Clustering Of Membrane Proteins Research Articles

Partitioning into ER membrane microdomains impacts autophagic protein turnover during cellular aging

Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.

Membrane protein clustering from tension and multibody interactions

The point-curvature model for membrane protein inclusions is shown to capture multibody interactions very well. Using this model, we find that the interplay between membrane tension and multibody interactions results in a collective attraction of oppositely curved inclusions tending to form antiferromagnetic structures with a square lattice. This attraction can produce a phase separation between curved and non-curved proteins, resulting in the clustering of curved proteins. We also show that the many-body repulsion between identical curved proteins is enhanced by membrane tension. This can lead to the dissolution of clusters stabilized by short-range forces when the tension is increased. These new phenomena are biologically relevant and could be investigated experimentally.

Curvature-induced clustering of cell adhesion proteins.

Cell adhesion proteins typically form stable clusters that anchor the cell membrane to its environment. Several works have suggested that cell membrane protein clusters can emerge from a local feedback between the membrane curvature and the density of proteins. Here, we investigate the effect of such a curvature-sensing mechanism in the context of cell adhesion proteins. We show how clustering emerges in an intermediate range of adhesion and curvature-sensing strengths. We identify key differences with the tilt-induced gradient sensing mechanism we previously proposed (Lin et al., arXiv:2307.03670).

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface.

The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.

Phytosterols reverse antiretroviral-induced hearing loss, with potential implications for cochlear aging.

Cholesterol contributes to neuronal membrane integrity, supports membrane protein clustering and function, and facilitates proper signal transduction. Extensive evidence has shown that cholesterol imbalances in the central nervous system occur in aging and in the development of neurodegenerative diseases. In this work, we characterize cholesterol homeostasis in the inner ear of young and aged mice as a new unexplored possibility for the prevention and treatment of hearing loss. Our results show that cholesterol levels in the inner ear are reduced during aging, an effect that is associated with an increased expression of the cholesterol 24-hydroxylase (CYP46A1), the main enzyme responsible for cholesterol turnover in the brain. In addition, we show that pharmacological activation of CYP46A1 with the antiretroviral drug efavirenz reduces the cholesterol content in outer hair cells (OHCs), leading to a decrease in prestin immunolabeling and resulting in an increase in the distortion product otoacoustic emissions (DPOAEs) thresholds. Moreover, dietary supplementation with phytosterols, plant sterols with structure and function similar to cholesterol, was able to rescue the effect of efavirenz administration on the auditory function. Altogether, our findings point towards the importance of cholesterol homeostasis in the inner ear as an innovative therapeutic strategy in preventing and/or delaying hearing loss.

Modulation of bacterial membrane proteins activity by clustering into plasma membrane nanodomains.

Recent research has demonstrated specific protein clustering within membrane subdomains in bacteria, challenging the long-held belief that prokaryotes lack these subdomains. This mini review provides examples of bacterial membrane protein clustering, discussing the benefits of protein assembly in membranes and highlighting how clustering regulates protein activity.

Expansion Microscopy Application for Calcium Protein Clustering Imaging in Cells and Brain Tissues.

Many biological studies require high-resolution imaging and subsequent analysis of cell organelles and molecules. Some membrane proteins form tight clusters, and this process is directly linked to their function. In most studies, these small protein clusters have been investigated by total internal reflection fluorescence (TIRF) microscopy, which enables imaging with high spatial resolution within 100 nm of the membrane surface. Recently developed expansion microscopy (ExM) makes it possible to achieve nanometer resolution using a conventional fluorescence microscope by physically expanding the sample. In this article, we describe implementation of ExM for imaging of protein clusters formed by the endoplasmic reticulum (ER) calcium sensor protein STIM1. This protein translocates during ER store depletion and forms clusters that support contact with plasma membrane (PM) calcium-channel proteins. ER calcium channels such as the type 1 inositol triphosphate receptor (IP3R) also form clusters, but their investigation by TIRF microscopy is impossible due to the large distance from the PM. In this article, we demonstrate how to investigate IP3R clustering using ExM in hippocampal brain tissues. We compare IP3R clustering in the CA1 area of the hippocampus of wild-type and 5xFAD Alzheimer's disease model mice. To facilitate future applications, we describe experimental protocols and image processing guidelines for application of ExM to membrane and ER protein clustering studies in cultured cells and brain tissues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Expansion microscopy application for protein cluster visualization in cells Alternate Protocol: Expansion microscopy application for protein cluster visualization in brain tissues Basic Protocol 2: Protein cluster analysis of expansion microscopy images using ImageJ and Icy software.

