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1.
PLoS Comput Biol ; 16(8): e1007962, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776920

RESUMO

Curvature is a fundamental morphological descriptor of cellular membranes. Cryo-electron tomography (cryo-ET) is particularly well-suited to visualize and analyze membrane morphology in a close-to-native state and molecular resolution. However, current curvature estimation methods cannot be applied directly to membrane segmentations in cryo-ET, as these methods cannot cope with some of the artifacts introduced during image acquisition and membrane segmentation, such as quantization noise and open borders. Here, we developed and implemented a Python package for membrane curvature estimation from tomogram segmentations, which we named PyCurv. From a membrane segmentation, a signed surface (triangle mesh) is first extracted. The triangle mesh is then represented by a graph, which facilitates finding neighboring triangles and the calculation of geodesic distances necessary for local curvature estimation. PyCurv estimates curvature based on tensor voting. Beside curvatures, this algorithm also provides robust estimations of surface normals and principal directions. We tested PyCurv and three well-established methods on benchmark surfaces and biological data. This revealed the superior performance of PyCurv not only for cryo-ET, but also for data generated by other techniques such as light microscopy and magnetic resonance imaging. Altogether, PyCurv is a versatile open-source software to reliably estimate curvature of membranes and other surfaces in a wide variety of applications.


Assuntos
Membrana Celular/fisiologia , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Software , Algoritmos , Animais , Células HeLa , Humanos , Camundongos , Saccharomyces cerevisiae
2.
Khirurgiia (Mosk) ; (7): 12-17, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32736458

RESUMO

OBJECTIVE: To determine the value of membrane protective effect in intestine and liver cells for the effectiveness of minimally invasive surgery for acute peritonitis. MATERIAL AND METHODS: Patients with acute peritonitis undergoing laparoscopic (n=60) and open (n=50) surgery are analyzed. Functional characteristics of liver and bowel, disorders of homeostasis were evaluated in early postoperative period. RESULTS: Reduced negative impact of surgical aggression on the state of liver and intestine is essential to improve treatment outcomes in patients with acute peritonitis undergoing minimally invasive surgery. Fast recovery of intestine inevitably results reduced release of endotoxins while restoration of liver function is associated with rapid elimination of these toxins. These processes prevent severe intoxication and facilitate accelerated recovery. Functional restoration of liver and bowel is associated with reduced oxidative stress during laparoscopic operations. It is also important because peritonitis causes activation of free-radical processes per se. Therefore, an additional source of oxidative phenomena is extremely undesirable in these cases. CONCLUSION: Laparoscopic surgery for acute peritonitis minimizes surgical aggression and is associated with more favorable recovery of liver and bowel function. Undoubtedly, these findings should be considered to choose surgical approach in this severe category of patients.


Assuntos
Procedimentos Cirúrgicos Minimamente Invasivos/efeitos adversos , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Peritonite/cirurgia , Doença Aguda , Membrana Celular/metabolismo , Membrana Celular/patologia , Membrana Celular/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Intestinos/fisiopatologia , Laparoscopia/efeitos adversos , Laparotomia/efeitos adversos , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Estresse Oxidativo/fisiologia , Peritonite/metabolismo , Peritonite/fisiopatologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Recuperação de Função Fisiológica , Toxinas Biológicas/biossíntese , Toxinas Biológicas/metabolismo
3.
Phys Rev Lett ; 125(6): 068101, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845697

RESUMO

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.


Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , Viscosidade
4.
Appl Environ Microbiol ; 86(16)2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32561580

