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1.
Invest Ophthalmol Vis Sci ; 63(1): 2, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34978559

RESUMO

Purpose: Our studies in mouse eye lenses demonstrate that ephrin-A5 and EphA2 are needed for normal epithelial cells and lens transparency. We sought to determine whether EphA2 and ephrin-A5 are important for lens morphometrics, nucleus formation, and refractive index. Methods: We performed tissue morphometric measurements, electron microscopy, Western blots, and interferometric measurements using an X-ray synchrotron beam source to measure the gradient of refractive index (GRIN) to compare mouse lenses with genetic disruption of EphA2 or ephrin-A5. Results: Morphometric analysis revealed that although there is no change in the overall lens volume, there is a change in lens shape in both EphA2-/- lenses and ephrin-A5-/- lenses. Surprisingly, EphA2-/- lenses had small and soft lens nuclei different from hard lens nuclei of control lenses. SEM images revealed changes in cell morphology of EphA2-/- fiber cells close to the center of the lens. Inner EphA2-/- lens fibers had more pronounced tongue-and-groove interdigitations and formed globular membrane morphology only in the deepest layers of the lens nucleus. We did not observe nuclear defects in ephrin-A5-/- lenses. There was an overall decrease in magnitude of refractive index across EphA2-/- lenses, which is most pronounced in the nucleus. Conclusions: This work reveals that Eph-ephrin signaling plays a role in fiber cell maturation, nuclear compaction, and lens shape. Loss of EphA2 disrupts the nuclear compaction resulting in a small lens nucleus. Our data suggest that Eph-ephrin signaling may be required for fiber cell membrane reorganization and compaction and for establishing a normal GRIN.


Assuntos
Núcleo do Cristalino/crescimento & desenvolvimento , Receptor EphA2/fisiologia , Refração Ocular/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Efrina-A5/fisiologia , Técnicas de Genotipagem , Interferometria , Núcleo do Cristalino/metabolismo , Núcleo do Cristalino/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Forma das Organelas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Raios X
2.
Biochim Biophys Acta Biomembr ; 1864(1): 183781, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34555419

RESUMO

Surface-active amphiphiles find applications in a wide range of areas of industry such as agrochemicals, personal care, and pharmaceuticals. In many of these applications, interaction with cell membranes is a key factor for achieving their purpose. How do amphiphiles interact with lipid membranes? What are their bases for membrane specificity? Which biophysical properties of membranes are susceptible to modulation by amphiphilic membrane-effectors? What aspects of this interaction are important for performing their function? In our work on membrane biophysics over the years, questions like these have arisen and we now share some of our findings and discuss them in this review. This topic was approached focusing on the membrane properties and their alterations rather than on the amphiphile structure requirements for their interaction. Here, we do not aim to provide a comprehensive list of the modes of action of amphiphiles of biological interest but to help in understanding them.


Assuntos
Membrana Celular/química , Lipídeos de Membrana/química , Tensoativos/química , Biofísica , Membrana Celular/ultraestrutura
3.
Biochim Biophys Acta Biomembr ; 1864(1): 183791, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34624277

RESUMO

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.


Assuntos
Aquaporinas/química , Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Fosfolipídeos/química , Aquaporinas/genética , Aquaporinas/ultraestrutura , Membrana Celular/genética , Membrana Celular/ultraestrutura , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/ultraestrutura , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfolipídeos/genética
4.
Biochim Biophys Acta Biomembr ; 1864(1): 183813, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34748743

RESUMO

Cellular membranes are fundamental building blocks regulating an extensive repertoire of biological functions. These structures contain lipids and membrane proteins that are known to laterally self-aggregate in the plane of the membrane, forming defined membrane nanoscale domains essential for protein activity. Membrane rafts are described as heterogeneous, dynamic, and short-lived cholesterol- and sphingolipid-enriched membrane nanodomains (10-200 nm) induced by lipid-protein and lipid-lipid interactions. Those membrane nanodomains have been extensively characterized using model membranes and in silico methods. However, despite the development of advanced fluorescence microscopy techniques, undoubted nanoscale visualization by imaging techniques of membrane rafts in the membrane of unperturbed living cells is still uncompleted, increasing the skepticism about their existence. Here, we broadly review recent biochemical and microscopy techniques used to investigate membrane rafts in living cells and we enumerate persistent open questions to answer before unlocking the mystery of membrane rafts in living cells.


