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1.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809364

RESUMO

The conserved VPS13 proteins constitute a new family of lipid transporters at membrane contact sites. These large proteins are suspected to bridge membranes and form a direct channel for lipid transport between organelles. Mutations in the 4 human homologs (VPS13A-D) are associated with a number of neurological disorders, but little is known about their precise functions or the relevant contact sites affected in disease. In contrast, yeast has a single Vps13 protein which is recruited to multiple organelles and contact sites. The yeast model system has proved useful for studying the function of Vps13 at different organelles and identifying the localization determinants responsible for its membrane targeting. In this review we describe recent advances in our understanding of VPS13 proteins with a focus on yeast research.


Assuntos
Transporte Biológico/genética , Membrana Celular/genética , Lipídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Membrana Celular/metabolismo , Humanos , Membranas Mitocondriais/metabolismo , Mutação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
2.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800499

RESUMO

While approximately 2000 mutations have been discovered in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), only a small amount (about 10%) is associated with clinical cystic fibrosis (CF) disease. The discovery of the association between CFTR and the hyperactive epithelial sodium channel (ENaC) has raised the question of the influence of ENaC on the clinical CF phenotype. ENaC disturbance contributes to the pathological secretion, and overexpression of one ENaC subunit, the ß-unit, can give a CF-like phenotype in mice with normal acting CFTR. The development of ENaC channel modulators is now in progress. Both CFTR and ENaC are located in the cell membrane and are influenced by its lipid configuration. Recent studies have emphasized the importance of the interaction of lipids and these proteins in the membranes. Linoleic acid deficiency is the most prevailing lipid abnormality in CF, and linoleic acid is an important constituent of membranes. The influence on sodium excretion by linoleic acid supplementation indicates that lipid-protein interaction is of importance for the clinical pathophysiology in CF. Further studies of this association can imply a simple clinical adjuvant in CF therapy.


Assuntos
Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ácido Linoleico/deficiência , Animais , Membrana Celular/genética , Membrana Celular/patologia , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Humanos , Ácido Linoleico/metabolismo , Camundongos
3.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33803919

RESUMO

Ion channels are well recognized to select ions to pass through the cell membrane in a wide variety of cells [...].


Assuntos
Membrana Celular/genética , Fenômenos Eletrofisiológicos/fisiologia , Canais Iônicos/genética , Potenciais da Membrana/genética , Humanos , Canais Iônicos/metabolismo , Íons/metabolismo
4.
Mol Cell ; 81(7): 1397-1410.e9, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33725486

RESUMO

Phospholipid scrambling in dying cells promotes phosphatidylserine exposure, a critical process for efferocytosis. We previously identified the Xkr family protein Xkr4 as a phospholipid-scrambling protein, but its activation mechanisms remain unknown. Here we show that Xkr4 is activated in two steps: dimer formation by caspase-mediated cleavage and structural change caused by activating factors. To identify the factors, we developed a new screening system, "revival screening," using a CRISPR sgRNA library. Applying this system, we identified the nuclear protein XRCC4 as the single candidate for the Xkr4 activator. Upon apoptotic stimuli, XRCC4, contained in the DNA repair complex, is cleaved by caspases, and its C-terminal fragment with an intrinsically disordered region is released into the cytoplasm. Protein interaction screening showed that the fragment interacts directly with the Xkr4 dimer to activate it. This study demonstrates that caspase-mediated cleavage releases a nuclear protein fragment for direct regulation of lipid dynamics on the plasma membrane.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteólise , Animais , Proteínas Reguladoras de Apoptose/genética , Caspases/genética , Linhagem Celular Tumoral , Membrana Celular/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Fosfolipídeos/genética , Multimerização Proteica
5.
Nat Commun ; 12(1): 1609, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707455

RESUMO

Histopathological images are used to characterize complex phenotypes such as tumor stage. Our goal is to associate features of stained tissue images with high-dimensional genomic markers. We use convolutional autoencoders and sparse canonical correlation analysis (CCA) on paired histological images and bulk gene expression to identify subsets of genes whose expression levels in a tissue sample correlate with subsets of morphological features from the corresponding sample image. We apply our approach, ImageCCA, to two TCGA data sets, and find gene sets associated with the structure of the extracellular matrix and cell wall infrastructure, implicating uncharacterized genes in extracellular processes. We find sets of genes associated with specific cell types, including neuronal cells and cells of the immune system. We apply ImageCCA to the GTEx v6 data, and find image features that capture population variation in thyroid and in colon tissues associated with genetic variants (image morphology QTLs, or imQTLs), suggesting that genetic variation regulates population variation in tissue morphological traits.


