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BioRxiv : the Preprint Server For... May 2024Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates...
Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates and regulators via short linear motifs (SLIMs), PxIxIT and LxVP. PxIxIT binding to CN is Ca independent and affects its distribution, while LxVP associates only with the active enzyme and promotes catalysis. 31 human proteins contain one or more composite 'LxVPxIxIT' motifs, whose functional properties have not been examined. Here we report studies of calcimembrin/C16orf74 (CLMB), a largely uncharacterized protein containing a composite motif that binds and directs CN to membranes. We demonstrate that CLMB associates with membranes via N-myristoylation and dynamic S-acylation and is dephosphorylated by CN on Thr44. The LxVP and PxIxIT portions of the CLMB composite sequence, together with Thr44 phosphorylation, confer high affinity PxIxIT-mediated binding to CN (KD∼8.9 nM) via an extended, LxVPxIxITxx(p)T sequence. This binding promotes CLMB-based targeting of CN to membranes, but also protects Thr44 from dephosphorylation. Thus, we propose that CN dephosphorylates CLMB in multimeric complexes, where one CLMB molecule recruits CN to membranes via PxIxIT binding, allowing others to engage through their LxVP motif for dephosphorylation. This unique mechanism makes dephosphorylation sensitive to CLMB:CN ratios and is supported by and analyses. CLMB overexpression is associated with poor prognoses for several cancers, suggesting that it promotes oncogenesis by shaping CN signaling.
PubMed: 38798520
DOI: 10.1101/2024.05.12.593783 -
Current Opinion in Cell Biology Jun 2024Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation... (Review)
Review
Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation requires caveolin, cavin, and specific lipid types. The recent advances in understanding the structure and assembly of caveolin and cavin complexes within the membrane context have clarified the fundamental processes underlying caveola biogenesis. In addition, the curvature of the caveola membrane is controlled by the regulatory proteins EHD2, pacsin2, and dynamin2, which also function to restrain the scission of caveolae from the plasma membrane (PM). Here, this is integrated with novel insights on caveolae as lipid and mechanosensing complexes that can dynamically flatten or disassemble to counteract mechanical, and oxidative stress.
Topics: Humans; Caveolae; Cell Membrane; Animals; Caveolins
PubMed: 38788266
DOI: 10.1016/j.ceb.2024.102371 -
The Journal of Clinical Investigation May 2024Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an anti-inflammatory phenotype. High laminar shear stress...
Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an anti-inflammatory phenotype. High laminar shear stress also induces flow-aligned cell elongation and front-rear polarity, but whether these are required for the anti-inflammatory phenotype is unclear. Here, we showed that Caveolin-1-rich microdomains polarize to the downstream end of ECs that are exposed to continuous high laminar flow. These microdomains were characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid-type 4 (TRPV4) ion channels were ubiquitously expressed on the plasma membrane but mediated localized Ca2+ entry only at these microdomains where they physically interacted with clustered Caveolin-1. These focal Ca2+ bursts activated endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we found that signaling at these domains required both cell body elongation and sustained flow. Finally, TRPV4 signaling at these domains was necessary and sufficient to suppress inflammatory gene expression, and exogenous activation of TRPV4 channels ameliorated the inflammatory response to stimuli both in vitro and in vivo. Our work revealed a polarized mechanosensitive signaling hub in arterial ECs that dampens inflammatory gene expression and promotes cell resilience.
PubMed: 38771648
DOI: 10.1172/JCI175057 -
Frontiers in Molecular Biosciences 2024Advances in molecular targeting of ion channels may open up new avenues for therapeutic approaches in cancer based on the cells' bioelectric properties. In addition to...
INTRODUCTION
Advances in molecular targeting of ion channels may open up new avenues for therapeutic approaches in cancer based on the cells' bioelectric properties. In addition to or models, models can provide deeper insight into the complex role of electrophysiology in cancer and reveal the impact of altered ion channel expression and the membrane potential on malignant processes. The A549 model is the first computational cancer whole-cell ion current model that simulates the bioelectric mechanisms of the human non-small cell lung cancer cell line A549 during the different phases of the cell cycle. This work extends the existing model with a detailed mathematical description of the store-operated Ca entry (SOCE) and the complex local intracellular calcium dynamics, which significantly affect the entire electrophysiological properties of the cell and regulate cell cycle progression.
