Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.     

Real-time cellular impedance measurements detect Ca channel-dependent oscillations of morphology in human H295R adrenoma cells

Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca²⁺ channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca²⁺ concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca²⁺-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles.     

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.     

Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx

Cell function can be modulated by the insertion and removal of ion channels from the cell surface. The mechanism used to keep channels quiescent prior to delivery to the cell surface is not known. In eggs, cortical vesicle exocytosis inserts voltage-gated calcium channels into the cell surface. Calcium influx through these channels triggers compensatory endocytosis. Secretory vesicles contain high concentrations of calcium and hydrogen ions. We propose that lumenal hydrogen ions inhibit vesicular calcium channel gating prior to exocytosis, discharge of lumenal protons upon vesicle-plasma membrane fusion enables calcium channel gating. Consistent with this hypothesis we find that cortical vesicle lumens are acidic, and exocytosis releases lumenal hydrogen ions. Acidic extracellular pH reversibly blocks endocytosis, and the windows of opportunity for inhibition with a calcium-channel blocker or hydrogen ions are indistinguishable. Calcium ionophore treatment circumvents the low pH block, suggesting that calcium influx, or an upstream step, is obstructed. Inhibition of calcium influx by preventing membrane depolarization is unlikely, as elevation of the extracellular potassium concentration failed to overcome the pH block, and low extracellular pH was found to depolarize the membrane potential. We conclude that low pH inhibits endocytosis at a step between membrane depolarization and calcium influx .     

植物胞吞和胞吐的耦合调控

真核细胞通过胞吞和胞吐作用将大分子和颗粒性物质运出或运送至质膜,其中包括一些具有重要生物学功能的蛋白质。胞吞和胞吐途径之间的耦合对维持质膜的完整性以及调控质膜蛋白的丰度和活性至关重要。动物中,突触小泡的胞吞和胞吐在时空上紧密耦合已被证明是持续神经传递的必要条件。近年来,随着对植物囊泡运输的深入研究,越来越多的证据表明,植物细胞的胞吞和胞吐间同样存在耦合调控,且在植物生长发育和对外界环境的响应中扮演重要角色。该文综述了植物协同调控胞吞和胞吐的生理学意义,并结合网格蛋白介导囊泡运输的最新研究进展探讨了其可能的耦合机制。     

Methods for patch clamp capacitance recordings from the calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.     

Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes

Pollen tubes are one of the fastest growing eukaryotic cells.Rapid anisotropic growth is supported by highly active exocytosisand endocytosis at the plasma membrane, but the subcellularlocalization of these sites is unknown. To understand molecularprocesses involved in pollen tube growth, it is crucial to identifythe sites of vesicle localization and trafficking. This reportpresents novel strategies to identify exocytic and endocyticvesicles and to visualize vesicle trafficking dynamics, usingpulse-chase labelling with styryl FM dyes and refraction-freehigh-resolution time-lapse differential interference contrastmicroscopy. These experiments reveal that the apex is the siteof endocytosis and membrane retrieval, while exocytosis occursin the zone adjacent to the apical dome. Larger vesicles areinternalized along the distal pollen tube. Discretely sizedvesicles that differentially incorporate FM dyes accumulatein the apical, subapical, and distal regions. Previous workestablished that pollen tube growth is strongly correlated withhydrodynamic flux and cell volume status. In this report, itis shown that hydrodynamic flux can selectively increase exocytosisor endocytosis. Hypotonic treatment and cell swelling stimulatedexocytosis and attenuated endocytosis, while hypertonic treatmentand cell shrinking stimulated endocytosis and inhibited exocytosis.Manipulation of pollen tube apical volume and membrane remodellingenabled fine-mapping of plasma membrane dynamics and definedthe boundary of the growth zone, which results from the orchestratedaction of endocytosis at the apex and along the distal tubeand exocytosis in the subapical region. This report providescrucial spatial and temporal details of vesicle traffickingand anisotropic growth. Key words: Endocytosis; exocytosis, hydrodynamics, lipophilic FM dyes, pollen tube growth, vesicle trafficking Received 14 September 2007; Revised 23 November 2007 Accepted 7 January 2008     

