Structural basis of pH-dependent activation in a CLC transporter.

CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H+ pathways and is required for transport. The more frequent formation of short water wires in the open H+ pathway enables Cl- pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.

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.

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers.

The CLC-ec1 chloride/proton antiporter is a membrane-embedded homodimer with subunits that can dissociate and associate, but the thermodynamic driving forces favor the assembled dimer at biological densities. Yet, the physical reasons for this stability are confounding as dimerization occurs via the burial of hydrophobic interfaces away from the lipid solvent. For binding of nonpolar surfaces in aqueous solution, the driving force is often attributed to the hydrophobic effect, but this should not apply in the membrane since there is very little water. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG°. To ensure that the reaction reached equilibrium at different temperatures, we utilized a Förster resonance energy transfer assay to report on relaxation kinetics of subunit exchange as a function of temperature. Equilibration times were then applied to measure CLC-ec1 dimerization isotherms at different temperatures using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in Escherichia coli-like membranes exhibits a nonlinear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects such as the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the nonbilayer defect required to solvate the monomeric state is one source of the observed change in heat capacity and indicates the existence of a generalizable driving force for protein association in membranes.

Roadmap for C9ORF72 in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis: Report on the C9ORF72 FTD/ALS Summit.

A summit held March 2023 in Scottsdale, Arizona (USA) focused on the intronic hexanucleotide expansion in the C9ORF72 gene and its relevance in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS; C9ORF72-FTD/ALS). The goal of this summit was to connect basic scientists, clinical researchers, drug developers, and individuals affected by C9ORF72-FTD/ALS to evaluate how collaborative efforts across the FTD-ALS disease spectrum might break down existing disease silos. Presentations and discussions covered recent discoveries in C9ORF72-FTD/ALS disease mechanisms, availability of disease biomarkers and recent advances in therapeutic development, and clinical trial design for prevention and treatment for individuals affected by C9ORF72-FTD/ALS and asymptomatic pathological expansion carriers. The C9ORF72-associated hexanucleotide repeat expansion is an important locus for both ALS and FTD. C9ORF72-FTD/ALS may be characterized by loss of function of the C9ORF72 protein and toxic gain of functions caused by both dipeptide repeat (DPR) proteins and hexanucleotide repeat RNA. C9ORF72-FTD/ALS therapeutic strategies discussed at the summit included the use of antisense oligonucleotides, adeno-associated virus (AAV)-mediated gene silencing and gene delivery, and engineered small molecules targeting RNA structures associated with the C9ORF72 expansion. Neurofilament light chain, DPR proteins, and transactive response (TAR) DNA-binding protein 43 (TDP-43)-associated molecular changes were presented as biomarker candidates. Similarly, brain imaging modalities (i.e., magnetic resonance imaging [MRI] and positron emission tomography [PET]) measuring structural, functional, and metabolic changes were discussed as important tools to monitor individuals affected with C9ORF72-FTD/ALS, at both pre-symptomatic and symptomatic disease stages. Finally, summit attendees evaluated current clinical trial designs available for FTD or ALS patients and concluded that therapeutics relevant to FTD/ALS patients, such as those specifically targeting C9ORF72, may need to be tested with composite endpoints covering clinical symptoms of both FTD and ALS. The latter will require novel clinical trial designs to be inclusive of all patient subgroups spanning the FTD/ALS spectrum.

The C9ORF72 Summit was held in March 2023 in Scottsdale, Arizona (USA). Some people who have the disease frontotemporal dementia or the disease amyotrophic lateral sclerosis have a change in one of their genes; the name of the gene is C9ORF72. People who carry this genetic difference usually inherited it from a parent. Researchers are improving their understanding of how the change in the C9ORF72 gene affects people, and efforts are being made to use this knowledge to develop treatments for amyotrophic lateral sclerosis and frontotemporal dementia. In addition to studying the cellular and molecular mechanisms of how the C9ORF72 mutation leads to cellular dysfunction and frontotemporal dementia and amyotrophic lateral sclerosis clinical symptoms, a large effort of the research community is aimed at developing measurements, called biomarkers, that could enhance therapy development efforts in multiple ways. Examples include monitoring of disease activity, identifying those at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, predicting which people might benefit from a particular treatment, and showing that a drug has had a biological effect. Markers that identify healthy people who are at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia could be used to test treatments that would start before a person shows any symptoms and hopefully would delay or even prevent their onset.