RESUMO

Physical agents, such as low electric voltage and current, have recently gained attention for antimicrobial treatment due to their bactericidal capability. Although microampere electric current was shown to suppress the growth of bacteria, it remains unclear to what extent the microampere current damaged the bacterial membrane. Here, we investigated the membrane damage and two-way leakage caused by microampere electric current (≤100 µA) with a short exposure time (30 min). Based on MitoTracker staining, propidium iodide staining, filtration assays, and quantitative single-molecule localization microscopy, we observed significant membrane damage, which allowed two-way leakage of ions, small molecules, and proteins. This study paves the way to new development of antimicrobial applications for ultralow electric voltage and current.IMPORTANCE Although electric voltage and current have been studied for a long time in terms of their ability to suppress the growth of bacteria and to kill bacteria, increasing interest has been aroused more recently due to the prevalence of antibiotic resistance of microbes in past decades. Toward understanding the antimicrobial mechanism of low electric voltage and current, previous studies showed that treating bacteria with milliampere electric currents (≥5 mA) for ≥72 h led to significant damage of the bacterial membrane, which likely resulted in leakage of cellular contents and influx of toxic substances through the damaged membrane. However, it remains unclear to what extent membrane damage and two-way (i.e., inward and outward) leakage are caused by lower (i.e., microampere) electric current in a shorter time frame. In this work, we set out to answer this question. We observed that the membrane damage was caused by microampere electric current in half an hour, which allowed two-way leakage of ions, small molecules, and proteins.


Assuntos
Membrana Celular/fisiologia , Condutividade Elétrica , Escherichia coli K12/fisiologia , Íons/metabolismo
5.
Am J Physiol Lung Cell Mol Physiol ; 319(2): L369-L379, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579851

RESUMO

Proper development of the respiratory bronchiole and alveolar epithelium proceeds through coordinated cross talk between the interface of epithelium and neighboring mesenchyme. Signals that facilitate and coordinate the cross talk as the bronchial forming canalicular stage transitions to construction of air-exchanging capillary-alveoli niche in the alveolar stage are poorly understood. Expressed within this decisive region, levels of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) inversely correlate with the maturation of the lung. The present study addresses the role of AIMP1 in lung development through the generation and characterization of Aimp1-/- mutant mice. Mating of Aimp1+/- produced offspring in expected Mendelian ratios throughout embryonic development. However, newborn Aimp1-/- pups exhibited neonatal lethality with mild cyanosis. Imaging both structure and ultrastructure of Aimp1-/- lungs showed disorganized bronchial epithelium, decreased type I but not type II cell differentiation, increased distal vessels, and disruption of E-cadherin deposition in cell-cell junctions. Supporting the in vivo findings of disrupted epithelial cell-cell junctions, in vitro biochemical experiments show that a portion of AIMP1 binds to phosphoinositides, the lipid anchor of proteins that have a fundamental role in both cellular membrane and actin cytoskeleton organization; a dramatic disruption in F-actin cytoskeleton was observed in Aimp1-/- mouse embryonic fibroblasts. Such observed structural defects may lead to disrupted cell-cell boundaries. Together, these results suggest a requirement of AIMP1 in epithelial cell differentiation in proper lung development.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Diferenciação Celular/fisiologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Pulmão/metabolismo , Pulmão/fisiologia , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Feminino , Junções Intercelulares/metabolismo , Junções Intercelulares/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
6.
Proc Natl Acad Sci U S A ; 117(19): 10278-10285, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341158

RESUMO

Neurons undergo nanometer-scale deformations during action potentials, and the underlying mechanism has been actively debated for decades. Previous observations were limited to a single spot or the cell boundary, while movement across the entire neuron during the action potential remained unclear. Here we report full-field imaging of cellular deformations accompanying the action potential in mammalian neuron somas (-1.8 to 1.4 nm) and neurites (-0.7 to 0.9 nm), using high-speed quantitative phase imaging with a temporal resolution of 0.1 ms and an optical path length sensitivity of <4 pm per pixel. The spike-triggered average, synchronized to electrical recording, demonstrates that the time course of the optical phase changes closely matches the dynamics of the electrical signal. Utilizing the spatial and temporal correlations of the phase signals across the cell, we enhance the detection and segmentation of spiking cells compared to the shot-noise-limited performance of single pixels. Using three-dimensional (3D) cellular morphology extracted via confocal microscopy, we demonstrate that the voltage-dependent changes in the membrane tension induced by ionic repulsion can explain the magnitude, time course, and spatial features of the phase imaging. Our full-field observations of the spike-induced deformations shed light upon the electromechanical coupling mechanism in electrogenic cells and open the door to noninvasive label-free imaging of neural signaling.