Exocytosis in plants

Exocytosis is the final event in the secretory pathway and requires the fusion of the secretory vesicle membrane with the plasma membrane. It results in the release to the outside of vesicle cargo from the cell interior and also the delivery of vesicle membrane and proteins to the plasma membrane. An electrophysiological assay that measures changes in membrane capacitance has recently been used to monitor exocytosis in plants. This complements information derived from earlier light and electron microscope studies, and allows both transient and irreversible fusion of single exocytotic vesicles to be followed with high resolution in protoplasts. It also provides a tool to investigate bulk exocytotic activity in single protoplasts under the influence of cytoplasmic modulators. This research highlights the role of intracellular Ca²⁺, GTP and pressure in the control of exocytosis in plants.In parallel to these functional studies, plant proteins with the potential to regulate exocytosis are being identified by molecular analysis. In this review we describe these electrophysiological and molecular advances, and emphasise the need for parallel biochemical work to provide a complete picture of the mechanisms controlling vesicle fusion at the plasma membrane of plant cells.     

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P₂ and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca²⁺ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P₂ complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca²⁺ concentrations is likely to be important for Ca²⁺-regulated secretion.     

Actin in membrane trafficking

Actin cytoskeleton remodeling provides the forces required for a variety of cellular processes based on membrane dynamics, such as endocytosis, exocytosis, and vesicular trafficking at the Golgi. All these events are coordinated by networks of associated proteins, and some of them are functionally connected with cell migration. The site and the duration of actin polymerization, in connection with vesicle budding and fusion, are tightly controlled by both small GTPases and the large GTPase dynamin. Recent advances in the understanding of the mechanisms coupling actin dynamics with membrane trafficking at the cell surface have been brought by the combined studies of actin polymerizing factors and of the endocytic/exocytic machinery.     

Aspects of signal transduction in stimulus exocytosis-coupling in Paramecium

This paper deals with the detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in a eukaryotic cell, Paramecium tetraurelia. There are at least three cellular compartments involved in the process: I) the plasma membrane, which contains secretagogue receptors and other transmembrane proteins, II) the cytoplasms, particularly in the region between the cell and secretory vesicle membranes, where molecules may influence interactions of the membranes, and III) the secretory vesicle itself. The ciliated protozoan Paramecium tetraurelia is very well suited for the study of signal transduction events associated with exocytosis because this eukaryotic cell contains thousands of docked secretory vesicles (trichocysts) below the cell membrane which can be induced to release synchronously when triggered with secretagogue. This ensures a high signal-to-noise ratio for events associated with this process. Upon release the trichocyst membrane fuses with the cell membrane and the trichocyst content undergoes a Ca2+-dependent irreversible expansion. Secretory mutants are available which are blocked at different points in the signal transduction pathway. Aspects of the three components mentioned above that will be discussed here include a) the properties of the vesicle content, its pH, and its membrane; b) the role of phosphorylation/dephosphorylation of a cytosolic 63-kilodalton (kDa)Mr protein in membrane fusion; and c) how influx of extracellular Ca2+ required for exocytosis may take place via exocytic Ca2+ channels which may be associated with specific membrane microdomains (fusion rosettes).     

The measurement of exocytosis in plant cells

Exocytosis is of vital importance to the growth and developmentof plant cells. It is a dynamic process in which vesicles bearingpolysaccharide precursors and proteins fuse with the plasmamembrane and release their contents. Equally important, newplasma membrane is delivered by exocytosis as secretory vesiclemembrane becomes incorporated. The requirements for polysaccharides,proteins and plasma membrane are very different in differentcell types, so there must be sophisticated mechanisms for ensuringdelivery of these materials to the correct cellular locationsat the appropriate time and, particularly in the case of membrane,their recovery and recycling. Currently, little is known ofthese mechanisms in plants, but new methods for measuring exocytosisare under development, and existing techniques have alreadycontributed data of considerable relevance. Here the methodsfor measuring exocytosis are described and evaluated, with emphasison the electrophysiological measurement of capacitance as arelatively non-invasive method, and on cell-free assays becauseof their potential importance in the identification of proteinsand other factors that control exocytosis in plant cells. Key words: Exocytosis, vesicle traffic, vesicle fusion, polysaccharides, cell wall, cell plate, root cap, secretion, patch-clamping     