Thermoplasmonic Vesicle Fusion Reveals Membrane Phase Segregation of Influenza Spike Proteins.

Many cellular processes involve the lateral organization of integral and peripheral membrane proteins into nanoscale domains. Despite the biological significance, the mechanisms that facilitate membrane protein clustering into nanoscale lipid domains remain enigmatic. In cells, the analysis of membrane protein phase affinity is complicated by the size and temporal nature of ordered and disordered lipid domains. To overcome these limitations, we developed a method for delivering membrane proteins from transfected cells into phase-separated model membranes that combines optical trapping with thermoplasmonic-mediated membrane fusion and confocal imaging. Using this approach, we observed clear phase partitioning into the liquid disordered phase following the transfer of GFP-tagged influenza hemagglutinin and neuraminidase from transfected cell membranes to giant unilamellar vesicles. The generic platform presented here allows investigation of the phase affinity of any plasma membrane protein which can be labeled or tagged with a fluorescent marker.

A novel FRET-based DNA nanosensor to quantify clustering forces of membrane proteins.

Membrane proteins play a crucial role in various biological phenomena such as endocytosis and signal transduction. These phenomena are heavily dependent on the clustering of membrane proteins. While the propensity of proteins to cluster on the cell membrane has been well established, the physical mechanisms that govern this behavior are far from clear. Furthermore, the tools to be able to measure the forces responsible for the clustering of membrane proteins have not been developed yet. To bridge this gap, we have combined elements from biophysics, cellular and chemical biology, and biochemistry, to design a FRET-based DNA nanosensor. Our results demonstrate that an increase in the stiffness of the sensor increases the distance between the two dyes, which is obtained via FRET efficiency measurements. The sensor, once calibrated for stiffness, will be combined with this distance measurement to obtain the force that drives the interaction between pairs of proteins. As a proof of concept, the sensor design and measurements are being done using the B-subunit of the Shiga Toxin (STxB). Following measurements on STxB proteins, this novel method shall then be applied to understand the clustering behaviour of other membrane proteins.

Nonequilibrium control of cell membrane organisation - active emulsions.

The organisation of lipids and proteins on the plasma membrane have conventionally been described using equilibrium physics principles. Over the years there is growing evidence for a nonequilibrium control of membrane composition arising from the interaction of membrane components with cortical components. A specific example of this is the interaction with cortical actomyosin. This gives rise to dynamic nano clusters of membrane proteins and mesoscale active emulsions of lipids. Such a nonequilibrium picture highlights the role of membrane asymmetry and transbilayer coupling. Specificity of organisation is also facilitated by having an elaboration of the actomyosin cortex with a diversity of force generators. In this talk I will highlight the new theoretical ideas that form the basis of this active view of the cell membrane.

Lifetime of actin-dependent protein nanoclusters

Protein nanoclusters (PNCs) are dynamic collections of a few proteins that spatially organize in nanometer-length clusters. PNCs are one of the principal forms of spatial organization of membrane proteins, and they have been shown or hypothesized to be important in various cellular processes, including cell signaling. PNCs show remarkable diversity in size, shape, and lifetime. In particular, the lifetime of PNCs can vary over a wide range of timescales. The diversity in size and shape can be explained by the interaction of the clustering proteins with the actin cytoskeleton or the lipid membrane, but very little is known about the processes that determine the lifetime of the nanoclusters. In this paper, using mathematical modeling of the cluster dynamics, we model the biophysical processes that determine the lifetime of actin-dependent PNCs. In particular, we investigated the role of actin aster fragmentation, which had been suggested to be a key determinant of the PNC lifetime, and we found that it is important only for a small class of PNCs. A simple extension of our model allowed us to investigate the kinetics of protein-ligand interaction near PNCs. We found an anomalous increase in the lifetime of ligands near PNCs, which agrees remarkably well with experimental data on RAS-RAF kinetics. In particular, analysis of the RAS-RAF data through our model provides falsifiable predictions and novel hypotheses that will not only shed light on the role of RAS-RAF kinetics in various cancers, but also will be useful in studying membrane protein clustering in general.