Assuntos
Potenciais de Ação , Membrana Celular/fisiologia , Interferometria/métodos , Neurônios/citologia , Neurônios/fisiologia , Animais , Imagem Molecular , Optogenética
7.
Appl Environ Microbiol ; 86(11)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32245757

RESUMO

In Candida glabrata, the transcription factor CgRds2 has been previously characterized as a regulator of glycerophospholipid metabolism, playing a crucial role in the response to osmotic stress. Here, we report that CgRds2 is also involved in the response to pH 2.0 stress. At pH 2.0, the deletion of CgRDS2 led to reduced cell growth and survival, by 33% and 57%, respectively, compared with those of the wild-type strain. These adverse phenotypes resulted from the downregulation of genes related to energy metabolism in the Cgrds2Δ strain at pH 2.0, which led to a 34% reduction of the intracellular ATP content and a 24% decrease in membrane permeability. In contrast, the overexpression of CgRDS2 rescued the growth defect of the Cgrds2Δ strain and increased cell survival at pH 2.0 by 17% compared with that of the wild-type strain, and this effect was accompanied by significant increases in ATP content and membrane permeability, by 42% and 19%, respectively. Furthermore, we found that the calcium/calmodulin-dependent protein kinase (CaMK) CgCmk1 physically interacts with the PAS domain of CgRds2, and CgCmk1 is required for CgRds2 activation and translocation from the cytoplasm to the nucleus under pH 2.0 stress. Moreover, CgCmk1 is critical for CgRds2 function in resistance to pH 2.0 stress, because cells of the Cgrds2-pas strain with a disrupted CgCmk1-CgRds2 interaction exhibited impaired energy metabolism and membrane permeability at pH 2.0. Therefore, our results indicate that CgCmk1 positively regulates CgRds2 and suggest that they promote resistance to low-pH stress by enhancing energy metabolism and membrane permeability in C. glabrata IMPORTANCE An acidic environment is the main problem in the production of organic acids in C. glabrata The present study reports that the calcium/calmodulin-dependent protein kinase CgCmk1 positively regulates CgRds2 to increase intracellular ATP content, membrane permeability, and resistance to low-pH stress. Hence, the transcription factor CgRds2 may be a potential target for improving the acid stress tolerance of C. glabrata during the fermentation of organic acids. The present study also establishes a new link between the calcium signaling pathway and energy metabolism.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Candida glabrata/fisiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Membrana Celular/fisiologia , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(17): 9621-9629, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284410

RESUMO

The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response.


Assuntos
Arabidopsis/fisiologia , Quitina/metabolismo , Plasmodesmos/fisiologia , Tabaco/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fenômenos Biomecânicos , Membrana Celular/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mecanotransdução Celular/fisiologia , Folhas de Planta/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio
10.
Curr Diab Rep ; 20(6): 20, 2020 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-32306181

RESUMO

PURPOSE OF REVIEW: Impairments in mitochondrial function in patients with insulin resistance and type 2 diabetes have been disputed for decades. This review aims to briefly summarize the current knowledge on mitochondrial dysfunction in metabolic tissues and to particularly focus on addressing a new perspective of mitochondrial dysfunction, the altered capacity of mitochondria to communicate with other organelles within insulin-resistant tissues. RECENT FINDINGS: Organelle interactions are temporally and spatially formed connections essential for normal cell function. Recent studies have shown that mitochondria interact with various cellular organelles, such as the endoplasmic reticulum, lysosomes and lipid droplets, forming inter-organelle junctions. We will discuss the current knowledge on alterations in these mitochondria-organelle interactions in insulin resistance and diabetes, with a focus on changes in mitochondria-lipid droplet communication as a major player in ectopic lipid accumulation, lipotoxicity and insulin resistance.


