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1.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639226

RESUMO

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.


Assuntos
Citoesqueleto de Actina/fisiologia , Membrana Celular/fisiologia , Fenômenos Fisiológicos Celulares , Cicatrização , Animais , Humanos , Análise de Célula Única
2.
Nat Methods ; 18(10): 1239-1246, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34608318

RESUMO

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.


Assuntos
Adenosina Trifosfatases/metabolismo , Fenômenos Biofísicos , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/fisiologia , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Membrana Celular/metabolismo , Escherichia coli , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química
3.
Cells ; 10(10)2021 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-34685758

RESUMO

The lipid matrix in cell membranes is a dynamic, bidimensional array of amphipathic molecules exhibiting mesomorphism, which contributes to the membrane fluidity changes in response to temperature fluctuation. As sessile organisms, plants must rapidly and accurately respond to environmental thermal variations. However, mechanisms underlying temperature perception in plants are poorly understood. We studied the thermal plasticity of membrane fluidity using three fluorescent probes across a temperature range of -5 to 41 °C in isolated microsomal fraction (MF), vacuolar membrane (VM), and plasma membrane (PM) vesicles from Arabidopsis plants. Results showed that PM were highly fluid and exhibited more phase transitions and hysteresis, while VM and MF lacked such attributes. These findings suggest that PM is an important cell hub with the capacity to rapidly undergo fluidity modifications in response to small changes of temperatures in ranges spanning those experienced in natural habitats. PM fluidity behaves as an ideal temperature detector: it is always present, covers the whole cell, responds quickly and with sensitivity to temperature variations, functions with a cell free-energy cost, and it is physically connected with potential thermal signal transducers to elicit a cell response. It is an optimal alternative for temperature detection selected for the plant kingdom.


Assuntos
Arabidopsis/fisiologia , Membrana Celular/fisiologia , Fluidez de Membrana/fisiologia , Arabidopsis/ultraestrutura , Membrana Celular/ultraestrutura , Corantes Fluorescentes/metabolismo , Temperatura , Vacúolos/metabolismo , Vacúolos/ultraestrutura
4.
Toxicol Lett ; 351: 145-154, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34509610

RESUMO

Fe3O4 nanoparticles are widely used in the diagnosis and treatment of diseases due to their superparamagnetism, but their toxicity in vivo, which can result in apoptosis or autophagy, cannot be ignored. It has been reported that polydopamine (PDA) modification can reduce the toxicity of Fe3O4 and increase its biocompatibility. However, more research is warranted to further improve the modification method. We therefore developed a new method to coat Fe3O4@PDA nanoparticles with the mesenchymal stem cell membrane (MSCM) and evaluated the toxicity of the modified particles in the lungs of mice. We found that the MSCM modification significantly reduced lung injury induced by Fe3O4 particles in mice. Compared with Fe3O4@PDA nanoparticles, co-modification with MSCM and PDA significantly reduced autophagy and apoptosis in mouse lung tissue, and reduced activation of autophagy mediated by the AMPK-ULK1 pathway axis. Thus, co-modification with MSCM and PDA prevents Fe3O4-induced pulmonary toxicity in mice by inhibiting autophagy, apoptosis, and oxidative stress.


Assuntos
Adenilato Quinase/metabolismo , Membrana Celular/efeitos dos fármacos , Compostos Férricos/toxicidade , Indóis/farmacologia , Pneumopatias/induzido quimicamente , Células-Tronco Mesenquimais/efeitos dos fármacos , Polímeros/farmacologia , Adenilato Quinase/genética , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Membrana Celular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos
5.
Cells ; 10(9)2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34572065

RESUMO

Both inherited and acquired cardiac arrhythmias are often associated with the abnormal functional expression of ion channels at the cellular level. The complex machinery that continuously traffics, anchors, organizes, and recycles ion channels at the plasma membrane of a cardiomyocyte appears to be a major source of channel dysfunction during cardiac arrhythmias. This has been well established with the discovery of mutations in the genes encoding several ion channels and ion channel partners during inherited cardiac arrhythmias. Fibrosis, altered myocyte contacts, and post-transcriptional protein changes are common factors that disorganize normal channel trafficking during acquired cardiac arrhythmias. Channel availability, described notably for hERG and KV1.5 channels, could be another potent arrhythmogenic mechanism. From this molecular knowledge on cardiac arrhythmias will emerge novel antiarrhythmic strategies.


