-
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 -
British Journal of Pharmacology Apr 2024ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches...
BACKGROUND AND PURPOSE
ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated.
EXPERIMENTAL APPROACH
Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca measurements were carried out in Fluo-4 loaded cells. Submembrane Ca was examined in PC12 cells transfected with a membrane-tethered Ca indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca influx through this receptor was measured using a P2X7 receptor-GCamp6 construct.
KEY RESULTS
ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca microdomains inhibited by A740003.
CONCLUSION AND IMPLICATIONS
Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca microdomains.
PubMed: 38679932
DOI: 10.1111/bph.16371 -
Cell Reports May 2024Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor...
Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both in vivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.
Topics: Receptors, Opioid, mu; Cilia; Animals; Mice; Humans; HEK293 Cells; Protein Transport
PubMed: 38678559
DOI: 10.1016/j.celrep.2024.114164 -
Journal of Cell Science Apr 2024Amyloid β (Aβ) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aβ disrupts AMPA receptor-mediated synaptic...
Amyloid β (Aβ) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aβ disrupts AMPA receptor-mediated synaptic plasticity, a key factor in early AD progression. Numerous studies propose that Aβ oligomers hinder synaptic plasticity, particularly long-term potentiation (LTP), by disrupting GluA1 (encoded by GRIA1) function, although the precise mechanism remains unclear. In this study, we demonstrate that Aβ mediates the accumulation of GM1 ganglioside in lipid raft domains of cultured cells, and GluA1 exhibits preferential localization in lipid rafts via direct binding to GM1. Aβ enhances the raft localization of GluA1 by increasing GM1 in these areas. Additionally, chemical LTP stimulation induces lipid raft-dependent GluA1 internalization in Aβ-treated neurons, resulting in reduced cell surface and postsynaptic expression of GluA1. Consistent with this, disrupting lipid rafts and GluA1 localization in rafts rescues Aβ-mediated suppression of hippocampal LTP. These findings unveil a novel functional deficit in GluA1 trafficking induced by Aβ, providing new insights into the mechanism underlying AD-associated cognitive dysfunction.
Topics: Amyloid beta-Peptides; Long-Term Potentiation; Receptors, AMPA; Membrane Microdomains; Alzheimer Disease; Animals; Hippocampus; G(M1) Ganglioside; Humans; Neurons; Rats; Mice; Protein Transport
PubMed: 38668720
DOI: 10.1242/jcs.261281 -
Biosensors Apr 2024C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates...
C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.
Topics: Fluorescence Resonance Energy Transfer; Biosensing Techniques; Humans; Animals; CSK Tyrosine-Protein Kinase; Rats; src-Family Kinases; Phosphorylation; Membrane Microdomains; src Homology Domains
PubMed: 38667199
DOI: 10.3390/bios14040206 -
Journal of Chemical Information and... May 2024The lipid raft subdomains in cancer cell membranes play a key role in signal transduction, biomolecule recruitment, and drug transmembrane transport. Augmented membrane...
The lipid raft subdomains in cancer cell membranes play a key role in signal transduction, biomolecule recruitment, and drug transmembrane transport. Augmented membrane rigidity due to the formation of a lipid raft is unfavorable for the entry of drugs, a limiting factor in clinical oncology. The short-chain ceramide (CER) has been reported to promote drug entry into membranes and disrupt lipid raft formation, but the underlying mechanism is not well understood. We recently explored the carrier-membrane fusion dynamics of PEG-DPPE micelles in delivering doxorubicin (DOX). Based on the phase-segregated membrane model composed of DPPC/DIPC/CHOL/GM1/PIP2, we aim to explore the dynamic mechanism of the PEG-DPPE micelle-encapsulating DOXs in association with the raft-included cell membrane modulated by C8 acyl tail CERs. The results show that the lipid raft remains integrated and DOX-resistant subjected to free DOXs and the micelle-encapsulating ones. Addition of CERs disorganizes the lipid raft by pushing CHOL aside from DPPC. It subsequently allows for a good permeability for PEG-DPPE micelle-encapsulated DOXs, which penetrate deeper as CER concentration increases. GM1 is significant in guiding drugs' redistributing between bilayer phases, and the anionic PIP2 further helps DOXs attain the inner bilayer surface. These results elaborate on the perturbing effect of CERs on lipid raft stability, which provides a new comprehensive approach for further design of drug delivery systems.