Assuntos
Comunicação Celular/fisiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina/fisiologia , Mitocôndrias/fisiologia , Doenças Mitocondriais/fisiopatologia , Organelas/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/fisiologia , Lisossomos/metabolismo , Lisossomos/fisiologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Organelas/metabolismo , Sobrepeso/metabolismo , Sobrepeso/fisiopatologia , Peroxissomos/metabolismo , Peroxissomos/fisiologia
11.
Int J Mol Sci ; 21(7)2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32276321

RESUMO

Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis. In addition to garbage disposal tools, they are now considered a cell-to-cell communication mechanism. Autophagy is a cellular process leading to sequestration of cytosolic cargoes for their degradation within lysosomes. However, the autophagic machinery is also involved in unconventional protein secretion and autophagy-dependent secretion, which are fundamental mechanisms for toxic protein disposal, immune signalling and pathogen surveillance. These cellular processes underline the crosstalk between the autophagic and the endosomal system and indicate an intersection between degradative and secretory functions. Further, they suggest that the molecular mechanisms underlying fusion, either with lysosomes or plasma membrane, are key determinants to maintain cell homeostasis upon stressing stimuli. When they fail, the accumulation of undigested substrates leads to pathological consequences, as indicated by the involvement of autophagic and lysosomal alteration in human diseases, namely lysosomal storage disorders, age-related neurodegenerative diseases and cancer. In this paper, we reviewed the current knowledge on the functional role of extracellular release pathways involving lysosomes and the autophagic- and endo-lysosomal systems, evaluating their implication in health and disease.


Assuntos
Autofagia , Exocitose , Vesículas Extracelulares/fisiologia , Lisossomos/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Endossomos/fisiologia , Exossomos/fisiologia , Humanos
12.
Exp Parasitol ; 212: 107871, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32147525

RESUMO

Toxocara spp. are responsible for causing toxocariasis, a zoonotic disease of global significance. In some countries of South America, toxocariasis is considered the most prevalent human helminthic infection. The objective of this study was to evaluate LIVE/DEAD® Viability/Cytotoxicity kit as an alternative method to analyze the viability of Toxacara cati larvae. Two control groups were used to confirm the usage of this methodology: 100 untreated T. cati larvae as a negative control (G1) and 100 T. cati larvae killed by thermal shock as a positive control (G2). Subsequently, the viability of T. cati larvae was assessed by the exclusion of the trypan blue dye and by LIVE/DEAD® Viability/Cytotoxicity kit, as well as observation of motility and morphology. In order to confirm the larvicidal effect, T. cati larvae G1 and G2 were inoculated in mice to evaluate their progression in vivo. As expected, G1 showed negative staining by Trypan blue and was stained green by LIVE/DEAD® Viability/Cytotoxicity kit in all the exposure periods. Moreover, G1 presented 100% of relative motility (RM) (score of 5). G2 group was stained blue by Trypan blue and red by LIVE/DEAD® Viability/Cytotoxicity kit, and had 0% RM (score zero) in 24 h of incubation period. In mice, G2 was not viable and, therefore, was not able to infect the animals. In mice inoculated with G1, however, larvae were recovered from all the evaluated organs, except eyes. These results demonstrate that the viability of T. cati larvae was accurately obtained by the LIVE/DEAD® Viability/Cytotoxicity kit, making it an alternative method for viability evaluation.


Assuntos
Toxocara/crescimento & desenvolvimento , Análise de Variância , Animais , Membrana Celular/fisiologia , Sobrevivência Celular , Cães , Feminino , Larva/citologia , Camundongos , Camundongos Endogâmicos BALB C , Coloração e Rotulagem , Toxocara/citologia , Toxocara/fisiologia , Toxocaríase/parasitologia , Azul Tripano
13.
Curr Opin Cell Biol ; 63: 125-134, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32088611

RESUMO

The endoplasmic reticulum (ER) forms an extensive network of membrane contact sites with intra-cellular organelles and the plasma membrane (PM). Interorganelle contacts have vital roles in membrane lipid and ion dynamics. In particular, ER-PM contacts are integral to numerous inter-cellular and intra-cellular signaling pathways including phosphoinositide lipid and calcium signaling, mechanotransduction, metabolic regulation, and cell stress responses. Accordingly, ER-PM contacts serve important signaling functions in excitable cells including neurons and muscle and endocrine cells. This review highlights recent advances in our understanding of the vital roles for ER-PM contacts in phosphoinositide and calcium signaling and how signaling pathways in turn regulate proteins that form and function at ER-PM contacts.


