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1.
Sci Adv ; 10(19): eadi9156, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38718108

RESUMO

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.


Assuntos
Endocitose , Exossomos , Tetraspanina 30 , Exossomos/metabolismo , Humanos , Tetraspanina 30/metabolismo , Biomarcadores/metabolismo , Sinteninas/metabolismo , Sinteninas/genética , Tetraspanina 28/metabolismo , Membrana Celular/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Tetraspanina 29/metabolismo
2.
Life Sci Alliance ; 7(7)2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38719747

RESUMO

The differential expression of plasma membrane proteins is integrally analyzed for their diagnosis, prognosis, and therapeutic applications in diverse clinical manifestations. Necessarily, distinct membrane protein enrichment methods and mass spectrometry platforms are employed for their global and relative quantitation. First of its kind to explore, we compiled membrane-associated proteomes in human and mouse systems into a database named, Resource of Experimental Membrane-Enriched Mass spectrometry-derived Proteome (REMEMProt). It currently hosts 14,626 proteins (9,507 proteins in Homo sapiens; 5,119 proteins in Mus musculus) with information on their membrane-protein enrichment methods, experimental/physiological context of detection in cells or tissues, transmembrane domain analysis, and their current attribution as biomarkers. Based on these annotations and the transmembrane domain analysis in proteins or their binary/complex protein-protein interactors, REMEMProt facilitates the assessment of the plasma membrane localization potential of proteins through batch query. A cross-study enrichment analysis platform is enabled in REMEMProt for comparative analysis of proteomes using novel/modified membrane enrichment methods and evaluation of methods for targeted enrichment of membrane proteins. REMEMProt data are made freely accessible to explore and download at https://rememprot.ciods.in/.


Assuntos
Biomarcadores , Bases de Dados de Proteínas , Proteínas de Membrana , Proteoma , Proteômica , Humanos , Proteoma/metabolismo , Proteínas de Membrana/metabolismo , Biomarcadores/metabolismo , Animais , Camundongos , Proteômica/métodos , Membrana Celular/metabolismo , Espectrometria de Massas/métodos
3.
Front Immunol ; 15: 1401294, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38720899

RESUMO

Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in trans on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in cis, but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in cis and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors.


Assuntos
Esfingolipídeos , Animais , Humanos , Esfingolipídeos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fagocitose , Fagócitos/imunologia , Fagócitos/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Membrana Celular/metabolismo , Ligação Proteica
4.
Acta Biochim Pol ; 71: 11999, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38721306

RESUMO

Candida glabrata is an important opportunistic human pathogen well known to develop resistance to antifungal drugs. Due to their numerous desirable qualities, antimicrobial lipopeptides have gained significant attention as promising candidates for antifungal drugs. In the present study, two bioactive lipopeptides (AF4 and AF5 m/z 1071.5 and 1085.5, respectively), coproduced and purified from Bacillus subtilis RLID12.1, consist of seven amino acid residues with lipid moieties. In our previous studies, the reversed phased-HPLC purified lipopeptides demonstrated broad-spectrum of antifungal activities against over 110 Candida albicans, Candida non-albicans and mycelial fungi. Two lipopeptides triggered membrane permeabilization of C. glabrata cells, as confirmed by propidium iodide-based flow cytometry, with PI uptake up to 99% demonstrating fungicidal effects. Metabolic inactivation in treated cells was confirmed by FUN-1-based confocal microscopy. Together, the results indicate that these lipopeptides have potentials to be developed into a new set of antifungals for combating fungal infections.


Assuntos
Antifúngicos , Bacillus subtilis , Candida glabrata , Permeabilidade da Membrana Celular , Lipopeptídeos , Testes de Sensibilidade Microbiana , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Lipopeptídeos/isolamento & purificação , Bacillus subtilis/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Antifúngicos/farmacologia , Antifúngicos/química , Antifúngicos/isolamento & purificação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo
5.
Arch Microbiol ; 206(6): 251, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38727840

RESUMO

The prevalence of Candida albicans infection has increased during the past few years, which contributes to the need for new, effective treatments due to the increasing concerns regarding antifungal drug toxicity and multidrug resistance. Butyl isothiocyanate (butylITC) is a glucosinolate derivative, and has shown a significant antifungal effect contrary to Candida albicans. Additionally, how butylITC affects the virulence traits of C. albicans and molecular mode of actions are not well known. Present study shows that at 17.36 mM concentration butylITC inhibit planktonic growth. butylITC initially slowed the hyphal transition at 0.542 mM concentration. butylITC hampered biofilm development, and inhibits biofilm formation at 17.36 mM concentration which was analysed using metabolic assay (XTT assay) and Scanning Electron Microscopy (SEM). In addition, it was noted that butylITC inhibits ergosterol biosynthesis. The permeability of cell membranes was enhanced by butylITC treatment. Moreover, butylITC arrests cells at S-phase and induces intracellular Reactive Oxygen Species (ROS) accumulation in C. albicans. The results suggest that butylITC may have a dual mode of action, inhibit virulence factors and modulate cellular processes like inhibit ergosterol biosynthesis, cell cycle arrest, induces ROS production which leads to cell death in C. albicans.


