RESUMO
Antimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Peptídeos Penetradores de Células/química , Corantes Fluorescentes/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência/métodos , Marcadores de Fotoafinidade/química , Espectrometria de Fluorescência/métodosRESUMO
Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Colesterol/metabolismo , Glicosaminoglicanos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Peptídeos Penetradores de Células/química , Ceramidas/análise , Cricetinae , Cricetulus , Endocitose , Microscopia Confocal , Dados de Sequência Molecular , Esfingomielinas/metabolismo , Triptofano/químicaRESUMO
Cell-penetrating peptides (CPPs) can cross cell membranes in a receptor-independent manner. Two main routes for their cellular uptake have been proposed: endocytosis and direct translocation through the cell membrane. The ability of a peptide to enter cells through direct translocation can be assessed by evaluating the amount of peptide crossing the membrane of liposomes. Most methods reported so far rely on the use of fluorescent probes, which, when attached to a CPP, often alter its physical/chemical properties. Herein, a matrix-assisted laser desorption/ionization time-of-flight MS-based method is described to quantify the amount of CPP taken up into lipid vesicles and to distinguish it from the amount that is bound or inserted in the membrane. For comparison, visualization of the uptake of the same, but fluorophore-labeled, peptides into giant vesicles and cells by fluorescence microscopy is also reported. We show that membrane charge density is an important factor for direct translocation. We also show that fluorophore-labeled peptides have a different translocation behavior and that they are more toxic to cells. Alternative methods to fluorescence, such as the one reported herein, should be favored when investigating the uptake mechanism of CPPs, as fluorescent dyes can alter short peptides' physical/chemical properties and their internalization capacities.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Lipossomos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Cricetinae , Cricetulus , Transporte Proteico , Espectrometria de FluorescênciaRESUMO
The fusion between two lipid membranes is a ubiquitous mechanism in cell traffic and pathogens invasion. Yet it is not well understood how two distinct bilayers overcome the energy barriers towards fusion and reorganize themselves to form a unique continuous bilayer. The magnitudes and numbers of these energy barriers are themselves an open question. To tackle these issues, we developed a new tool that allows to control the forces applied between two supported lipid bilayers (SLBs) deposited on superparamagnetic beads. By applying a magnetic field, the beads self-organize along field lines in chains of beads and compress the two membranes on the contact zone. Using the diffusion of fluorescently labelled lipids from one bilayer to the other allows us to identify fusion of the bilayers in contact. We applied increasing forces on SLBs and increased the occurrence of fusion. This experimental system allows the simultaneous study of tens of facing bilayers in a single experiment and mitigates the stochasticity of the fusion process. It is thus a powerful tool to test the various parameters involved in the membrane fusion process.
Assuntos
Bicamadas Lipídicas , Fusão de Membrana , Bicamadas Lipídicas/metabolismo , Difusão , Membranas/metabolismo , Nanopartículas Magnéticas de Óxido de FerroRESUMO
Membrane permeabilization is achieved via numerous techniques involving the use of molecular agents such as peptides used in antimicrobial therapy. Although high efficiency is reached, the permeabilization mechanism remains global with a noticeable lack of control. To achieve localized control and more gradual increase in membrane perturbation, we have developed hydrophobically modified poly(acrylic acid) amphiphilic copolymers with light-responsive azobenzene hydrophobic moieties. We present evidence for light triggered membrane permeabilization in the presence azobenzene-modified polymers (AMPs). Exposure to UV or blue light reversibly switches the polarity of the azobenzene (cis-trans isomerization) in AMPs, hence controlling AMP-loaded lipid vesicles permeabilization via in situ activation. Release of encapsulated probes was studied by microscopy on isolated AMP-loaded giant unilamellar vesicles (pol-GUVs). We show that in pH and ionic strength conditions that are biologically relevant pol-GUVs are kept impermeable when they contain predominantly cis-AMPs but become leaky with no membrane breakage upon exposure to blue light due to AMPs switch to a trans-apolar state. In addition, we show that AMPs induce destabilization of plasma membranes when added to mammal cells in their trans-apolar state, with no loss of cell viability. These features make AMPs promising tools for remote control of cell membrane permeabilization in mild conditions.
Assuntos
Resinas Acrílicas/química , Permeabilidade da Membrana Celular , Luz , Lipídeos de Membrana/química , Resinas Acrílicas/síntese química , Animais , Compostos Azo/química , Células COS , Sobrevivência Celular , Células Cultivadas , Chlorocebus aethiops , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Concentração Osmolar , Propriedades de SuperfícieRESUMO
Proteo-giant liposomes were electroformed from a mixture of lecithin vesicles and inside-out vesicles from erythrocytes. After addition of Mg-ATP in the vicinity of the proteo-giant liposomes, small buds appeared on the liposome surfaces, which--via an increase in lipids in the outer monolayer--demonstrated the active transport of lipids from the inner to the outer monolayer, indicating flippase activity.