Assuntos
Sinalização do Cálcio/fisiologia , Membrana Celular/fisiologia , Retículo Endoplasmático/fisiologia , Fosfatidilinositóis/metabolismo , Animais , Transporte Biológico/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mecanotransdução Celular/fisiologia , Membranas Mitocondriais/metabolismo , Neurônios/metabolismo
14.
Biochim Biophys Acta Biomembr ; 1862(5): 183213, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057755

RESUMO

Picosecond pulse trains (psPTs) are emerging as a new characteristic diagnostic and therapeutic tool in biomedical fields. To specifically determine the stimulus provided to cells, in this article, we use a molecular dynamics (MD) model to show the molecular mechanisms of electroporation induced by symmetrical bipolar psPTs and predict a bipolar cancellation for the studied picosecond pulses. Electric field conditions that do not cause electroporation reveal that the interfacial water molecules continuously flip and redirect as the applied bipolar psPT reverses, and the molecules cannot keep moving in one direction or leave the lipid-water interface. Based on our simulation results, we determine the threshold for electroporation with symmetrical bipolar psPTs. For a fixed electric field intensity, a lower repetition frequency leads to more rapid electroporation. For a fixed repetition frequency, a higher electric field intensity leads to more rapid electroporation. We found that the water dipole relaxation time decreases as the electric field magnitude increases. Additionally, the influences of the symmetrical bipolar psPT intensity and frequency on the pore formation time are presented. Discrete nanoscale pores can form with the applied psPT at terahertz (THz) repetition frequency. When the psPT amplitude increases or the frequency decreases, the number of water bridges will increase. Moreover, for the first time, the molecular mechanism of bipolar cancellation for the studied picosecond pulse is discussed preliminarily. Our results indicate that the influence of the unipolar picosecond pulse on the interfacial water dipoles will accumulate in one direction, but the bipolar picosecond pulse does not cause this effect.


Assuntos
Eletroporação/métodos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Eletricidade , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/fisiologia
15.
FASEB J ; 34(3): 4653-4669, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32017270

RESUMO

Transmissible gastroenteritis virus (TGEV) is a swine enteropathogenic coronavirus that causes significant economic losses in swine industry. Current studies on TGEV internalization mainly focus on viral receptors, but the internalization mechanism is still unclear. In this study, we used single-virus tracking to obtain the detailed insights into the dynamic events of the TGEV internalization and depict the whole sequential process. We observed that TGEVs could be internalized through clathrin- and caveolae-mediated endocytosis, and the internalization of TGEVs was almost completed within ~2 minutes after TGEVs attached to the cell membrane. Furthermore, the interactions of TGEVs with actin and dynamin 2 in real time during the TGEV internalization were visualized. To our knowledge, this is the first report that single-virus tracking technique is used to visualize the entire dynamic process of the TGEV internalization: before the TGEV internalization, with the assistance of actin, clathrin, and caveolin 1 would gather around the virus to form the vesicle containing the TGEV, and after ~60 seconds, dynamin 2 would be recruited to promote membrane fission. These results demonstrate that TGEVs enter ST cells via clathrin- and caveolae-mediated endocytic, actin-dependent, and dynamin 2-dependent pathways.


Assuntos
Gastroenterite Suína Transmissível/metabolismo , Gastroenterite Suína Transmissível/virologia , Vírus da Gastroenterite Transmissível/patogenicidade , Actinas/metabolismo , Animais , Cavéolas/metabolismo , Caveolina 1/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Membrana Celular/virologia , Clatrina/metabolismo , Dinamina II/metabolismo , Endocitose/fisiologia , Fusão de Membrana/fisiologia , Suínos , Internalização do Vírus
16.
J Neurosci ; 40(11): 2246-2258, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32001613