Assuntos
Arritmias Cardíacas/patologia , Membrana Celular/fisiologia , Canal de Potássio ERG1/metabolismo , Canais Iônicos/fisiologia , Canal de Potássio Kv1.5/metabolismo , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Transporte Biológico , Humanos
6.
Biochemistry ; 60(37): 2761-2772, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34492183

RESUMO

Glutathione peroxidase 4 (GPx4) serves as the only enzyme that protects membranes through the reduction of lipid hydroperoxides, preventing membrane oxidative damage and cell death through ferroptosis. Recently, GPx4 has gained attention as a therapeutic target for cancer through inhibition and as a target for inflammatory diseases through activation. In addition, GPx4 isoforms perform several distinct moonlighting functions including cysteine cross-linking of protamines during sperm cell chromatin remodeling, a function for which molecular and structural details are undefined. Despite the importance in biology, disease, and potential for drug development, little is known about GPx4 functional interactions at high resolution. This study presents the first NMR assignments of GPx4, and the electrostatic interaction of GPx4 with the membrane is characterized. Mutagenesis reveals the cationic patch residues that are key to membrane binding and stabilization. The cationic patch is observed to be important in binding headgroups of highly anionic cardiolipin. A novel lipid binding site is observed adjacent to the catalytic site and may enable protection of lipid-headgroups from oxidative damage. Arachidonic acid is also found to engage with GPx4, while cholesterol did not display any interaction. The cationic patch residues were also found to enable DNA binding, the first observation of this interaction. Electrostatic DNA binding explains a mechanism for the nuclear isoform of GPx4 to target DNA-bound protamines and to potentially reduce oxidatively damaged DNA. Together, these results highlight the importance of electrostatics in the function of GPx4 and illuminate how the multifunctional enzyme is able to fill multiple biological roles.


Assuntos
Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/ultraestrutura , Sítios de Ligação , Domínio Catalítico , Morte Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , DNA/metabolismo , DNA/fisiologia , Ferroptose , Glutationa Peroxidase/metabolismo , Humanos , Peróxidos Lipídicos/metabolismo , Imageamento por Ressonância Magnética/métodos , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/fisiologia , Estresse Oxidativo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/fisiologia , Ligação Proteica , Isoformas de Proteínas/metabolismo , Eletricidade Estática
7.
Immunol Res ; 69(6): 496-519, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34410575

RESUMO

The SARS-CoV-2 S protein on the membrane of infected cells can promote receptor-dependent syncytia formation, relating to extensive tissue damage and lymphocyte elimination. In this case, it is challenging to obtain neutralizing antibodies and prevent them through antibodies effectively. Considering that, in the current study, structural domain search methods are adopted to analyze the SARS-CoV-2 S protein to find the fusion mechanism. The results show that after the EF-hand domain of S protein bound to calcium ions, S2 protein had CaMKII protein activities. Besides, the CaMKII_AD domain of S2 changed S2 conformation, facilitating the formation of HR1-HR2 six-helix bundles. Apart from that, the Ca2+-ATPase of S2 pumped calcium ions from the virus cytoplasm to help membrane fusion, while motor structures of S drove the CaATP_NAI and CaMKII_AD domains to extend to the outside and combined the viral membrane and the cell membrane, thus forming a calcium bridge. Furthermore, the phospholipid-flipping-ATPase released water, triggering lipid mixing and fusion and generating fusion pores. Then, motor structures promoted fusion pore extension, followed by the cytoplasmic contents of the virus being discharged into the cell cytoplasm. After that, the membrane of the virus slid onto the cell membrane along the flowing membrane on the gap of the three CaATP_NAI. At last, the HR1-HR2 hexamer would fall into the cytoplasm or stay on the cell membrane. Therefore, the CaMKII_like system of S protein facilitated membrane fusion for further inducing syncytial multinucleated giant cells.


Assuntos
COVID-19/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Células Gigantes/metabolismo , Fusão de Membrana/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Membrana Celular/fisiologia , Membrana Celular/virologia , Células Gigantes/virologia , Humanos , SARS-CoV-2 , Alinhamento de Sequência , Internalização do Vírus
8.
Nutrients ; 13(8)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34444861