Assuntos
Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica/genética , Expressão Gênica/genética , Neoplasias/patologia , Locos de Características Quantitativas/genética , Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Membrana Celular/genética , Membrana Celular/fisiologia , Matriz Extracelular/genética , Matriz Extracelular/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genética
6.
Nat Commun ; 12(1): 931, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568658

RESUMO

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.


Assuntos
Cavéolas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Cavéolas/química , Caveolina 1/genética , Caveolina 1/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Camundongos , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Eletricidade Estática
7.
J Steroid Biochem Mol Biol ; 207: 105827, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33497793

RESUMO

Progesterone modulates many processes in the body, acting through nuclear receptors (nPR) in various organs and tissues. However, a number of effects are mediated by membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ (PAQR) receptor family. These receptors are found in most tissues and immune cells. They are expressed in various cancer cells and appear to play an important role in the development of tumors. The role of mPRs in the development of insulin resistance and metabolic syndrome has also attracted attention. Since progesterone efficiently binds to both nPRs and mPRs, investigation of the functions of the mPRs both at the level of the whole body and at the cell level requires ligands that selectively interact with mPRs, but not with nPRs, with an affinity comparable with that of the natural hormone. The development of such ligands faces difficulties primarily due to the lack of data on the three-dimensional structure of the ligand-binding site of mPR. This review is the first attempt to summarize available data on the structures of compounds interacting with mPRs and analyze them in terms of the differences in binding to membrane and nuclear receptors. Based on the identified main structural fragments of molecules, which affect the efficiency of binding to mPRs and are responsible for the selectivity of interactions, we propose directions of modification of the steroid scaffold to create new selective mPRs ligands.


Assuntos
Síndrome Metabólica/genética , Progesterona/genética , Receptores de Superfície Celular/genética , Receptores de Progesterona/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/genética , Humanos , Ligantes , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Progesterona/metabolismo , Progestinas/genética , Receptores de Superfície Celular/metabolismo
8.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443205

RESUMO

The type 6 secretion system (T6SS) is a dynamic organelle encoded by many gram-negative bacteria that can be used to kill competing bacterial prey species in densely occupied niches. Some predatory species, such as Vibrio cholerae, use their T6SS in an untargeted fashion while in contrast, Pseudomonas aeruginosa assembles and fires its T6SS apparatus only after detecting initial attacks by other bacterial prey cells; this targeted attack strategy has been termed the T6SS tit-for-tat response. Molecules that interact with the P. aeruginosa outer membrane such as polymyxin B can also trigger assembly of T6SS organelles via a signal transduction pathway that involves protein phosphorylation. Recent work suggests that a phospholipase T6SS effector (TseL) of V. cholerae can induce T6SS dynamic activity in P. aeruginosa when delivered to or expressed in the periplasmic space of this organism. Here, we report that inhibiting expression of essential genes involved in outer membrane biogenesis can also trigger T6SS activation in P. aeruginosa Specifically, we developed a CRISPR interference (CRISPRi) system to knock down expression of bamA, tolB, and lptD and found that these knockdowns activated T6SS activity. This increase in T6SS activity was dependent on the same signal transduction pathway that was previously shown to be required for the tit-for-tat response. We conclude that outer membrane perturbation can be sensed by P. aeruginosa to activate the T6SS even when the disruption is generated by aberrant cell envelope biogenesis.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Genes Essenciais/fisiologia , Proteínas Periplásmicas/metabolismo , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo VI/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/genética , Membrana Celular/patologia , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes Essenciais/genética , Genótipo , Proteínas Periplásmicas/genética , Fenótipo , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , RNA-Seq , Transdução de Sinais/genética , Estresse Fisiológico , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimento
9.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477953

RESUMO

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.


Assuntos
Amiloide/genética , Proteínas Amiloidogênicas/genética , Ataxina-3/genética , Doença de Machado-Joseph/genética , Membrana Celular/genética , Proliferação de Células/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Humanos , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Proteínas do Tecido Nervoso/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia
10.
Adv Protein Chem Struct Biol ; 123: 163-191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33485482