Assuntos
Membrana Celular/ultraestrutura , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Humanos , Transporte de Íons/genética , Microdomínios da Membrana/química , Microdomínios da Membrana/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Esfingolipídeos/química , Esfingolipídeos/genética
5.
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
6.
Nat Commun ; 12(1): 5434, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521845

RESUMO

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.


Assuntos
Criptocromos/genética , Exossomos/metabolismo , Receptores da Transferrina/genética , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Criptocromos/metabolismo , Exossomos/ultraestrutura , Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fusão de Membrana/genética , Microscopia de Fluorescência , Optogenética/métodos , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/ultraestrutura , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
7.
Elife ; 102021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34543184

RESUMO

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. 'Zap-and-freeze' electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Axônios/enzimologia , Membrana Celular/enzimologia , Hipocampo/enzimologia , Vesículas Sinápticas/enzimologia , Sinaptotagminas/metabolismo , Animais , Axônios/ultraestrutura , Membrana Celular/ultraestrutura , Células Cultivadas , Exocitose , Hipocampo/ultraestrutura , Lipoilação , Camundongos Knockout , Simulação de Acoplamento Molecular , Plasticidade Neuronal , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Ratos Sprague-Dawley , Transmissão Sináptica , Vesículas Sinápticas/ultraestrutura , Sinaptotagminas/genética , Fatores de Tempo
8.
Elife ; 102021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34490842

RESUMO

We investigate the structural and orientational variability of the membrane-embedded T cell receptor (TCR) - CD3 complex in extensive atomistic molecular dynamics simulations based on the recent cryo-EM structure determined by Dong et al., 2019. We find that the TCR extracellular (EC) domain is highly variable in its orientation by attaining tilt angles relative to the membrane normal that range from 15° to 55°. The tilt angle of the TCR EC domain is both coupled to a rotation of the domain and to characteristic changes throughout the TCR - CD3 complex, in particular in the EC interactions of the Cß FG loop of the TCR, as well as in the orientation of transmembrane helices. The concerted motions of the membrane-embedded TCR - CD3 complex revealed in our simulations provide atomistic insights on conformational changes of the complex in response to tilt-inducing forces on antigen-bound TCRs.


Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismo , Complexo CD3/ultraestrutura , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Complexo Receptor-CD3 de Antígeno de Linfócitos T/ultraestrutura , Receptores de Antígenos de Linfócitos T alfa-beta/ultraestrutura , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/ultraestrutura
9.
Pharmacol Res ; 172: 105820, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34403732