Assuntos
Eritrócitos/enzimologia , Eritrócitos/metabolismo , Lecitinas/metabolismo , Lipossomas Unilamelares/metabolismo , Trifosfato de Adenosina/metabolismo , Fluorescência , Magnésio/metabolismo , Microscopia de Fluorescência , Sódio/metabolismo , Estresse Mecânico , Tensão SuperficialRESUMO
The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Células Cromafins/metabolismo , Depsipeptídeos , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Transporte/genética , Bovinos , Células Cromafins/ultraestrutura , Exocitose/efeitos dos fármacos , Exocitose/genética , Microscopia Eletrônica , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Células PC12 , Peptídeos Cíclicos/farmacologia , Ratos , Vesículas Secretórias/ultraestrutura , Tiazóis/farmacologia , Tiazolidinas , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
In endocrine cells, plasma membrane (PM)-bound secretory granules must undergo a number of maturation stages (i.e., priming) to become fusion-competent. Despite identification of several molecules involved in binding granules to the PM and priming them, the exact nature of events occurring at the PM still largely remains a mystery. In stimulated BON cells, we used evanescent wave microscopy to study trajectories of granules shortly before their exocytoses, which provided a physical description of vesicle-PM interactions at an unprecedented level of detail, and directly lead to an original mechanistic model. In these cells, tethered (T), nonfusogenic, vesicles are prevented from converting to fusogenic, docked (D) ones in resting conditions. Upon elevation of calcium, T-vesicles perform a 21-nm step toward the PM to become D, and fuse approximately 3 s thereafter. Our ability to directly visualize different modes of PM-attachment paves the way for clarifying the exact role of various molecules implicated in attachment and priming of granules in future studies.
Assuntos
Tumor Carcinoide/patologia , Tumor Carcinoide/fisiopatologia , Membrana Celular/ultraestrutura , Exocitose , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Linhagem Celular Tumoral , Humanos , Movimento (Física)RESUMO
Secretion of hormones and other bioactive substances is a fundamental process for virtually all multicellular organisms. Using total internal reflection fluorescence microscopy (TIRFM), we have studied the calcium-triggered exocytosis of single, fluorescently labeled large, dense core vesicles in the human neuroendocrine BON cell line. Three types of exocytotic events were observed: (1) simple fusions (disappearance of a fluorescent spot by rapid diffusion of the dye released to the extracellular space), (2) "orphan" fusions for which only rapid dye diffusion, but not the parent vesicle, could be detected, and (3) events with incomplete or multi-step disappearance of a fluorescent spot. Although all three types were reported previously, only the first case is clearly understood. Here, thanks to a combination of two-color imaging, variable angle TIRFM, and novel statistical analyses, we show that the latter two types of events are generated by the same basic mechanism, namely shape retention of fused vesicle ghosts which become targets for sequential fusions with deeper lying vesicles. Overall, approximately 25% of all exocytotic events occur via sequential fusion. Secondary vesicles, located 200-300 nm away from the cell membrane are as fusion ready as primary vesicles located very near the cell membrane. These findings call for a fundamental shift in current models of regulated secretion in endocrine cells. Previously, sequential fusion had been studied mainly using two-photon imaging. To the best of our knowledge, this work constitutes the first quantitative report on sequential fusion using TIRFM, despite its long running and widespread use in studies of secretory mechanisms.
Assuntos
Exocitose/fisiologia , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Células Neuroendócrinas/citologia , Células Neuroendócrinas/fisiologia , Vesículas Transportadoras/fisiologia , Vesículas Transportadoras/ultraestrutura , Linhagem Celular , HumanosRESUMO
We have observed large pores in the membrane of giant vesicles in an aqueous medium. The lifetime of the pores can reach 2 min and their size (a few micrometers) enables their visualization by fluorescence microscopy. These pores are obtained thanks to a destabilization of the membrane due to the synergistic action of a cone-shaped and nitrobenzodiazole (NBD) labeled phospholipid illuminated in the presence of dithionite. The opening of the pore occurs immediately after illumination starts so that it can be accurately triggered. A concomitant decrease of the vesicle radius is observed; we interpret it as a solubilization of the membrane. Depending on the rate of this solubilization, long- or short-lived pores were observed. At the transition between both regimes for a 30 microm vesicle, the solubilization rate was about 1/300 s{-1} . In order to interpret these observations, we have revisited the current model of pore opening to take into account this solubilization. This proposed model along with simulations enables us to prove that solubilization explains why the large long-lived pores are observed even in an aqueous medium. The model also predicts the solubilization rate at the transition between a single long-lived pore and a cascade of short-lived pores.