Topics: Humans; Ceramides; Doxorubicin; Membrane Microdomains; Micelles; Molecular Dynamics Simulation; Phosphatidylethanolamines; Polyethylene Glycols
PubMed: 38652138
DOI: 10.1021/acs.jcim.4c00170 -
Journal of the American Chemical Society May 2024Lipid rafts, which are dynamic nanodomains in the plasma membrane, play a crucial role in intermembrane processes by clustering together and growing in size within the...
Lipid rafts, which are dynamic nanodomains in the plasma membrane, play a crucial role in intermembrane processes by clustering together and growing in size within the plane of the membrane while also aligning with each other across different membranes. However, the physical origin of layer by layer alignment of lipid rafts remains to be elucidated. Here, by using fluorescence imaging and synchrotron X-ray reflectivity in a phase-separated multilayer system, we find that the alignment of raft-mimicking L domains is regulated by the distance between bilayers. Molecular dynamics simulations reveal that the aligned state is energetically preferred when the intermembrane distance is small due to its ability to minimize the volume of surface water, which has fewer water hydrogen bonds (HBs) compared to bulk water. Our results suggest that water HB-driven alignment of lipid rafts plays a role as a precursor of intermembrane processes such as cell-cell fusion, virus entry, and signaling.
Topics: Water; Membrane Microdomains; Hydrogen Bonding; Molecular Dynamics Simulation; Lipid Bilayers
PubMed: 38652033
DOI: 10.1021/jacs.4c00544 -
Nature Reviews. Molecular Cell Biology Jul 2024
Topics: Endocytosis; Caveolae; Dynamins; Animals; Humans
PubMed: 38649754
DOI: 10.1038/s41580-024-00735-x -
FEBS Letters May 2024The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA...
The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA sequences binds both the amino acid arginine and lipid membranes, we constructed a bifunctional RNA 10Arg aptamer specific for arginine and lipid vesicles. The preference of RNA 10Arg for lipid rafts was visualized and confirmed using FRET microscopy in neuroblastoma cells. The selection-amplification (SELEX) method using a doped (with the other three nucleotides) pool of RNA 10Arg sequences yielded several RNA 10Arg(D) sequences, and the affinities of these RNAs both to arginine and liposomes are improved in comparison to pre-doped RNA. Generation of these bispecific aptamers supports the hypothesis that an RNA molecule can bind both to RNA-binding proteins (RBPs) through arginine within the RBP-binding site and to membrane lipid rafts, thus facilitating RNA loading into exosomes and other extracellular vesicles.
Topics: Arginine; Humans; Liposomes; Membrane Microdomains; Aptamers, Nucleotide; Cell Line, Tumor; RNA-Binding Proteins; Base Sequence; RNA; Exosomes; Fluorescence Resonance Energy Transfer
PubMed: 38649155
DOI: 10.1002/1873-3468.14880 -
Lipids in Health and Disease Apr 2024Disturbances in cholesterol homeostasis have been associated with ASD. Lipid rafts are central in many transmembrane signaling pathways (including mTOR) and changes in...
Disturbances in cholesterol homeostasis have been associated with ASD. Lipid rafts are central in many transmembrane signaling pathways (including mTOR) and changes in raft cholesterol content affect their order function. Cholesterol levels are controlled by several mechanisms, including endoplasmic reticulum associated degradation (ERAD) of the rate limiting HMGCoA reductase. A new approach to increase cholesterol via temporary ERAD blockade using a benign bacterial toxin-derived competitor for the ERAD translocon is suggested.A new lock and key model for cholesterol/lipid raft dependent signaling is proposed in which the rafts provide both the afferent and efferent 'tumblers' across the membrane to allow 'lock and key' receptor transmembrane signals.
Topics: Humans; Autism Spectrum Disorder; Cholesterol; Endoplasmic Reticulum-Associated Degradation; Membrane Microdomains
PubMed: 38643132
DOI: 10.1186/s12944-024-02075-3