RESUMO

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enter the host cells through the interaction between its receptor binding domain (RBD) of spike glycoprotein with angiotensin-converting enzyme 2 (ACE2) receptor on the plasma membrane of host cell. Neutralizing antibodies and peptide binders of RBD can block viral infection, however, the concern of accessibility and affordability of viral infection inhibitors has been raised. Here, we report the identification of natural compounds as potential SARS-CoV-2 entry inhibitors using the molecular docking-based virtual screening coupled with bilayer interferometry (BLI). From a library of 1871 natural compounds, epigallocatechin gallate (EGCG), 20(R)-ginsenoside Rg3 (RRg3), 20(S)-ginsenoside Rg3 (SRg3), isobavachalcone (Ibvc), isochlorogenic A (IscA) and bakuchiol (Bkc) effectively inhibited pseudovirus entry at concentrations up to 100 µM. Among these compounds, four compounds, EGCG, Ibvc, salvianolic acid A (SalA), and isoliensinine (Isl), were effective in inhibiting SARS-CoV-2-induced cytopathic effect and plaque formation in Vero E6 cells. The EGCG was further validated with no observable animal toxicity and certain antiviral effect against SARS-CoV-2 pseudovirus mutants (D614G, N501Y, N439K & Y453F). Interestingly, EGCG, Bkc and Ibvc bind to ACE2 receptor in BLI assay, suggesting a dual binding to RBD and ACE2. Current findings shed some insight into identifications and validations of SARS-CoV-2 entry inhibitors from natural compounds.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Antivirais/química , Produtos Biológicos/química , COVID-19/tratamento farmacológico , Inibidores Enzimáticos/química , SARS-CoV-2/enzimologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Antivirais/farmacologia , Ligação Competitiva , Produtos Biológicos/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Chalconas/farmacologia , Ácido Clorogênico/análogos & derivados , Ácido Clorogênico/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Ginsenosídeos/farmacologia , Humanos , Interferometria , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Fenóis/farmacologia , Ligação Proteica
10.
J Cell Biol ; 220(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34382996

RESUMO

Cell-cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h- isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h- cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h- cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h- cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.


Assuntos
Membrana Celular/metabolismo , Schizosaccharomyces/metabolismo , Membrana Celular/ultraestrutura , Fusão de Membrana , Microscopia Eletrônica de Varredura , Schizosaccharomyces/citologia
11.
ACS Appl Mater Interfaces ; 13(33): 39018-39029, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34397215

RESUMO

Targeting nanoparticles as drug delivery platforms is crucial to facilitate their cellular entry. Docking of nanoparticles by targeting ligands on cell membranes is the first step for the initiation of cellular uptake. As a model system, we studied brain microvascular endothelial cells, which form the anatomical basis of the blood-brain barrier, and the tripeptide glutathione, one of the most effective targeting ligands of nanoparticles to cross the blood-brain barrier. To investigate this initial docking step between glutathione and the membrane of living brain endothelial cells, we applied our recently developed innovative optical method. We present a microtool, with a task-specific geometry used as a probe, actuated by multifocus optical tweezers to characterize the adhesion probability and strength of glutathione-coated surfaces to the cell membrane of endothelial cells. The binding probability of the glutathione-coated surface and the adhesion force between the microtool and cell membrane was measured in a novel arrangement: cells were cultured on a vertical polymer wall and the mechanical forces were generated laterally and at the same time, perpendicularly to the plasma membrane. The adhesion force values were also determined with more conventional atomic force microscopy (AFM) measurements using functionalized colloidal probes. The optical trapping-based method was found to be suitable to measure very low adhesion forces (≤ 20 pN) without a high level of noise, which is characteristic for AFM measurements in this range. The holographic optical tweezers-directed functionalized microtools may help characterize the adhesion step of nanoparticles initiating transcytosis and select ligands to target nanoparticles.


Assuntos
Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Glutationa/metabolismo , Nanopartículas/metabolismo , Pinças Ópticas , Fenômenos Biofísicos , Barreira Hematoencefálica/metabolismo , Encéfalo , Adesão Celular , Membrana Celular/ultraestrutura , Células Endoteliais/citologia , Galactosamina/química , Humanos , Ligantes , Microscopia de Força Atômica , Nanopartículas/química , Polietilenoglicóis/química , Polímeros/metabolismo , Propriedades de Superfície , Transcitose
12.
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
13.
J Vet Sci ; 22(5): e59, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34423597