RESUMO

The ependyma of the adult spinal cord is a latent stem cell niche that is reactivated by spinal cord injury contributing new cells to the glial scar. The cellular events taking place in the early stages of the reaction of the ependyma to injury remain little understood. Ependymal cells are functionally heterogeneous with a mitotically active subpopulation lining the lateral domains of the central canal (CC) that are coupled via gap junctions. Gap junctions and connexin hemichannels are key regulators of the biology of neural progenitors during development and in adult neurogenic niches. Thus, we hypothesized that communication via connexins in the CC is developmentally regulated and may play a part in the reactivation of this latent stem cell niche after injury. To test these possibilities, we combined patch-clamp recordings of ependymal cells with immunohistochemistry for various connexins in the neonatal and the adult (P > 90) normal and injured spinal cord of male and female mice. We find that coupling among ependymal cells is downregulated as postnatal development proceeds but increases after injury, resembling the immature CC. The increase in gap junction coupling in the adult CC was paralleled by upregulation of connexin 26, which correlated with the resumption of proliferation and a reduction of connexin hemichannel activity. Connexin blockade reduced the injury-induced proliferation of ependymal cells. Our findings suggest that connexins are involved in the early reaction of ependymal cells to injury, representing a potential target to improve the contribution of the CC stem cell niche to repair.SIGNIFICANCE STATEMENT Ependymal cells in the adult spinal cord are latent progenitors that react to injury to support some degree of endogenous repair. Understanding the mechanisms by which these progenitor-like cells are regulated in the aftermath of spinal cord injury is critical to design future manipulations aimed at improving healing and functional recovery. Gap junctions and connexin hemichannels are key regulators of the biology of neural progenitors during development and in adult neurogenic niches. We find here that connexin signaling in the ependyma changes after injury of the adult spinal cord, functionally resembling the immature active-stem cell niche of neonatal animals. Our findings suggest that connexins in ependymal cells are potential targets to improve self-repair of the spinal cord.


Assuntos
Conexinas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Nicho de Células-Tronco/fisiologia , Fatores Etários , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Membrana Celular/fisiologia , Permeabilidade da Membrana Celular , Conexinas/antagonistas & inibidores , Epêndima/citologia , Epêndima/crescimento & desenvolvimento , Feminino , Corantes Fluorescentes/farmacocinética , Junções Comunicantes/fisiologia , Hidrogéis , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/antagonistas & inibidores , Técnicas de Patch-Clamp , Peptídeos/química , Peptídeos/farmacologia , Poloxâmero/farmacologia , Distribuição Aleatória
17.
Sci Rep ; 10(1): 68, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919394

RESUMO

Electroporation is defined as cell membrane permeabilization under the application of electric fields. The mechanism of hydrophilic pore formation is not yet well understood. When cells are exposed to electric fields, electrical stresses act on their surfaces. These electrical stresses play a crucial role in cell membrane structural changes, which lead to cell permeabilization. These electrical stresses depend on the dielectric properties of the cell, buffer solution, and the applied electric field characteristics. In the current study, the effect of electric field frequency on the electrical stresses distribution on the cell surface and cell deformation is numerically and experimentally investigated. As previous studies were mostly focused on the effect of electric fields on a group of cells, the present study focused on the behavior of a single cell exposed to an electric field. To accomplish this, the effect of cells on electrostatic potential distribution and electric field must be considered. To do this, Fast immersed interface method (IIM) was used to discretize the governing quasi-electrostatic equations. Numerical results confirmed the accuracy of fast IIM in satisfying the internal electrical boundary conditions on the cell surface. Finally, experimental results showed the effect of applied electric field on cell deformation at different frequencies.


Assuntos
Membrana Celular/fisiologia , Eletroforese/métodos , Estresse Fisiológico , Eletricidade , Eritrócitos/citologia , Eritrócitos/fisiologia , Humanos , Microfluídica/instrumentação , Modelos Biológicos , Análise de Célula Única
18.
Mol Biol Cell ; 31(3): 143-148, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999511

RESUMO

While the organization of inanimate systems such as gases or liquids is predominantly thermodynamically driven-a mixture of two gases will tend to mix until they reach equilibrium-biological systems frequently exhibit organization that is far from a well-mixed equilibrium. The anisotropies displayed by cells are evident in some of the dynamic processes that constitute life including cell development, movement, and division. These anisotropies operate at different length-scales, from the meso- to the nanoscale, and are proposed to reflect self-organization, a characteristic of living systems that is becoming accessible to reconstitution from purified components, and thus a more thorough understanding. Here, some examples of self-organization underlying cellular anisotropies at the cellular level are reviewed, with an emphasis on Rho-family GTPases operating at the plasma membrane. Given the technical challenges of studying these dynamic proteins, some of the successful approaches that are being employed to study their self-organization will also be considered.