Assuntos
Antifúngicos , Biofilmes , Candida albicans , Membrana Celular , Isotiocianatos , Estresse Oxidativo , Espécies Reativas de Oxigênio , Candida albicans/efeitos dos fármacos , Candida albicans/fisiologia , Biofilmes/efeitos dos fármacos , Antifúngicos/farmacologia , Isotiocianatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Testes de Sensibilidade Microbiana , Ciclo Celular/efeitos dos fármacos , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Ergosterol/metabolismo
6.
Methods Mol Biol ; 2799: 55-77, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727903

RESUMO

NMDA-type ionotropic glutamate receptors are critically involved in many brain functions and are implicated in a variety of brain disorders. Seven NMDA receptor subunits exist (GluN1, GluN2A-D, and GluN3A-B) that assemble into tetrameric receptor subtypes with distinct functional properties and physiological roles. The majority NMDA receptors are composed of two GluN1 and two GluN2 subunits, which can assemble into four diheteromeric receptors subtypes composed of GluN1 and one type of GluN2 subunit (e.g., GluN1/2A), and presumably also six triheteromeric receptor subtypes composed of GluN1 and two different GluN2 subunits (e.g., GluN1/2A/2B). Furthermore, the GluN1 subunit exists as eight splice variants (e.g., GluN1-1a and GluN1-1b isoforms), and two different GluN1 isoforms can co-assemble to also form triheteromeric NMDA receptors (e.g., GluN1-1a/1b/2A). Here, we describe a method to faithfully express triheteromeric NMDA receptors in heterologous expression systems by controlling the identity of two of the four subunits. This method overcomes the problem that co-expression of three different NMDA receptor subunits generates two distinct diheteromeric receptor subtypes as well as one triheteromeric receptor subtype, thereby confounding studies that require a homogenous population of triheteromeric NMDA receptors. The method has been applied to selectively express recombinant triheteromeric GluN1/2A/2B, GluN1/2A/2C, GluN1/2B/2D, GluN1-1a/GluN1-1b/2A, GluN1-1a/GluN1-1b/2B receptors with negligible co-expression of the respective diheteromeric receptor subtypes. This method therefore enables quantitative evaluation of functional and pharmacological properties of triheteromeric NMDA receptors, some of which are abundant NMDA receptor subtypes in the adult brain.


Assuntos
Isoformas de Proteínas , Subunidades Proteicas , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Humanos , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células HEK293 , Animais , Membrana Celular/metabolismo , Expressão Gênica
7.
Phys Rev E ; 109(4-1): 044403, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38755805

RESUMO

Membrane tubes are essential structural features in cells that facilitate biomaterial transport and inter- and intracellular signaling. The shape of these tubes can be regulated by the proteins that surround and adhere to them. We study the stability of a biomembrane tube coated with proteins by combining linear stability analysis, out-of-equilibrium hydrodynamic calculations, and numerical solutions of a Helfrich-like membrane model. Our analysis demonstrates that both long- and short-wavelength perturbations can destabilize the tubes. Numerical simulations confirm the derived linear stability criteria and yield the nonlinearly perturbed vesicle shapes. Our study highlights the interplay between membrane shape and protein density, where the shape instability concurs with a redistribution of proteins into a banded pattern.


Assuntos
Membrana Celular , Modelos Biológicos , Membrana Celular/metabolismo , Membrana Celular/química , Hidrodinâmica , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química
8.
Development ; 151(9)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38713014

RESUMO

Lipid distribution in an organism is mediated by the interplay between lipoprotein particles, lipoprotein receptors and class B scavenger receptors of the CD36 family. CD36 is a multifunctional protein mediating lipid uptake, mobilization and signaling at the plasma membrane and inside of the cell. The CD36 protein family has 14 members in Drosophila melanogaster, which allows for the differentiated analysis of their functions. Here, we unravel a role for the so far uncharacterized scavenger receptor Bez in lipid export from Drosophila adipocytes. Bez shares the lipid binding residue with CD36 and is expressed at the plasma membrane of the embryonic, larval and adult fat body. Bez loss of function lowers the organismal availability of storage lipids and blocks the maturation of egg chambers in ovaries. We demonstrate that Bez interacts with the APOB homolog Lipophorin at the plasma membrane of adipocytes and trace the Bez-dependent transfer of an alkyne-labeled fatty acid from adipocytes to Lipophorin. Our study demonstrates how lipids are distributed by scavenger receptor-lipoprotein interplay and contribute to the metabolic control of development.