RESUMO

BACKGROUND: Antimicrobial peptides (AMPs) have been identified as promising compounds for consideration as novel antimicrobial agents. OBJECTIVES: This study analyzed the efficacy of cecropin B against Haemophilus parasuis isolates through scanning electron microscopy (SEM) and atomic force microscopy (AFM) experiments. RESULTS: Cecropin B exhibited broad inhibition activity against 15 standard Haemophilus parasuis (HPS) strains and 5 of the clinical isolates had minimum inhibition concentrations (MICs) ranging from 2 to 16 µg/mL. Microelectrophoresis and hexadecane adsorption assays indicated that the more hydrophobic and the higher the isoelectric point (IEP) of the strain, the more sensitive it was to cecropin B. Through SEM, multiple blisters of various shapes and dents on the cell surface were observed. Protrusions and leakage were detected by AFM. CONCLUSIONS: Based on the results, cecropin B could inhibit HPS via a pore-forming mechanism by interacting with the cytoplasmic membrane of bacteria. Moreover, as cecropin B concentration increased, the bacteria membrane was more seriously damaged. Thus, cecropin B could be developed as an effective anti-HPS agent for use in clinical applications.


Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cecropinas/farmacologia , Haemophilus parasuis/citologia , Microscopia de Força Atômica/veterinária , Microscopia Eletrônica de Varredura/veterinária , Membrana Celular/ultraestrutura , Haemophilus parasuis/ultraestrutura , Testes de Sensibilidade Microbiana/veterinária
14.
Science ; 373(6552)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34437126

RESUMO

Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.


Assuntos
Apolipoproteínas L/metabolismo , Membrana Celular/metabolismo , Citosol/microbiologia , Bactérias Gram-Negativas/fisiologia , Interferon gama/imunologia , Apolipoproteínas L/química , Apolipoproteínas L/genética , Membrana Externa Bacteriana/metabolismo , Bacteriólise , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Células Cultivadas , Detergentes/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Edição de Genes , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Negativas/ultraestrutura , Humanos , Imunidade Inata , Lipoproteínas/química , Viabilidade Microbiana , Antígenos O/metabolismo , Domínios Proteicos , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Salmonella typhimurium/ultraestrutura , Solubilidade
15.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34241635

RESUMO

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios/metabolismo , Via Secretória/genética , Sinapses/metabolismo , Transmissão Sináptica/genética , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Feminino , Corantes Fluorescentes/química , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Hipocampo/citologia , Hipocampo/metabolismo , Luz , Masculino , Imagem Molecular/métodos , Neurônios/citologia , Cultura Primária de Células , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/ultraestrutura , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
16.
Nat Commun ; 12(1): 4639, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330922

RESUMO

The silica cell wall of diatoms, a widespread group of unicellular microalgae, is an exquisite example for the ability of organisms to finely sculpt minerals under strict biological control. The prevailing paradigm for diatom silicification is that this is invariably an intracellular process, occurring inside specialized silica deposition vesicles that are responsible for silica precipitation and morphogenesis. Here, we study the formation of long silicified extensions that characterize many diatom species. We use cryo-electron tomography to image silica formation in situ, in 3D, and at a nanometer-scale resolution. Remarkably, our data suggest that, contradictory to the ruling paradigm, these intricate structures form outside the cytoplasm. In addition, the formation of these silica extensions is halted at low silicon concentrations that still support the formation of other cell wall elements, further alluding to a different silicification mechanism. The identification of this unconventional strategy expands the suite of mechanisms that diatoms use for silicification.


Assuntos
Parede Celular/metabolismo , Diatomáceas/metabolismo , Espaço Extracelular/metabolismo , Dióxido de Silício/metabolismo , Ciclo Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/ultraestrutura , Microscopia Crioeletrônica/métodos , Diatomáceas/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura
17.
J Neurosci ; 41(33): 7003-7014, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34266899

RESUMO

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.


