged permeation pathway. However, the mechanisms of in-membrane assembly of thermodynamically stable channels are largely unknown. Here, we demonstrate a key advance by reporting the dimerization equilibrium reaction of an inverted-topology, homodimeric fluoride channel Fluc in lipid bilayers. While the wild-type channel is a long-lived dimer, we leverage a known mutation, N43S, that weakens Na+ binding in a buried site at the interface, thereby unlocking the complex for reversible association in lipid bilayers. Single-channel recordings show that Na+ binding is required for fluoride conduction while single-molecule microscopy experiments demonstrate that N43S Fluc exists in a dynamic monomer-dimer equilibrium in the membrane, even following removal of Na+. Quantifying the thermodynamic stability while titrating Na+ indicates that dimerization occurs first, providing a membrane-embedded binding site where Na+ binding weakly stabilizes the complex. To understand how these subunits form stable assemblies while presenting charged surfaces to the membrane, we carried out molecular dynamics simulations, which show the formation of a thinned membrane defect around the exposed dimerization interface. In simulations where subunits are permitted to encounter each other while preventing protein contacts, we observe spontaneous and selective association at the native interface, where stability is achieved by mitigation of the membrane defect. These results suggest a model wherein membrane-associated forces drive channel assembly in the native orientation while subsequent factors, such as Na+ binding, result in channel activation.

Disrupted mechanobiology links the molecular and cellular phenotypes in familial dilated cardiomyopathy.

Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a major indicator for heart transplant. The disease is frequently caused by mutations of sarcomeric proteins; however, it is not well understood how these molecular mutations lead to alterations in cellular organization and contractility. To address this critical gap in our knowledge, we studied the molecular and cellular consequences of a DCM mutation in troponin-T, ΔK210. We determined the molecular mechanism of ΔK210 and used computational modeling to predict that the mutation should reduce the force per sarcomere. In mutant cardiomyocytes, we found that ΔK210 not only reduces contractility but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and disease). These results help link the molecular and cellular phenotypes and implicate alterations in mechanosensing as an important factor in the development of DCM.

A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes.

Membrane proteins are often observed as higher-order oligomers, and in some cases in multiple stoichiometric forms, raising the question of whether dynamic oligomerization can be linked to modulation of function. To better understand this potential regulatory mechanism, there is an ongoing effort to quantify equilibrium reactions of membrane protein oligomerization directly in membranes. Single-molecule photobleaching analysis is particularly useful for this as it provides a binary readout of fluorophores attached to protein subunits at dilute conditions. However, any quantification of stoichiometry also critically requires knowing the probability that a subunit is fluorescently labeled. Since labeling uncertainty is often unavoidable, we developed an approach to estimate labeling yields using the photobleaching probability distribution of an intrinsic dimeric control. By iterative fitting of an experimental dimeric photobleaching probability distribution to an expected dimer model, we estimate the fluorophore labeling yields and find agreement with direct measurements of labeling of the purified protein by UV-VIS absorbance before reconstitution. Using this labeling prediction, similar estimation methods are applied to determine the dissociation constant of reactive CLC-ec1 dimerization constructs without prior knowledge of the fluorophore labeling yield. Finally, we estimate the operational range of subunit labeling yields that allows for discrimination of monomer and dimer populations across the reactive range of mole fraction densities. Thus, our study maps out a practical method for quantifying fluorophore labeling directly from single-molecule photobleaching data, improving the ability to quantify reactive membrane protein stoichiometry in membranes.

Dimerization mechanism of an inverted-topology ion channel in membranes.

Many ion channels are multi-subunit complexes with a polar permeation pathway at the oligomeric interface, but their mechanisms of assembly into functional, thermodynamically stable units within the membrane are largely unknown. Here we characterize the assembly of the inverted-topology, homodimeric fluoride channel Fluc, leveraging a known mutation, N43S, that weakens Na + binding to the dimer interface, thereby unlocking the complex. While single-channel recordings show Na + is required for activation, single-molecule photobleaching and bulk Förster Resonance Energy Transfer experiments in lipid bilayers demonstrate that N43S Fluc monomers and dimers exist in dynamic equilibrium, even without Na + . Molecular dynamics simulations indicate this equilibrium is dominated by a differential in the lipid-solvation energetics of monomer and dimer, which stems from hydrophobic exposure of the polar ion pathway in the monomer. These results suggest a model wherein membrane-associated forces induce channel assembly while subsequent factors, in this case Na + binding, result in channel activation. Teaser: Membrane morphology energetics foster inverted-topology Fluc channels to form dimers, which then become active upon Na + binding.

The Role of the Membrane in Transporter Folding and Activity.

The synthesis, folding, and function of membrane transport proteins are critical factors for defining cellular physiology. Since the stability of these proteins evolved amidst the lipid bilayer, it is no surprise that we are finding that many of these membrane proteins demonstrate coupling of their structure or activity in some way to the membrane. More and more transporter structures are being determined with some information about the surrounding membrane, and computational modeling is providing further molecular details about these solvation structures. Thus, the field is moving towards identifying which molecular mechanisms - lipid interactions, membrane perturbations, differential solvation, and bulk membrane effects - are involved in linking membrane energetics to transporter stability and function. In this review, we present an overview of these mechanisms and the growing evidence that the lipid bilayer is a major determinant of the fold, form, and function of membrane transport proteins in membranes.

DDR2 controls breast tumor stiffness and metastasis by regulating integrin mediated mechanotransduction in CAFs.

Biomechanical changes in the tumor microenvironment influence tumor progression and metastases. Collagen content and fiber organization within the tumor stroma are major contributors to biomechanical changes (e., tumor stiffness) and correlated with tumor aggressiveness and outcome. What signals and in what cells control collagen organization within the tumors, and how, is not fully understood. We show in mouse breast tumors that the action of the collagen receptor DDR2 in CAFs controls tumor stiffness by reorganizing collagen fibers specifically at the tumor-stromal boundary. These changes were associated with lung metastases. The action of DDR2 in mouse and human CAFs, and tumors in vivo, was found to influence mechanotransduction by controlling full collagen-binding integrin activation via Rap1-mediated Talin1 and Kindlin2 recruitment. The action of DDR2 in tumor CAFs is thus critical for remodeling collagen fibers at the tumor-stromal boundary to generate a physically permissive tumor microenvironment for tumor cell invasion and metastases.

Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12-15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).