Rapid Endocytosis and Vesicle Recycling in Neuroendocrine Cells

Endocytosis is a crucial process for neuroendocrine cells that ensures membrane homeostasis, vesicle recycling, and hormone release reliability. Different endocytic mechanisms have been described in chromaffin cells, such as clathrin-dependent slow endocytosis and clathrin-independent rapid endocytosis. Rapid endocytosis, classically measured in terms of a fast decrease in membrane capacitance, exhibits two different forms, “rapid compensatory endocytosis” and “excess retrieval.” While excess retrieval seems to be associated with formation of long-lasting endosomes, rapid compensatory endocytosis is well correlated with exocytotic activity, and it is regarded as a mechanism associated to rapid vesicle recycling during normal secretory activity. It has been suggested that rapid compensatory endocytosis may be related to the prevalence of a transient fusion mode of exo-endocytosis. In the latter mode, the fusion pore, a nanometric-sized channel formed at the onset of exocytosis, remains open for a few hundred milliseconds and later abruptly closes, releasing a small amount of transmitters. By this mechanism, endocrine cell selectively releases low molecular weight transmitters, and rapidly recycles the secretory vesicles. In this article, we discuss the cellular and molecular mechanisms that define the different forms of exocytosis and endocytosis and their impact on vesicle recycling pathways.     

Still life: Pollen tube growth observed in millisecond resolution

Our recent work used novel methods to localize and track discrete vesicle populations in pollen tubes undergoing oscillatory growth. The results show that clathrin-dependent endocytosis occurs along the shank of the pollen tube, smooth vesicle endocytosis occurs at the tip, and exocytosis occurs in the subapical region. Here, growth of tobacco and lily pollen tubes is examined in greater temporal resolution using refraction-free high-resolution time-lapse differential interference contrast microscopy. Images were collected at 0.21 s intervals for 10 min, sequentially examined for millisecond details, compressed into video format and then examined for details of growth dynamics. The subapical growth zone is structurally fluid, with vesicle insertion into the plasma membrane, construction of new cell surface and cellular expansion. Incorporation of new membrane and wall materials causes localized disruption at the cell surface that precedes the start of the growth cycle by 3.44 ± 0.39 s in tobacco, and 1.02 ± 0.01 s in lily pollen tubes. Vesicle deposition increases after the start of the growth cycle and supports expansion of the growth zone. Growth reorientation involves a shift in the position and angle of the growth zone. In summary, these results support a new model of pollen tube growth.Key words: growth zone, oscillation, exocytosis, growth reorientation, differential interference contrast microscopy, refraction-free     

Kinetic analysis of the triggered exocytosis/endocytosis secretory cycle in cultured bovine adrenal medullary cells

Cultured bovine adrenal medullary cells are an excellent preparation for quantitative analysis of the secretory exocytosis/endocytosis cycle. In this paper we examine the kinetics of endocytosis after stimulation of secretion. Membrane retrieval was monitored by uptake of the fluid phase marker horseradish peroxidase. Horseradish peroxidase was found to be suitable because it can be washed off completely, assayed quantitatively, and its uptake increases linearly with concentration. If this marker is present during stimulation, the rate of uptake is initially slower than catecholamine secretion but faster at a later time, suggesting that the formation of endocytotic vesicles follows exocytosis. To monitor the time-dependent concentration of secretory vesicle-plasma membrane fusion product (omega-profiles), secretion was halted at various time intervals after stimulation and the excess membrane allowed to transform into endocytotic vesicles in the presence of horseradish peroxidase. By adding horseradish peroxidase at various times after inhibition of secretion, the time course of membrane retrieval could be measured directly. All our results are consistent with a two-step kinetic model in which exocytosis and membrane retrieval are consecutive events. The estimated volumes of the compartments involved are roughly equal. The rate of endocytosis is strongly temperature-dependent but unaffected by extracellular calcium in the range of 10(-8)-2.5 X 10(-3) M, suggesting that calcium is not required at the site of endocytotic membrane fusion. Membrane retrieval is also unaffected by Lanthanum (1 mM) but is slowed by hypertonic media.     