Assuntos
Antígenos CD36 , Proteínas de Drosophila , Drosophila melanogaster , Corpo Adiposo , Metabolismo dos Lipídeos , Ovário , Animais , Feminino , Ovário/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Antígenos CD36/metabolismo , Antígenos CD36/genética , Corpo Adiposo/metabolismo , Receptores Depuradores/metabolismo , Receptores Depuradores/genética , Membrana Celular/metabolismo , Adipócitos/metabolismo , Lipoproteínas/metabolismo
9.
Eur J Med Chem ; 271: 116449, 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38691893

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a widespread pathogen causing clinical infections and is multi-resistant to many antibiotics, making it urgent need to develop novel antibacterials to combat MRSA. Herein, we designed and prepared a series of novel osthole amphiphiles 6a-6ad by mimicking the structures and function of antimicrobial peptides (AMPs). Antibacterial assays showed that osthole amphiphile 6aa strongly inhibited S. aureus and 10 clinical MRSA isolates with MIC values of 1-2 µg/mL, comparable to that of the commercial antibiotic vancomycin. Additionally, 6aa had the advantages of rapid bacteria killing without readily developing drug resistance, low toxicity, good membrane selectivity, and good plasma stability. Mechanistic studies indicated that 6aa possesses good membrane-targeting ability to bind to phosphatidylglycerol (PG) on the bacterial cell membranes, thereby disrupting the cell membranes and causing an increase in intracellular ROS as well as leakage of proteins and DNA, and accelerating bacterial death. Notably, in vivo activity results revealed that 6aa exhibits strong anti-MRSA efficacy than vancomycin as well as a substantial reduction in MRSA-induced proinflammatory cytokines, including TNF-α and IL-6. Given the impressive in vitro and in vivo anti-MRSA efficacy of 6aa, which makes it a potential candidate against MRSA infections.


Assuntos
Antibacterianos , Cumarínicos , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Cumarínicos/química , Cumarínicos/farmacologia , Cumarínicos/síntese química , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Humanos , Relação Dose-Resposta a Droga , Camundongos , Tensoativos/farmacologia , Tensoativos/química , Tensoativos/síntese química
10.
J Agric Food Chem ; 72(19): 10853-10861, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38708871

RESUMO

The purpose of this study was to investigate the antibacterial activity and mechanism of action of osthole against Listeria monocytogenes. The antibacterial activity of osthole was evaluated by determining the minimum inhibitory concentration (MIC) and growth curve. Cell morphology, membrane permeability, membrane integrity, bacterial physiology, and metabolism were explored using different methods to elucidate the mechanism of action of osthole. It was shown that the MIC of osthole against L. monocytogenes was 62.5 µg/mL and it inhibited the growth of L. monocytogenes effectively in a concentration-dependent manner. Scanning electron microscopy (SEM) images demonstrated morphology changes of L. monocytogenes, including rough surface, cell shrinkage, and rupture. It was found that extracellular conductivity and macromolecule content were increased significantly in the presence of osthole, indicating the disruption of cell membrane integrity and permeability. Laser confocal microscopy results supported the conclusion that osthole caused severe damage to the cell membrane. It was also noticed that osthole depleted intracellular adenosine triphosphate (ATP), inhibited Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity, and promoted the accumulation of intracellular reactive oxygen species (ROS), leading to cell death. This study suggests that osthole is a promising antibacterial agent candidate against L. monocytogenes, and it shows potential in the prevention and control of foodborne pathogens.


Assuntos
Antibacterianos , Cumarínicos , Listeria monocytogenes , Testes de Sensibilidade Microbiana , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Antibacterianos/farmacologia , Antibacterianos/química , Cumarínicos/farmacologia , Cumarínicos/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo
11.
Methods Mol Biol ; 2807: 61-76, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743221

RESUMO

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.


Assuntos
Membrana Celular , HIV-1 , Microscopia de Fluorescência , Imagem Individual de Molécula , Linfócitos T , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/fisiologia , Humanos , Membrana Celular/metabolismo , Membrana Celular/virologia , Imagem Individual de Molécula/métodos , Linfócitos T/virologia , Linfócitos T/metabolismo , Microscopia de Fluorescência/métodos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
12.
Biochem Biophys Res Commun ; 716: 149954, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38704887

RESUMO

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.