Correlated STORM-homoFRET imaging reveals highly heterogeneous membrane receptor structures

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

Architecture of the human erythrocyte ankyrin-1 complex.

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.

Environmental Cues Contribute to Dynamic Plasma Membrane Organization of Nanodomains Containing Flotillin-1 and Hypersensitive Induced Reaction-1 Proteins in Arabidopsis thaliana.

Plasma membranes are heterogeneous and contain multiple functional nanodomains. Although several signaling proteins have been shown to function by moving into or out of nanodomains, little is known regarding the effects of environmental cues on nanodomain organization. In this study, we investigated the heterogeneity and organization of distinct nanodomains, including those containing Arabidopsis thaliana flotillin-1 (AtFlot1) and hypersensitive induced reaction-1 proteins (AtHIR1), in response to biotic and abiotic stress. Variable-angle total internal reflection fluorescence microscopy coupled with single-particle tracking (SPT) revealed that AtFlot1 and AtHIR1 exhibit different lateral dynamics and inhabit different types of nanodomains. Furthermore, via SPT and fluorescence correlation spectroscopy, we observed lower density and intensity of AtFlot1 fluorescence in the plasma membrane after biotic stress. In contrast, the density and intensity of signal indicating AtHIR1 markedly increased in response to biotic stress. In response to abiotic stress, the density and intensity of both AtFlot1 and AtHIR1 signals decreased significantly. Importantly, SPT coupled with fluorescence recovery after photobleaching revealed that biotic and abiotic stress can regulate the dynamics of AtFlot1; however, only the abiotic stress can regulate AtHIR1 dynamics. Taken together, these findings suggest that a plethora of highly distinct nanodomains coexist in the plasma membrane (PM) and that different nanodomains may perform distinct functions in response to biotic and abiotic stresses. These phenomena may be explained by the spatial clustering of plasma membrane proteins with their associated signaling components within dedicated PM nanodomains.

ARHGAP1 Transported with Influenza Viral Genome Ensures Integrity of Viral Particle Surface through Efficient Budozone Formation.

ABSTRACTInfluenza viral particles are assembled at the plasma membrane concomitantly with Rab11a-mediated endocytic transport of viral ribonucleoprotein complexes (vRNPs). The mechanism of spatiotemporal regulation of viral budozone formation and its regulatory molecules on the endocytic vesicles remain unclear. Here, we performed a proximity-based proteomics approach for Rab11a and found that ARHGAP1, a Rho GTPase-activating protein, is transported through the Rab11a-mediated apical transport of vRNP. ARHGAP1 stabilized actin filaments in infected cells for the lateral clustering of hemagglutinin (HA) molecules, a viral surface membrane protein, to the budozone. Disruption of the HA clustering results in the production of virions with low HA content, and such virions were less resistant to protease and had enhanced antigenicity, presumably because reduced clustering of viral membrane proteins exposes hidden surfaces. Collectively, these results demonstrate that Rab11a-mediated endocytic transport of ARHGAP1 with vRNPs stimulates budozone formation to ensure the integrity of virion surface required for viral survival.

In Situ DNA Self-Assembly on the Cell Surface Drives Unidirectional Clustering of Membrane Proteins for the Modulation of Cell Behaviors.

Cell membrane proteins play a pivotal role in regulating intracellular signal transductions and cell behaviors. Many membrane proteins form clusters in order to initiate downstream signaling pathways for the modulation of cell behaviors. Developing rational methods to program the in situ clustering of designated membrane proteins on the cell surface to form large assemblies remains challenging. Here we use the membrane-anchored DNA hybridization chain reaction (HCR) to induce DNA self-assembly on the live cell surface and drive the unidirectional clustering of membrane proteins for the modulation of cell behaviors. Reactive DNA strands are specifically anchored onto the membrane proteins of interest by using DNA aptamers. Upon activation, the chain reaction between the protein-anchored DNA strands drives the assembly of membrane proteins forming one-dimensional clusters. We demonstrate both homogeneous and heterogeneous clustering of membrane proteins on multiple cell types that exhibit a potent capability for modulating cell behaviors including migration, proliferation, and survival.