质膜的损伤修复及相关模型

生物膜的磷脂双分子层将细胞与外界环境分开。大部分细胞会在机械损伤或化学应激下引发质膜损伤,如果不及时修复将会导致细胞死亡。胞外钙离子通过伤口进入细胞,作为损伤的最初信号,会诱发一系列的修复反应。随后,胞内细胞器也释放钙离子,并产生系列细胞行为来应对损伤,维护质膜的完整性。本文介绍了在损伤修复过程的胞吞作用、胞吐作用、胞外小泡脱落等细胞行为。综述了补丁模型、修复帽模型和大损伤修复的模型特点。补丁模型是最早的修复模型,提出后不断得到完善。细胞除了需要在损伤处聚集小泡、融合形成补丁外,还需通过胞吐、胞吞和出芽（小泡脱落）等方式参与伤口修复。本文简要介绍参与质膜修复的重要蛋白质如钙蛋白酶、dysferlin、MG53、膜联蛋白、突触结合蛋白(Syt-VⅡ)、ESCRTⅢ、酸性鞘磷脂酶、细胞骨架蛋白质等在修复过程中的作用。     

Regulated exocytosis per partes

This Special Issue (SI) of Cell Calcium focuses on regulated exocytosis, a recent evolutionary invention of eukaryotic cells. This essential cellular process consists of several stages: (i) the delivery of membrane bound vesicles to specific plasma membrane sites, (ii) where the merger between the vesicle and the plasma membranes occurs, (iii) leading to the formation of an aqueous channel through which vesicle content starts to be discharged to the cell exterior, (iv) after the full incorporation of the vesicle membrane into the plasma membrane, the added vesicle membrane is retrieved back into the cytoplasm by endocytosis. (v) When a fusion pore opens it may close again, a process known as transient fusion pore opening (also kiss-and-run exocytosis). In some cell types these stages are extremely shortlived, as in some neurons, and thus relatively inaccessible to experimentation. In other cell types the transition between these stages is orders of magnitude slower and can be studied in more detail. However, despite the intense investigations of this critical biological process over the last decades, the molecular mechanisms underlying regulated exocytosis have yet to be fully resolved. We thus still lack a comprehensive physiological insight into the nature of the progressive and coupled stages of exocytosis. Such a molecular-level understanding would help to fully reconstruct this process in vitro, as well as identify potential therapeutic targets for a range of diseases and dysfunctions. There are 18 papers in this SI which have been organized into three sections: Rapid regulated exocytosis and calcium homeostasis with an introduction by Erwin Neher, Molecular mechanisms of regulated exocytosis, and Cell models for regulated exocytosis. Here we briefly outline and integrate the messages of these sections.     

Redfield revisited: variability of C:N:P in marine microalgae and its biochemical basis

Cytological events immediately following plasmogamy in Durvillaea potatorum are described. Eggs contain several types of cytoplasmic vesicle differing in size and appearance. Histochemical tests and measurements are used to characterise and distinguish different types of vesicle containing phenolic compounds, lipids and polysaccharides. Within 2 min of plasmogamy, small phenolic vesicles located just below the egg membrane undergo mass synchronised exocytosis. The contents of these vesicles are discharged as phenolic bodies on the outside of the membrane. Secretion of phenolic bodies precedes secretion of the primary zygote wall by several minutes. Limited secretion of phenolics also occurs in unfertilised eggs. Peripheral phenolic vesicles are distinguishable from physodes, which also contain phenolic compounds but which are significantly larger and tend to be localised around the egg nucleus. The possible functional significance of the phenolic bodies is discussed. Coated pits and vesicles are common in zygotes, and their presence is evidence for endocytosis.     

Real-time cellular impedance measurements detect Ca(2+) channel-dependent oscillations of morphology in human H295R adrenoma cells

Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca(2+) channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca(2+) concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca(2+)-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Rapid recovery of releasable vesicles and formation of nonreleasable endosomes follow intense exocytosis in chromaffin cells

Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of 20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K⁺ and 2 mM Ca²⁺) provoking intense exocytosis (51 ± 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 ± 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in <2 min. The remaining nonreleasable fraction was distributed mainly as fluorescent spots (0.7 µm) selectively labeled by 40- to 70-kDa dextrans and was suppressed by a phosphatidylinositol-3-phosphate kinase inhibitor, suggesting that it had been formed by a bulk retrieval mechanism. We concluded that chromaffin cells can rapidly recycle significant fractions of their total vesicle population, and that this pathway prevails when cholinergic agonists are used as secretagogues. When exocytosis exceeded 20% of plasma membrane, an additional mechanism was activated, which was unable to produce secretory vesicles in our experimental time frame but appeared crucial to maintaining membrane surface homeostasis under extreme conditions. endocytosis; mouse chromaffin cells; calcium signal; FM1-43; ADVASEP-7; bromophenol blue