RESUMO

So far, there is no consistent and convincing theory explaining the pathogenesis of migraines. Vascular disorders, the effect of oxidative stress on neurons, and the contribution of magnesium-calcium deficiencies in triggering cortical depression and abnormal glutaminergic neurotransmission are taken into account. However, there are no reliable publications confirming the role of dietary deficits of magnesium and latent tetany as factors triggering migraine attacks. The aim of the study was to evaluate the influence of latent magnesium deficiency assessed with the electrophysiological tetany test on the course of migraine. The study included: a group of 35 patients (29 women and six men; in mean age 41 years) with migraine and a control group of 24 (17 women and seven men; in mean age 39 years) healthy volunteers. Migraine diagnosis was based on the International Headache Society criteria, 3rd edition. All patients and controls after full general and neurological examination were subjected to a standard electrophysiological ischemic tetany test. Moreover, the level of magnesium in blood serum was tested and was in the normal range in all patients. Then, the incidence of a positive tetany EMG test results in the migraine group and the results in the subgroups with and without aura were compared to the results in the control group. Moreover, the relationship between clinical markers of spasmophilia and the results of the tetany test was investigated in the migraine group. As well as the relationship between migraine frequency and tetany test results. There was no statistically significant difference in the occurrence of the electrophysiological exponent of spasmophilia between the migraine and control group. Neither correlation between the occurrence of clinical symptoms nor the frequency of migraine attacks and the results of the tetany test was stated (p > 0.05). However, there was an apparent statistical difference between the subgroup of migraine patients with aura in relation to the control group (p < 0.05). The result raises hope to find a trigger for migraine attacks of this clinical form, the more that this factor may turn out to be easy to supplement with dietary supplementation.


Assuntos
Eletromiografia/métodos , Deficiência de Magnésio/fisiopatologia , Transtornos de Enxaqueca/etiologia , Período Refratário Eletrofisiológico , Tetania/fisiopatologia , Adulto , Estudos de Casos e Controles , Causalidade , Membrana Celular/fisiologia , Feminino , Humanos , Magnésio/sangue , Deficiência de Magnésio/complicações , Deficiência de Magnésio/diagnóstico , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/sangue , Estado Nutricional , Potássio/sangue , Tetania/complicações , Tetania/diagnóstico , Adulto Jovem
9.
Neuron ; 109(19): 3119-3134.e5, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34411513

RESUMO

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis = exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation.


Assuntos
Células Cromafins/fisiologia , Células Cromafins/ultraestrutura , Endocitose/fisiologia , Células Neuroendócrinas/fisiologia , Células Neuroendócrinas/ultraestrutura , Animais , Sinalização do Cálcio , Bovinos , Fusão Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Sistemas Computacionais , Dinaminas/fisiologia , Exocitose/fisiologia , Fusão de Membrana , Cultura Primária de Células , Vesículas Sinápticas/metabolismo
10.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34338279

RESUMO

The conserved exocyst complex regulates plasma membrane-directed vesicle fusion in eukaryotes. However, its role in stem cell proliferation has not been reported. Germline stem cell (GSC) proliferation in the nematode Caenorhabditis elegans is regulated by conserved Notch signaling. Here, we reveal that the exocyst complex regulates C. elegans GSC proliferation by modulating Notch signaling cell autonomously. Notch membrane density is asymmetrically maintained on GSCs. Knockdown of exocyst complex subunits or of the exocyst-interacting GTPases Rab5 and Rab11 leads to Notch redistribution from the GSC-niche interface to the cytoplasm, suggesting defects in plasma membrane Notch deposition. The anterior polarity (aPar) protein Par6 is required for GSC proliferation, and for maintaining niche-facing membrane levels of Notch and the exocyst complex. The exocyst complex biochemically interacts with the aPar regulator Par5 (14-3-3ζ) and Notch in C. elegans and human cells. Exocyst components are required for Notch plasma membrane localization and signaling in mammalian cells. Our study uncovers a possibly conserved requirement of the exocyst complex in regulating GSC proliferation and in maintaining optimal membrane Notch levels.


Assuntos
Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Membrana Celular/metabolismo , Proliferação de Células/fisiologia , Células Germinativas/metabolismo , Células Germinativas/fisiologia , Nicho de Células-Tronco/fisiologia , Proteínas 14-3-3/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular/fisiologia , Membrana Celular/fisiologia , Citoplasma/metabolismo , Citoplasma/fisiologia , Eucariotos/metabolismo , Eucariotos/fisiologia , Fusão de Membrana/fisiologia , Morfogênese/fisiologia , Transdução de Sinais/fisiologia
11.
Nat Commun ; 12(1): 4527, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312384

RESUMO

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.


Assuntos
Membrana Celular/fisiologia , Opsinas/metabolismo , Optogenética/métodos , Células Piramidais/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Furões/genética , Furões/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Opsinas/genética , Técnicas de Patch-Clamp/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes
12.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200063

RESUMO

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.