Assuntos
Anisotropia , Fenômenos Fisiológicos Celulares/fisiologia , Células/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Termodinâmica , Proteínas rho de Ligação ao GTP/metabolismo
19.
Development ; 147(4)2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31988188

RESUMO

Dendrites develop elaborate morphologies in concert with surrounding glia, but the molecules that coordinate dendrite and glial morphogenesis are mostly unknown. C. elegans offers a powerful model for identifying such factors. Previous work in this system examined dendrites and glia that develop within epithelia, similar to mammalian sense organs. Here, we focus on the neurons BAG and URX, which are not part of an epithelium but instead form membranous attachments to a single glial cell at the nose, reminiscent of dendrite-glia contacts in the mammalian brain. We show that these dendrites develop by retrograde extension, in which the nascent dendrite endings anchor to the presumptive nose and then extend by stretching during embryo elongation. Using forward genetic screens, we find that dendrite development requires the adhesion protein SAX-7/L1CAM and the cytoplasmic protein GRDN-1/CCDC88C to anchor dendrite endings at the nose. SAX-7 acts in neurons and glia, while GRDN-1 acts in glia to non-autonomously promote dendrite extension. Thus, this work shows how glial factors can help to shape dendrites, and identifies a novel molecular mechanism for dendrite growth by retrograde extension.


Assuntos
Encéfalo/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Proteínas dos Microfilamentos/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Neuroglia/fisiologia , Alelos , Animais , Caenorhabditis elegans/fisiologia , Membrana Celular/fisiologia , Citoplasma/fisiologia , Dendritos/fisiologia , Epitélio/fisiologia , Neurogênese , Isoformas de Proteínas , Células Receptoras Sensoriais/fisiologia
20.
mSphere ; 5(1)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996424

RESUMO

Saccharomyces cerevisiae has been frequently used to study biogenesis, functionality, and intracellular transport of various renal proteins, including ion channels, solute transporters, and aquaporins. Specific mutations in genes encoding most of these renal proteins affect kidney function in such a way that various disease phenotypes ultimately occur. In this context, human kidney anion exchanger 1 (kAE1) represents an important bicarbonate/chloride exchanger which maintains the acid-base homeostasis in the human body. Malfunctions in kAE1 lead to a pathological phenotype known as distal renal tubular acidosis (dRTA). Here, we evaluated the potential of baker's yeast as a model system to investigate different cellular aspects of kAE1 physiology. For the first time, we successfully expressed yeast codon-optimized full-length versions of tagged and untagged wild-type kAE1 and demonstrated their partial localization at the yeast plasma membrane (PM). Finally, pH and chloride measurements further suggest biological activity of full-length kAE1, emphasizing the potential of S. cerevisiae as a model system for studying trafficking, activity, and/or degradation of mammalian ion channels and transporters such as kAE1 in the future.IMPORTANCE Distal renal tubular acidosis (dRTA) is a common kidney dysfunction characterized by impaired acid secretion via urine. Previous studies revealed that α-intercalated cells of dRTA patients express mutated forms of human kidney anion exchanger 1 (kAE1) which result in inefficient plasma membrane targeting or diminished expression levels of kAE1. However, the precise dRTA-causing processes are inadequately understood, and alternative model systems are helpful tools to address kAE1-related questions in a fast and inexpensive way. In contrast to a previous study, we successfully expressed full-length kAE1 in Saccharomyces cerevisiae Using advanced microscopy techniques as well as different biochemical and functionality assays, plasma membrane localization and biological activity were confirmed for the heterologously expressed anion transporter. These findings represent first important steps to use the potential of yeast as a model organism for studying trafficking, activity, and degradation of kAE1 and its mutant variants in the future.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Membrana Celular/fisiologia , Saccharomyces cerevisiae , Proteína 1 de Troca de Ânion do Eritrócito/genética , Transporte Biológico , Vetores Genéticos , Microrganismos Geneticamente Modificados , Plasmídeos , Saccharomyces cerevisiae/genética , Transformação Genética
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