METHODS
The initial model was extended by a multicompartmental approach, addressing the heterogenous calcium profile and dynamics in the ER-PM junction provoked by local calcium entry of store-operated calcium channels (SOCs) and uptake by SERCA pumps. Changes of cytosolic calcium levels due to diffusion from the ER-PM junction, release from the ER by RyR channels and IP3 receptors, as well as corresponding PM channels were simulated and the dynamics evaluated based on calcium imaging data. The model parameters were fitted to available data from two published experimental studies, showing the function of CRAC channels and indirectly of IP3R, RyR and PMCA via changes of the cytosolic calcium levels.
RESULTS
The proposed calcium description accurately reproduces the dynamics of calcium imaging data and simulates the SOCE mechanisms. In addition, simulations of the combined A549-SOCE model in distinct phases of the cell cycle demonstrate how Ca - dynamics influence responding channels such as KCa, and consequently modulate the membrane potential accordingly.
DISCUSSION
Local calcium distribution and time evolution in microdomains of the cell significantly impact the overall electrophysiological properties and exert control over cell cycle progression. By providing a more profound description, the extended A549-SOCE model represents an important step on the route towards a valid model for oncological research and supported development of novel therapeutic strategies.
PubMed: 38770217
DOI: 10.3389/fmolb.2024.1394398 -
Langmuir : the ACS Journal of Surfaces... May 2024Diverse collections of lipids self-assemble into domains within biological membranes, and these domains are typically organized in both the transverse and lateral...
Diverse collections of lipids self-assemble into domains within biological membranes, and these domains are typically organized in both the transverse and lateral directions of the membrane. The ability of the membrane to link these domains across the membrane's interior grants cells control over features on the external cellular surface. Numerous hypothesized factors drive the cross-membrane (or transverse) coupling of lipid domains. In this work we seek to isolate these transverse lipid-lipid influences in a simple model system using droplet interface bilayers (DIBs) to better understand the associated mechanics. DIBs enable symmetric and asymmetric combinations of domain-forming lipid mixtures within a model bilayer, and the evolving energetics of the membrane may be tracked using drop-shape analysis. We find that symmetric distributions of domain-forming lipids produce long-lasting, gradual shifts in the DIB membrane energetics that are not observed in asymmetric distributions of the lipids where the domain-forming lipids are only within one leaflet. The approach selected for this work provides experimental measurement of the mismatch penalty associated with antiregistered lipid domains as well as measurements of the influence of rafts on DIB behaviors with suggestions for their future use as a model platform.
Topics: Lipid Bilayers; Membrane Microdomains; Phosphatidylcholines
PubMed: 38753461
DOI: 10.1021/acs.langmuir.4c00958 -
Immunity Jun 2024Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and...
Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.
Topics: Animals; Humans; Mice; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Claudins; Gene Expression Regulation, Neoplastic; Immunological Synapses; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Membrane Microdomains; Mice, Inbred C57BL; Pancreatic Neoplasms; T-Lymphocytes, Cytotoxic; Tumor Microenvironment
PubMed: 38749447
DOI: 10.1016/j.immuni.2024.04.021 -
International Journal of Molecular... Apr 2024Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for...
Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.
Topics: Epithelial Sodium Channels; Humans; Endocytosis; Neutrophils; Hypertension; Cell Membrane; Membrane Lipids; Protein Serine-Threonine Kinases; Male; Female; Immediate-Early Proteins; Middle Aged; Membrane Microdomains
PubMed: 38732158
DOI: 10.3390/ijms25094939 -
International Journal of Molecular... Apr 2024The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the...
The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.
Topics: Animals; Sphingomyelin Phosphodiesterase; TRPM Cation Channels; Mice; Humans; CHO Cells; Cricetulus; beta-Cyclodextrins; HEK293 Cells; Disease Models, Animal; Membrane Microdomains; Pain; Cholesterol; Male; Analgesics; Pregnenolone; Cell Survival
PubMed: 38731855
DOI: 10.3390/ijms25094637 -
The European Physical Journal. E, Soft... May 2024The aggregation or clustering of proteins and other macromolecules plays an important role in the formation of large-scale molecular assemblies within cell membranes....