Unraveling the impact of disrupted nucleocytoplasmic transport systems in C9orf72-associated ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two adult-onset neurodegenerative diseases that are part of a common disease spectrum due to clinical, genetic, and pathological overlap. A prominent genetic factor contributing to both diseases is a hexanucleotide repeat expansion in a non-coding region of the C9orf72 gene. This mutation in C9orf72 leads to nuclear depletion and cytoplasmic aggregation of Tar DNA-RNA binding protein 43 (TDP-43). TDP-43 pathology is characteristic of the majority of ALS cases, irrespective of disease causation, and is present in ~50% of FTD cases. Defects in nucleocytoplasmic transport involving the nuclear pore complex, the Ran-GTPase cycle, and nuclear transport factors have been linked with the mislocalization of TDP-43. Here, we will explore and discuss the implications of these system abnormalities of nucleocytoplasmic transport in C9orf72-ALS/FTD, as well as in other forms of familial and sporadic ALS.

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers.

The CLC-ec1 chloride/proton antiporter is a membrane embedded homodimer where subunits can dissociate and associate, but the thermodynamic driving forces favor the assembled form at biological densities. Yet, the physical reasons for this stability are confounding since binding occurs via the burial of hydrophobic protein interfaces yet the hydrophobic effect should not apply since there is little water within the membrane. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG° . To ensure that the reaction reached equilibrium under changing conditions, we utilized a Förster Resonance Energy Transfer based assay to report on the relaxation kinetics of subunit exchange as a function of temperature. These equilibration times were then applied to measure CLC-ec1 dimerization isotherms as a function of temperature using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in E. coli membranes exhibits a non-linear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects including the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the non-bilayer defect required to solvate the monomeric state is the molecular source of this large change in heat capacity and is a major and generalizable driving force for protein association 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.

Loss of C9orf72 perturbs the Ran-GTPase gradient and nucleocytoplasmic transport, generating compositionally diverse Importin ß-1 granules.

A hexanucleotide (GGGGCC)n repeat expansion in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), eliciting toxic effects through generation of RNA foci, dipeptide repeat proteins, and/or loss of C9orf72 protein. Defects in nucleocytoplasmic transport (NCT) have been implicated as a pathogenic mechanism underlying repeat expansion toxicity. Here, we show that loss of C9orf72 disrupts the Ran-GTPase gradient and NCT in vitro and in vivo. NCT disruption in vivo is enhanced by the presence of compositionally different types of cytoplasmic Importin ß-1 granule that exhibit neuronal subtype-specific properties. We show that the abundance of Importin ß-1 granules is increased in the context of C9orf72 deficiency, disrupting interactions with nuclear pore complex proteins. These granules appear to associate with the nuclear envelope and are co-immunoreactive for G3BP1 and K63-ubiquitin. These findings link loss of C9orf72 protein to gain-of-function mechanisms and defects in NCT.

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.

Generation of five induced pluripotent stem cells lines from four members of the same family carrying a C9orf72 repeat expansion and one wild-type member.

The most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) is the expansion of a G4C2 hexanucleotide repeat in the C9orf72 gene. The size of the repeat expansion is highly variable and a cut-off of 30 repeats has been suggested as the lower pathological limit. Repeat size variability has been observed intergenerationally and intraindividually in tissues from different organs and within the same tissue, suggesting instability of the pathological repeat expansion. In order to study this genomic instability, we established iPSCs from five members of the same family of which four carried a C9orf72 repeat expansion and one was wild-type.