Reelin has antidepressant-like effects after repeated or singular peripheral injections

Chronic stress is a significant risk factor for depression onset. The effects of chronic stress can be studied preclinically using a corticosterone (CORT)-administration paradigm that results in a phenotype of depressive-like behavior associated with neurochemical abnormalities in brain regions like the hippocampus. We have recently shown that intrahippocampal infusions of Reelin have a fast effect in normalizing CORT-induced behavioral and neurochemical alterations. Reelin is also expressed in multiple peripheral systems and is found in blood plasma which prompted us to investigate whether peripheral intravenous (i.v.) Reelin injections could also result in antidepressant (ATD)-like actions. Repeated i.v. injections of Reelin were effective in rescuing the CORT-induced increases in forced-swim-test immobility in male and female rats, decreases in Reelin-immunopositive cells in the dentate gyrus subgranular zone, the expression of hippocampal GABAAβ2/3, GluA1, and GluN2B receptors, and serotonin transporter (SERT) membrane protein clustering (MPC) in blood lymphocytes. However, Reelin had only a partial effect on the number and maturation rate of dentate gyrus newborn cells. CORT and Reelin did not affect open field test behavior. After evaluating the effects of multiple Reelin injections, we demonstrated that a single Reelin injection administered at the end of CORT treatment could rescue in 24 h the behavioral (forced-swim-test and object-in-place test), as well as SERT MPC and neurochemical effects of CORT. These findings show that i.v. injections of Reelin have fast ATD-like effects associated with the restoration of hippocampal neurochemical deficits. Although additional mechanistic and pharmacokinetic studies are necessary, our data open the possibility to develop Reelin-based therapeutics with putative fast-ATD activity.

Global well-posedness for volume–surface reaction–diffusion systems

We study the global existence of classical solutions to volume–surface reaction–diffusion systems with control of mass. Such systems appear naturally from modeling evolution of concentrations or densities appearing both in a volume domain and its surface, and therefore have attracted considerable attention. Due to the characteristic volume–surface coupling, global existence of solutions to general systems is a challenging issue. In particular, the duality method, which is powerful in dealing with mass conserved systems in domains, is not applicable on its own. In this paper, we introduce a new family of [Formula: see text]-energy functions and combine them with a suitable duality method for volume–surface systems, to ultimately obtain global existence of classical solutions under a general assumption called the intermediate sum condition. For systems that conserve mass, but do not satisfy this condition, global solutions are shown under a quasi-uniform condition, that is, under the assumption that the diffusion coefficients are close to each other. In the case of mass dissipation, we also show that the solution is bounded uniformly in time by studying the system on each time-space cylinder of unit size, and showing that the solution is sup-norm bounded independently of the cylinder. Applications of our results include global existence and boundedness for systems arising from membrane protein clustering or activation of Cdc42 in cell polarization.

A novel FRET-based DNA nanosensor to quantify clustering forces of membrane proteins

Membrane proteins play a crucial role in various biological phenomena such as endocytosis and signal transduction. These phenomena are heavily dependent on the clustering of membrane proteins. While the propensity of proteins to cluster on the cell membrane has been well established, the physical mechanisms that govern this behavior are far from clear. Furthermore, the tools to be able to measure the forces responsible for the clustering of membrane proteins have not been developed yet. To bridge this gap, we have combined elements from biophysics, cellular and chemical biology, and biochemistry, to develop a highly sensitive FRET-based DNA nanosensor.

From spatial to temporal patterns: cooperativity across ion channels in supercritical membranes

Theoretically predicted by Fisher and de Gennes in 1978, critical Casimir forces are the thermodynamic analogues of the Casimir-Polder attraction forces due to quantum fluctuations of confined electromagnetic field in vacuum. In the context of lipid membranes close to a miscibility transition, critical Casimir interactions are thought to be responsible for clustering of membrane proteins. Here we explore the hypothesis that these effective statistical-field mediated interactions can explain the observed ion channel cooperativity that has been suggested to promote multistability of the membrane potential and thus “cellular memory”.