Assuntos
Espinhas Dendríticas/ultraestrutura , Hipocampo/ultraestrutura , Potenciação de Longa Duração , Densidade Pós-Sináptica/ultraestrutura , Animais , Axônios/ultraestrutura , Biolística , Membrana Celular/ultraestrutura , Espinhas Dendríticas/fisiologia , Feminino , Glutamatos/efeitos da radiação , Processamento de Imagem Assistida por Computador , Indóis/efeitos da radiação , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Fotoquímica
18.
Molecules ; 26(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206777

RESUMO

Previous studies have revealed the numerous biological activities of the fruits of Illicium verum; however, the activities of its leaves and twigs have remained undiscovered. The study aimed to investigate the phytochemical components and antibacterial activity of the various extracts from the leaves and twigs of Illicium verum. The herbal extracts were prepared by supercritical CO2 extraction (SFE) and 95% ethanol extraction, followed by partition extraction based on solvent polarity. Analysis of antimicrobial activity was conducted through the usage of nine clinical antibiotic- resistant isolates, including Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. Among the tested samples, the SFE extracts exhibited broader and stronger antibacterial activities against the test strains, with a range of MIC between 0.1-4.0 mg/mL and MBC between 0.2-4.5 mg/mL. Observations made through scanning electron microscopy revealed potential mechanism of the antimicrobial activities involved disruption of membrane integrity of the test pathogens. Evaluation of the chemical composition by gas chromatography-mass spectrometry indicated the presence of anethole, anisyl aldehyde, anisyl acetone and anisyl alcohol within the SFE extracts, demonstrating significant correlations with the antibacterial activities observed. Therefore, the leaves and twigs of Illicium verum hold great potential in being developed as new natural antibacterial agents.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Illicium/química , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/ultraestrutura , Antibacterianos/análise , Anti-Infecciosos/análise , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Gasosa , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Extratos Vegetais/química , Folhas de Planta/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura
19.
J Bacteriol ; 203(17): e0015021, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34152201

RESUMO

Bacterial rod-shaped cells experiencing irreparable chromosome damage should filament without other morphological changes. Thymineless death (TLD) strikes thymidine auxotrophs denied external thymine/thymidine (T) supplementation. Such T-starved cells cannot produce the DNA precursor dTTP and therefore stop DNA replication. Stalled replication forks in T-starved cells were always assumed to experience mysterious chromosome lesions, but TLD was recently found to happen even without origin-dependent DNA replication, with the chromosome still remaining the main TLD target. T starvation also induces morphological changes, as if thymidine prevents cell envelope or cytoplasm problems that otherwise translate into chromosome damage. Here, we used transmission electron microscopy (TEM) to examine cytoplasm and envelope changes in T-starved Escherichia coli cells, using treatment with a DNA gyrase inhibitor as a control for "pure" chromosome death. Besides the expected cell filamentation in response to both treatments, we see the following morphological changes specific for T starvation and which might lead to chromosome damage: (i) significant cell widening, (ii) nucleoid diffusion, (iii) cell pole damage, and (iv) formation of numerous cytoplasmic bubbles. We conclude that T starvation does impact both the cytoplasm and the cell envelope in ways that could potentially affect the chromosome. IMPORTANCE Thymineless death is a dramatic and medically important phenomenon, the mechanisms of which remain a mystery. Unlike most other auxotrophs in the absence of the required supplement, thymidine-requiring E. coli mutants not only go static in the absence of thymidine, but rapidly die of chromosomal damage of unclear nature. Since this chromosomal damage is independent of replication, we examined fine morphological changes in cells undergoing thymineless death in order to identify what could potentially affect the chromosome. Here, we report several cytoplasm and cell envelope changes that develop in thymidine-starved cells but not in gyrase inhibitor-treated cells (negative control) that could be linked to subsequent irreparable chromosome damage. This is the first electron microscopy study of cells undergoing "genetic death" due to irreparable chromosome lesions.


Assuntos
Membrana Celular/ultraestrutura , Citoplasma/ultraestrutura , Escherichia coli/metabolismo , Timina/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Replicação do DNA , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Viabilidade Microbiana , Microscopia Eletrônica , Timidina/metabolismo
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