Assuntos
Membrana Celular , Colesterol , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Células Vero , Chlorocebus aethiops , Colesterol/metabolismo , Animais , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Membrana Celular/metabolismo , Membrana Celular/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Humanos , Proteínas de Transporte/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Ligação Proteica
13.
J Phys Chem B ; 128(19): 4735-4740, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38706129

RESUMO

Arc-shaped BIN/Amphiphysin/Rvs (BAR) domain proteins generate curvature by binding to membranes and induce membrane tubulation at sufficiently large protein coverages. For the amphiphysin N-BAR domain, Le Roux et al., Nat. Commun. 2021, 12, 6550, measured a threshold coverage of 0.44 ± 0.097 for nanotubules emerging from the supported lipid bilayer. In this article, we systematically investigate membrane tubulation induced by arc-shaped protein-like particles with coarse-grained modeling and simulations and determine the threshold coverages at different particle-particle interaction strengths and membrane spontaneous curvatures. In our simulations, the binding of arc-shaped particles induces a membrane shape transition from spherical vesicles to tubules at a particle threshold coverage of about 0.5, which is rather robust to variations of the direct attractive particle interactions or spontaneous membrane curvature in the coarse-grained model. Our study suggests that threshold coverages of around or slightly below 0.5 are a general requirement for membrane tubulation by arc-shaped BAR domain proteins.


Assuntos
Bicamadas Lipídicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Domínios Proteicos , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo
14.
Sci Rep ; 14(1): 10214, 2024 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702489

RESUMO

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.


Assuntos
Membrana Celular , Criopreservação , Ácidos Graxos , Fluidez de Membrana , Espermatozoides , Animais , Masculino , Gatos , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Ácidos Graxos/metabolismo , Ácidos Graxos/análise , Membrana Celular/metabolismo , Criopreservação/métodos , Motilidade dos Espermatozoides/fisiologia , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Análise do Sêmen/veterinária
15.
Commun Biol ; 7(1): 549, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38724689

RESUMO

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.


Assuntos
Actinas , Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Tecido Nervoso , Pseudópodes , Proteínas Supressoras de Tumor , Pseudópodes/metabolismo , Actinas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Membrana Celular/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética
16.
Methods Mol Biol ; 2800: 27-34, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38709475

RESUMO

The plasma membrane is a vital component in cellular processes, and its structure has a significant impact on cellular behavior. The physical characteristics of the extracellular environment, along with the presence of surface pores, can influence the formation of membrane protrusions. Nanoporous surfaces have demonstrated their capacity to induce membrane protrusions in both adherent and non-adherent cells. This chapter presents a methodology that utilizes a nanoporous substrate with nanotopographical constraints to effectively stimulate the formation of membrane protrusions in cells.


Assuntos
Propriedades de Superfície , Porosidade , Humanos , Extensões da Superfície Celular/ultraestrutura , Extensões da Superfície Celular/metabolismo , Membrana Celular/metabolismo , Adesão Celular , Animais
17.
Sci Rep ; 14(1): 10561, 2024 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-38719884

RESUMO

This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1-D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and FT-Raman spectroscopy, the research aims to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties suggest these halogenated flavonoids potential candidates for exploring their potential for medical use.


Assuntos
Flavonoides , Lipídeos de Membrana , Flavonoides/química , Flavonoides/farmacologia , Flavonoides/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/química , Membrana Celular/metabolismo , Halogenação , Citotoxinas/química , Citotoxinas/farmacologia , Citotoxinas/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Análise Espectral Raman , Espectroscopia de Infravermelho com Transformada de Fourier , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral
18.
Eur Phys J E Soft Matter ; 47(5): 30, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38720027

RESUMO

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.


Assuntos
Membrana Celular , Difusão , Membrana Celular/metabolismo , Membrana Celular/química , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Modelos Biológicos
19.
J Nanobiotechnology ; 22(1): 231, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38720360

RESUMO

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.


Assuntos
Biomarcadores Tumorais , Membrana Celular , Separação Celular , Nanopartículas de Magnetita , Células Neoplásicas Circulantes , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Humanos , Nanopartículas de Magnetita/química , Separação Celular/métodos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/química , Biomarcadores Tumorais/sangue , Receptor ErbB-2 , Molécula de Adesão da Célula Epitelial/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias
20.
J Cell Sci ; 137(10)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38770683

RESUMO

Membrane trafficking, a fundamental cellular process encompassing the transport of molecules to specific organelles, endocytosis at the plasma membrane and protein secretion, is crucial for cellular homeostasis and signalling. Cancer cells adapt membrane trafficking to enhance their survival and metabolism, and understanding these adaptations is vital for improving patient responses to therapy and identifying therapeutic targets. In this Review, we provide a concise overview of major membrane trafficking pathways and detail adaptations in these pathways, including COPII-dependent endoplasmic reticulum (ER)-to-Golgi vesicle trafficking, COPI-dependent retrograde Golgi-to-ER trafficking and endocytosis, that have been found in cancer. We explore how these adaptations confer growth advantages or resistance to cell death and conclude by discussing the potential for utilising this knowledge in developing new treatment strategies and overcoming drug resistance for cancer patients.


Assuntos
Carcinogênese , Membrana Celular , Neoplasias , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endocitose , Transporte Proteico , Complexo de Golgi/metabolismo
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