CAPTURE ALS: the comprehensive analysis platform to understand, remedy and eliminate ALS.

The absence of disease modifying treatments for amyotrophic lateral sclerosis (ALS) is in large part a consequence of its complexity and heterogeneity. Deep clinical and biological phenotyping of people living with ALS would assist in the development of effective treatments and target specific biomarkers to monitor disease progression and inform on treatment efficacy. The objective of this paper is to present the Comprehensive Analysis Platform To Understand Remedy and Eliminate ALS (CAPTURE ALS), an open and translational platform for the scientific community currently in development. CAPTURE ALS is a Canadian-based platform designed to include participants' voices in its development and through execution. Standardized methods will be used to longitudinally characterize ALS patients and healthy controls through deep clinical phenotyping, neuroimaging, neurocognitive and speech assessments, genotyping and multisource biospecimen collection. This effort plugs into complementary Canadian and international initiatives to share common resources. Here, we describe in detail the infrastructure, operating procedures, and long-term vision of CAPTURE ALS to facilitate and accelerate translational ALS research in Canada and beyond.

The spectrum of disease and tau pathology of nodding syndrome in Uganda.

Nodding syndrome is an enigmatic recurrent epidemic neurologic disease that affects children in East Africa. The illness begins with vertical nodding of the head and can progress to grand mal seizures and death after several years. The most recent outbreak of nodding syndrome occurred in northern Uganda. We now describe the clinicopathologic spectrum of nodding syndrome in northern Uganda. The neuropathologic findings of 16 children or young adults with fatal nodding syndrome were correlated with the onset, duration and progression of their neurological illness. The affected individuals ranged in age from 14 to 25 years at the time of death with a duration of illness ranging from 6-15 years. All 16 cases had chronic seizures. In 10 cases, detailed clinical histories were available and showed that three individuals had a clinical course that was predominantly characterized by epilepsy, whereas the other seven individuals had progressive cognitive, behavioural and motor decline, in addition to epilepsy. The main neuropathologic findings included: tau pathology (16/16 cases), cerebellar degeneration (11/16 cases) and white matter degeneration (7/16 cases). The tau pathology was characterized by filamentous tau-positive deposits in the form of neurofibrillary tangles, pre-tangles and dot-like grains and threads in the neuropil. All cases showed some degree of tau pathology in the neocortex and in the locus coeruleus with frequent involvement of the substantia nigra and tegmental nuclei and lesser involvement of other grey matter sites, but there was a lack of glial tau pathology. The tau pathology in the neocortex showed a multifocal superficial laminar pattern. We conclude that nodding syndrome is a clinicopathological entity associated consistently with tau pathology, but our observations did not establish the cause of the disease, or an explanation for the tau pathology.

Single-nucleus multiomic atlas of frontal cortex in amyotrophic lateral sclerosis with a deep learning-based decoding of alternative polyadenylation mechanisms.

The understanding of how different cell types contribute to amyotrophic lateral sclerosis (ALS) pathogenesis is limited. Here we generated a single-nucleus transcriptomic and epigenomic atlas of the frontal cortex of ALS cases with C9orf72 (C9) hexanucleotide repeat expansions and sporadic ALS (sALS). Our findings reveal shared pathways in C9-ALS and sALS, characterized by synaptic dysfunction in excitatory neurons and a disease-associated state in microglia. The disease subtypes diverge with loss of astrocyte homeostasis in C9-ALS, and a more substantial disturbance of inhibitory neurons in sALS. Leveraging high depth 3'-end sequencing, we found a widespread switch towards distal polyadenylation (PA) site usage across ALS subtypes relative to controls. To explore this differential alternative PA (APA), we developed APA-Net, a deep neural network model that uses transcript sequence and expression levels of RNA-binding proteins (RBPs) to predict cell-type specific APA usage and RBP interactions likely to regulate APA across disease subtypes.