The aggregation or clustering of proteins and other macromolecules plays an important role in the formation of large-scale molecular assemblies within cell membranes. Examples of such assemblies include lipid rafts, and postsynaptic domains (PSDs) at excitatory and inhibitory synapses in neurons. PSDs are rich in scaffolding proteins that can transiently trap transmembrane neurotransmitter receptors, thus localizing them at specific spatial positions. Hence, PSDs play a key role in determining the strength of synaptic connections and their regulation during learning and memory. Recently, a two-dimensional (2D) diffusion-mediated aggregation model of PSD formation has been developed in which the spatial locations of the clusters are determined by a set of fixed anchoring sites. The system is kept out of equilibrium by the recycling of particles between the cell membrane and interior. This results in a stationary distribution consisting of multiple clusters, whose average size can be determined using an effective mean-field description of the particle concentration around each anchored cluster. In this paper, we derive corrections to the mean-field approximation by applying the theory of diffusion in singularly perturbed domains. The latter is a powerful analytical method for solving two-dimensional (2D) and three-dimensional (3D) diffusion problems in domains where small holes or perforations have been removed from the interior. Applications range from modeling intracellular diffusion, where interior holes could represent subcellular structures such as organelles or biological condensates, to tracking the spread of chemical pollutants or heat from localized sources. In this paper, we take the bounded domain to be the cell membrane and the holes to represent anchored clusters. The analysis proceeds by partitioning the membrane into a set of inner regions around each cluster, and an outer region where mean-field interactions occur. Asymptotically matching the inner and outer stationary solutions generates an asymptotic expansion of the particle concentration, which includes higher-order corrections to mean-field theory that depend on the positions of the clusters and the boundary of the domain. Motivated by a recent study of light-activated protein oligomerization in cells, we also develop the analogous theory for cluster formation in a three-dimensional (3D) domain. The details of the asymptotic analysis differ from the 2D case due to the contrasting singularity structure of 2D and 3D Green's functions.
Topics: Diffusion; Cell Membrane; Membrane Microdomains; Models, Biological
PubMed: 38720027
DOI: 10.1140/epje/s10189-024-00425-8 -
Frontiers in Cellular and Infection... 2024cytolethal distending toxin (Cdt) is capable of intoxicating lymphocytes macrophages, mast cells and epithelial cells. Following Cdt binding to cholesterol, in the...
cytolethal distending toxin (Cdt) is capable of intoxicating lymphocytes macrophages, mast cells and epithelial cells. Following Cdt binding to cholesterol, in the region of membrane lipid rafts, the CdtB and CdtC subunits are internalized and traffic to intracellular compartments. These events are dependent upon, cellugyrin, a critical component of synaptic like microvesicles (SLMV). Target cells, such as Jurkat cells, rendered unable to express cellugyrin are resistant to Cdt-induced toxicity. Similar to Cdt, SARS-CoV-2 entry into host cells is initiated by binding to cell surface receptors, ACE-2, also associated with cholesterol-rich lipid rafts; this association leads to fusion and/or endocytosis of viral and host cell membranes and intracellular trafficking. The similarity in internalization pathways for both Cdt and SARS-CoV-2 led us to consider the possibility that cellugyrin was a critical component in both processes. Cellugyrin deficient Calu-3 cells (Calu-3) were prepared using Lentiviral particles containing shRNA; these cells were resistant to infection by VSV/SARS-CoV-2-spike pseudotype virus and partially resistant to VSV/VSV-G pseudotype virus. Synthetic peptides representing various regions of the cellugyrin protein were prepared and assessed for their ability to bind to Cdt subunits using surface plasmon resonance. Cdt was capable of binding to a region designated the middle outer loop (MOL) which corresponds to a region extending into the cytoplasmic surface of the SLMV. SARS-CoV-2 spike proteins were assessed for their ability to bind to cellugyrin peptides; SARS-CoV-2 full length spike protein preferentially binds to a region within the SLMV lumen, designated intraluminal loop 1A. SARS-CoV-2-spike protein domain S1, which contains the receptor binding domains, binds to cellugyrin N-terminus which extends out from the cytoplasmic surface of SLMV. Binding specificity was further analyzed using cellugyrin scrambled peptide mutants. We propose that SLMV represent a component of a common pathway that facilitates pathogen and/or pathogen-derived toxins to gain host cell entry.
Topics: Humans; Bacterial Toxins; Virus Internalization; SARS-CoV-2; Synaptogyrins; COVID-19; Jurkat Cells; Aggregatibacter actinomycetemcomitans; Angiotensin-Converting Enzyme 2; Endocytosis; Spike Glycoprotein, Coronavirus; Membrane Microdomains
PubMed: 38698905
DOI: 10.3389/fcimb.2024.1334224