Assuntos
Bicamadas Lipídicas/química , Lipossomos/química , Fluidez de Membrana , Modelos Químicos , Modelos Moleculares , Fosfolipídeos/química , Água/química , Simulação por Computador , Cinética , Microdomínios da Membrana/química , Porosidade , Soluções , Fatores de TempoRESUMO
The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high µM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Animais , Células CHO , Cricetulus , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Estrutura Molecular , Espectrometria de FluorescênciaRESUMO
Giant unilamellar vesicles (diameter of a few tens of micrometers) are commonly produced by hydration of a dried lipidic film. After addition of the aqueous solution, two major protocols are used: (i) the gentle hydration method where the vesicles spontaneously form and (ii) the electroformation method where an ac electric field is applied. Electroformation is known to improve the rate of unilamellarity of the vesicles though it imposes more restricting conditions for the lipidic composition of the vesicles. Here we further characterize these methods by using fluorescence microscopy. It enables not only a sensitive detection of the defects but also an evaluation of the quantity of lipids in these defects. A classification of the defects is proposed and statistics of their relative importance in regard to both methods and lipid composition are presented: it shows for example that 80% of the vesicles obtained by electroformation from 98% 1,2-dioleoyl-sn-glycero-3-phosphocholine are devoid of significant defects against only 40% of the vesicles with the gentle hydration method. It is also shown that the presence of too many negatively charged lipids does not favor the formation of unilamellar vesicles with both methods. For the gentle hydration, we checked if the negatively charged lipids were inserted in the vesicles membrane in the same proportion as that of the lipid mixture from which they are formed. The constant incorporation of a negatively charged labeled lipid despite an increasing presence of negatively charged 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] tends to confirm that the composition of vesicles is indeed close to that of the initial mixture.
Assuntos
Eletroquímica/métodos , Microscopia de Fluorescência/métodos , Biotecnologia , Glucose/química , Bicamadas Lipídicas/química , Lipídeos/química , Lipossomos/química , Substâncias Macromoleculares , Fosfatidilcolinas/química , Fosfatidilserinas/química , Ligação Proteica , Rodaminas/farmacologia , Temperatura , Fatores de Tempo , Água/químicaRESUMO
BON cells are human carcinoid cells that secrete serotonin (5-HT) and various peptides. Secretion of [(3)H]5-HT by cell cultures was investigated. Acetylcholine (Ach) stimulated secretion through a somatostatin-sensitive muscarinic pathway, whereas isoproterenol was inefficient. [(3)H]5-HT secretion also was induced by Ca(2+) in the presence of the ionophore A-23187 or after digitonin permeabilization. These two processes were insensitive to stomatostatin. Ba(2+) induced an efficient somatostatin-sensitive [(3)H]5-HT secretory response. Secretion also was analyzed at the single-cell level, using carbon fiber amperometry and evanescent-field fluorescence microscopy, after labeling the secretory vesicles by transfection of the cells with a NPY-GFP construct. Both techniques revealed slow kinetics of secretory responses, suggesting that ready-to-fuse vesicles do not accumulate in these cells. Single secretory vesicles were imaged either in resting conditions or after addition of Ca(2+) ions to digitonin-permeabilized cells. The three-dimensional movements of the vesicles before exocytosis were analyzed. The mean velocity of vesicles that released their content was lower than that of silent ones. Even in the case of mobile vesicles, exocytosis often was preceded by a period of arrest lasting at least 15 seconds, consistent with a docking/priming step.
Assuntos
Tumor Carcinoide , Células Enterocromafins/metabolismo , Neoplasias Pancreáticas , Serotonina/metabolismo , Linhagem Celular Tumoral/metabolismo , HumanosRESUMO
Analysis of trajectories of dynamical biological objects, such as breeding ants or cell organelles, is essential to reveal the interactions they develop with their environments. Many previous works used a global characterization based on parameters calculated for entire trajectories. In cases where transient behavior was detected, this usually concerned only a particular type, such as confinement or directed motion. However, these approaches are not appropriate in situations in which the tracked objects may display many different types of transient motion. We have developed a method to exhaustively analyze different kinds of transient behavior that the tracked objects may exhibit. The method discriminates stalled periods, constrained and directed motions from random dynamics by evaluating the diffusion coefficient, the mean-square displacement curvature, and the trajectory asymmetry along individual trajectories. To detect transient motions of various durations, these parameters are calculated along trajectories using a rolling analysis window whose width is variable. The method was applied to the study of secretory vesicle dynamics in the subplasmalemmal region of human carcinoid BON cells. Analysis of transitions between transient motion periods, combined with plausible assumptions about the origin of each motion type, leads to a model of dynamical subplasmalemmal organization.
Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Movimento/fisiologia , Vesículas Secretórias/fisiologia , Vesículas Secretórias/ultraestrutura , Linhagem Celular , HumanosRESUMO
The transbilayer diffusion of unlabeled ceramides with different acyl chains (C6-Cer, C10-Cer, and C16-Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (approximately 0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37 degrees C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after approximately 15 min of incubation at 20 degrees C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.