RESUMO

ATP-binding cassette (ABC) superfamily comprises membrane transporters that power the active transport of substrates across biological membranes. These proteins harness the energy of nucleotide binding and hydrolysis to fuel substrate translocation via an alternating-access mechanism. The primary structural blueprint is relatively conserved in all ABC transporters. A transport-competent ABC transporter is essentially made up of two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). While the NBDs are conserved in their primary sequence and form at their interface two nucleotide-binding sites (NBSs) for ATP binding and hydrolysis, the TMDs are variable among different families and form the translocation channel. Transporters catalyzing the efflux of substrates from the cells are called exporters. In humans, they range from A to G subfamilies, with the B, C and G subfamilies being involved in chemoresistance. The recently elucidated structures of ABCG5/G8 followed by those of ABCG2 highlighted a novel structural fold that triggered extensive research. Notably, suppressor genetics in the orthologous yeast Pleiotropic Drug Resistance (PDR) subfamily proteins have pointed to a crosstalk between TMDs and NBDs modulating substrate export. Considering the structural information provided by their neighbors from the G subfamily, these studies provide mechanistic keys and posit a functional role for the non-hydrolytic NBS found in several ABC exporters. The present chapter provides an overview of structural and functional aspects of ABCG proteins with a special emphasis on the yeast PDR systems.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Modelos Moleculares , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Sítios de Ligação , Membrana Celular/genética , Humanos
11.
Nat Commun ; 12(1): 374, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446636

RESUMO

During autophagy the enzyme Atg3 catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region of human Atg3 (hAtg3) communicates information from the N-terminal membrane curvature-sensitive amphipathic helix (AH), which presumably targets the enzyme to the tip of phagophore, to the C-terminally located catalytic core for LC3-PE conjugation. Mutations in the putative communication region greatly reduce or abolish the ability of hAtg3 to catalyze this conjugation in vitro and in vivo, and alter the membrane-bound conformation of the wild-type protein, as reported by NMR. Collectively, our results demonstrate that the N-terminal conserved region of hAtg3 works in concert with its geometry-selective AH to promote LC3-PE conjugation only on the target membrane, and substantiate the concept that highly curved membranes drive spatial regulation of the autophagosome biogenesis during autophagy.


Assuntos
Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membrana Celular/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/genética , Biocatálise , Membrana Celular/genética , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Enzimas de Conjugação de Ubiquitina/genética
12.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118876, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007331

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathological phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liquid-liquid phase separation (LLPS), however, the molecular mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.


Assuntos
Grânulos Citoplasmáticos/genética , Polirribossomos/genética , Ribonucleoproteínas/genética , Estresse Fisiológico/genética , Compartimento Celular/genética , Membrana Celular/genética , Citoplasma/genética , Humanos , Microextração em Fase Líquida , RNA Mensageiro/genética
13.
Methods Mol Biol ; 2178: 439-467, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33128765

RESUMO

G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs: (1) cell-free in vitro translation, (2) HEK cells, and (3) Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration, and can be analyzed with standard biophysical techniques and biochemical assays.


Assuntos
Cromatografia de Afinidade , Expressão Gênica , Receptores Acoplados a Proteínas-G , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Sistema Livre de Células , Escherichia coli , Células HEK293 , Humanos , Receptores Acoplados a Proteínas-G/biossíntese , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
14.
Methods Mol Biol ; 2233: 3-17, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222124

RESUMO

Determination of protein stoichiometry in living cells is key to understanding basic biological processes. This is particularly important for receptor-mediated endocytosis, a highly regulated mechanism that requires the sequential assembly of numerous factors. Here, we describe a quantitative approach to analyze receptor clustering dynamics at the plasma membrane. Our workflow combines TIRF live imaging of a CRISPR-Cas9-edited cell line expressing a GFP-tagged receptor in a physiological relevant environment, a calibration technique for single-molecule analysis of GFP, and detection and tracking with an open-source software. This method allows to determine the number of receptor molecules at the plasma membrane in real time.


Assuntos
Membrana Celular/ultraestrutura , Endocitose/genética , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Linhagem Celular , Membrana Celular/genética , Humanos
15.
Methods Mol Biol ; 2233: 139-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222133

RESUMO

Acrosome reaction is an exocytic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane, releasing the acrosomal content. Given the importance of the acrosome secretion in fertilization, many different methods have been developed to detect the acrosome reaction of sperm. In this chapter, we describe detailed practical procedures to assess the acrosomal status of human spermatozoa. To do this, we resorted to light optical and epifluorescence microscopy, flow cytometry, and transmission electron microscopy. We also itemize the protocol for real-time measurements of the acrosome reaction by confocal microscopy. Further, we discuss the level of complexity, costs, and the reasons why a researcher should choose each technique.This chapter is designed to provide the user with sufficient background to measure acrosomal exocytosis in human sperm.