Assuntos
Archaea/fisiologia , Membrana Celular/fisiologia , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fluidez de Membrana , Esqualeno/análogos & derivados , Archaea/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Esqualeno/farmacologia , Temperatura
13.
Nat Rev Endocrinol ; 17(9): 560-571, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34194011

RESUMO

The pharmacological treatment of pituitary tumours is based on the use of stable analogues of somatostatin and dopamine. The analogues bind to somatostatin receptor types 2 and 5 (SST2 and SST5) and dopamine receptor type 2 (DRD2), respectively, and generate signal transduction cascades in cancerous pituitary cells that culminate in the inhibition of hormone secretion, cell growth and invasion. Drug resistance occurs in a subset of patients and can involve different steps at different stages, such as following receptor activation by the agonist or during the final biological responses. Although the expression of somatostatin and dopamine receptors in cancer cells is a prerequisite for these drugs to reach a biological effect, their presence does not guarantee the success of the therapy. Successful therapy also requires the proper functioning of the machinery of signal transduction and the finely tuned regulation of receptor desensitization, internalization and intracellular trafficking. The present Review provides an updated overview of the molecular factors underlying the pharmacological resistance of pituitary tumours. The Review discusses the experimental evidence that supports a role for receptors and intracellular proteins in the function of SSTs and DRD2 and their clinical importance.


Assuntos
Adenoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Hipofisárias/tratamento farmacológico , Adenoma/patologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Hipofisárias/patologia , Transdução de Sinais/efeitos dos fármacos
14.
Nat Struct Mol Biol ; 28(7): 583-593, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239122

RESUMO

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Membrana Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Nanoestruturas , Ressonância Magnética Nuclear Biomolecular , Fosfatidiletanolaminas/química , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo
16.
Elife ; 102021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34212861

RESUMO

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1, and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Membrana Celular/fisiologia , Citoesqueleto/fisiologia , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Miosinas/genética , Miosinas/metabolismo , Fosfoproteínas Fosfatases/genética , Proteína Quinase C/genética , Quinases Ativadas por p21/genética
17.
PLoS Comput Biol ; 17(7): e1008525, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34264926

RESUMO

Cells polarize their movement or growth toward external directional cues in many different contexts. For example, budding yeast cells grow toward potential mating partners in response to pheromone gradients. Directed growth is controlled by polarity factors that assemble into clusters at the cell membrane. The clusters assemble, disassemble, and move between different regions of the membrane before eventually forming a stable polarity site directed toward the pheromone source. Pathways that regulate clustering have been identified but the molecular mechanisms that regulate cluster mobility are not well understood. To gain insight into the contribution of chemical noise to cluster behavior we simulated clustering using the reaction-diffusion master equation (RDME) framework to account for molecular-level fluctuations. RDME simulations are a computationally efficient approximation, but their results can diverge from the underlying microscopic dynamics. We implemented novel concentration-dependent rate constants that improved the accuracy of RDME-based simulations, allowing us to efficiently investigate how cluster dynamics might be regulated. Molecular noise was effective in relocating clusters when the clusters contained low numbers of limiting polarity factors, and when Cdc42, the central polarity regulator, exhibited short dwell times at the polarity site. Cluster stabilization occurred when abundances or binding rates were altered to either lengthen dwell times or increase the number of polarity molecules in the cluster. We validated key results using full 3D particle-based simulations. Understanding the mechanisms cells use to regulate the dynamics of polarity clusters should provide insights into how cells dynamically track external directional cues.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Simulação por Computador , Modelos Biológicos , Algoritmos , Membrana Celular/fisiologia , Biologia Computacional , Difusão , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Processos Estocásticos
18.
Nat Commun ; 12(1): 3999, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183670

RESUMO

Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigate how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Flagelos/metabolismo , Salmonella typhimurium/genética
19.
Nat Struct Mol Biol ; 28(7): 604-613, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34168372

RESUMO

The Ca2+-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca2+ regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca2+ site (CaICD) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The CaICD site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca2+ binding to the CaTMD site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca2+ sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents.


Assuntos
Membrana Celular/fisiologia , Canais de Cátion TRPM/metabolismo , Peixe-Zebra/metabolismo , Animais , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Técnicas de Patch-Clamp , Conformação Proteica , Canais de Cátion TRPM/antagonistas & inibidores
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