A C-Terminally Truncated TDP-43 Splice Isoform Exhibits Neuronal Specific Cytoplasmic Aggregation and Contributes to TDP-43 Pathology in ALS.

Neuronal cytoplasmic aggregation and ubiquitination of TDP-43 is the most common disease pathology linking Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 pathology is characterized by the presence of low molecular weight TDP-43 species generated through proteolytic cleavage and/or abnormal RNA processing events. In addition to N-terminally truncated TDP-43 species, it has become evident that C-terminally truncated variants generated through alternative splicing in exon 6 also contribute to the pathophysiology of ALS/FTLD. Three such variants are listed in UCSD genome browser each sharing the same C-terminal unique sequence of 18 amino acids which has been shown to contain a putative nuclear export sequence. Here we have identified an additional C-terminally truncated variant of TDP-43 in human spinal cord tissue. This variant, called TDP43C-spl, is generated through use of non-canonical splice sites in exon 6, skipping 1,020 bp and encoding a 272 aa protein lacking the C-terminus with the first 256 aa identical to full-length TDP-43 and the same 18 amino acid C-terminal unique sequence. Ectopic expression studies in cells revealed that TDP43C-spl was localized to the nucleus in astrocytic and microglial cell lines but formed cytoplasmic ubiquitinated aggregates in neuronal cell lines. An antibody raised to the unique 18 amino acid sequence showed elevated levels of C-terminally truncated variants in ALS spinal cord tissues, and co-labeled TDP-43 pathology in disease affected spinal motor neurons. The retention of this 18 amino acid sequence among several C-terminally truncated TDP-43 variants suggests important functional relevance. Our studies of TDP43C-spl suggest this may be related to the selective vulnerability of neurons to TDP-43 pathology and cell-subtype differences in nuclear export.

Localization atomic force microscopy.

Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.

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.

TDP-43 stabilizes G3BP1 mRNA: relevance to amyotrophic lateral sclerosis/frontotemporal dementia.

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. Ras-GAP SH3-domain-binding protein 1 (G3BP1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3' untranslated region. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data indicate that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states.

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study, we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium toward the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

A cell's outer membrane is made of molecules called lipids, which band together to form a flexible thin film, just two molecules thick. This membrane is dotted with proteins that transport materials in to and out of cells. Most of these membrane proteins join with other proteins to form structures known as oligomers. Except, how membrane-bound proteins assemble into oligomers ­ the physical forces driving these molecules to take shape ­ remains unclear. This is partly because the structural, physical and chemical properties of fat-like lipid membranes are radically different to the cell's watery interior. Consequently, the conditions under which membrane oligomers form are distinct from those surrounding proteins inside cells. Membrane proteins are also more difficult to study and characterize than water-soluble proteins inside the cell, and yet many therapeutic drugs such as antibiotics specifically target membrane proteins. Overall, our understanding of how the unique properties of lipid membranes affect the formation of protein structures embedded within, is lacking and warrants further investigation. Now, Chadda, Bernhardt et al. focused on one membrane protein, known as CLC, which tends to exist in pairs ­ or dimers. To understand why these proteins form dimers (a process called dimerization) Chadda, Bernhardt et al. first used computer simulations, and then validated the findings in experimental tests. These complementary approaches demonstrated that the main reason CLC proteins 'dimerize' lies in their interaction with the lipid membrane, and not the attraction of one protein to its partner. When CLC proteins are on their own, they deform the surrounding membrane and create structural defects that put the membrane under strain. But when two CLC proteins join as a dimer, this membrane strain disappears ­ making dimerization the more stable and energetically favorable option. Chadda, Bernhardt et al. also showed that with the addition of a few certain lipids, specifically smaller lipids, cell membranes become more tolerant of protein-induced structural changes. This might explain how cells could use various lipids to fine-tune the activity of membrane proteins by controlling how oligomers form. However, the theory needs to be examined further. Altogether, this work has provided fundamental insights into the physical forces shaping membrane-bound proteins, relevant to researchers studying cell biology and pharmacology alike.