Assuntos
Reação Acrossômica/genética , Membrana Celular/ultraestrutura , Exocitose/genética , Espermatozoides/ultraestrutura , Acrossomo/metabolismo , Membrana Celular/genética , Humanos , Masculino , Espermatozoides/patologia , Zona Pelúcida/metabolismo
16.
Methods Mol Biol ; 2233: 203-222, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222137

RESUMO

Exocytosis, the fusion of vesicles with the plasma membrane, can be measured with the patch-clamp technique as increases in membrane capacitance. Here we provide detailed information on how to monitor white adipocyte exocytosis using this method. We describe how to isolate the stromal vascular fraction of cells (SVF) within adipose tissue and how to differentiate SVF and cultured 3T3-L1 cells into adipocytes suitable for patch-clamp studies. We also give detailed protocols of how to record and analyze exocytosis in the differentiated cells.


Assuntos
Adipócitos Brancos/ultraestrutura , Membrana Celular/genética , Exocitose/genética , Técnicas de Patch-Clamp/métodos , Células 3T3-L1 , Adipócitos Brancos/metabolismo , Animais , Membrana Celular/ultraestrutura , Camundongos
17.
Methods Mol Biol ; 2233: 253-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222140

RESUMO

Proteins destined to be exposed to the extracellular space enter the secretory pathway at the level of the endoplasmic reticulum. Proteins are then transported to the Golgi apparatus and addressed to their destination compartment, such as the plasma membrane for exocytic cargos. Exocytosis constitutes the last step of the anterograde transport of secretory cargos. Exocytic vesicles fuse with the plasma membrane, releasing soluble proteins to the extracellular milieu and transmembrane proteins to the plasma membrane. In order to monitor local exocytosis of cargos, we describe in this chapter how to perform synchronization of the anterograde transport of an exocytic cargo of interest using the retention using selective hooks (RUSH) assay in combination with selective protein immobilization (SPI). SPI is based on the coating of coverslips with anti-green fluorescent protein (GFP) antibodies, which capture the GFP-tagged RUSH cargos once exposed to the cell surface after its release by the addition of biotin.


Assuntos
Exocitose/genética , Complexo de Golgi/genética , Biologia Molecular/métodos , Via Secretória/genética , Animais , Membrana Celular/genética , Retículo Endoplasmático/genética , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Transporte Proteico/genética , Vesículas Secretórias/genética
18.
Methods Mol Biol ; 2233: 287-300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222142

RESUMO

Ca2+ regulates a variety of cellular processes that are essential to maintain cell integrity and function. Different methods have been used to study these processes by increasing intracellular Ca2+ levels. Here, we describe a protocol to initiate Ca2+-dependent membrane-related events, using laser ablation by near-infrared irradiation. This creates a rupture in the plasma membrane that allows the extracellular Ca2+ to enter the cell and thereby induce a receptor-independent Ca2+ increase. We report laser ablation protocols to study two different Ca2+-induced processes in human endothelial cells-membrane resealing and exocytosis of secretory granules called Weibel-Palade bodies (WPBs). Thus, laser ablation represents a technique that permits the analysis of different Ca2+-regulated processes at high spatiotemporal resolution in a controlled manner.


Assuntos
Células Endoteliais/metabolismo , Exocitose/genética , Terapia a Laser/métodos , Fator de von Willebrand/genética , Cálcio/metabolismo , Membrana Celular/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Corpos de Weibel-Palade/genética
19.
Biochim Biophys Acta Biomembr ; 1863(1): 183486, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33069635

RESUMO

Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the "house-keeping" one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in S. cerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels.


Assuntos
Candida albicans/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Potássio/metabolismo , Candida albicans/genética , Proteínas de Transporte de Cátions/genética , Membrana Celular/genética , Proteínas Fúngicas/genética , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
20.
Methods Mol Biol ; 2191: 49-63, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865738

RESUMO

Two-electrode voltage clamp (TEVC) is a preferred electrophysiological technique used to study gating kinetics and ion selectivity of light-activated channelrhodopsins (ChRs). The method uses two intracellular microelectrodes to hold, or clamp, the membrane potential at a specific value and measure the flow of ions across the plasma membrane. Here, we describe the use of TEVC and a simple solution exchange protocol to measure cation selectivity and analyze gating kinetics of the C1C2 chimera expressed in Xenopus laevis oocytes. Detailed instructions on how to process the collected data and interpret the results are also provided.


Assuntos
Channelrhodopsins/química , Biologia Molecular/métodos , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Membrana Celular/genética , Channelrhodopsins/genética , Ativação do Canal Iônico , Cinética , Potenciais da Membrana/genética , Microeletrodos , Oócitos/química , Oócitos/crescimento & desenvolvimento , Xenopus laevis/genética
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