Cell-attached Capacitance Measurements Research Articles

Phosphatidylserine is critical for vesicle fission during clathrin-mediated endocytosis.

Phosphatidylserine (PS), a negatively charged phospholipid present predominantly at the inner leaflet of the plasma membrane, has been widely implicated in many cellular processes including membrane trafficking. Along this line, PS has been demonstrated to be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin-mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate in the present study that the fission-pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission during CME. Furthermore, our results show that the PS-mediated effect on the fission-pore duration is Ca2+ -dependent and abolished in the absence of synaptotagmin 1 (Syt1), implying that Syt1 is necessary for the stimulatory role of PS in vesicle fission during CME. Consistently, a Syt1 mutant with a defective PS-Syt1 interaction increases the fission-pore duration. Taken together, our study suggests that PS-Syt1 interaction may be critical in regulating fission dynamics during CME.

Dynamin regulates the fusion pore of endo- and exocytotic vesicles as revealed by membrane capacitance measurements

BackgroundDynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosis. MethodsThus we here employed high-resolution cell-attached capacitance measurements and super-resolution structured illumination microscopy to directly examine single vesicle interactions with the plasmalemma in cultured rat astrocytes treated with distinct pharmacological modulators of dynamin activity. Fluorescent dextrans and the lipophilic plasmalemmal marker DiD were utilized to monitor uptake and distribution of vesicles in the peri-plasmalemmal space and in the cell cytosol. ResultsDynamin inhibition with Dynole™-34-2 and Dyngo™-4a prevented vesicle internalization into the cytosol and decreased fusion pore conductance of vesicles that remained attached to the plasmalemma via a narrow fusion pore that lapsed into a state of repetitive opening and closing - flickering. In contrast, the dynamin activator Ryngo™-1-23 promoted vesicle internalization and favored fusion pore closure by prolonging closed and shortening open fusion pore dwell times. Immunocytochemical staining revealed dextran uptake into dynamin-positive vesicles and increased dextran uptake into Syt4- and VAMP2-positive vesicles after dynamin inhibition, indicating prolonged retention of these vesicles at the plasmalemma. ConclusionsOur results have provided direct evidence for a role of dynamin in regulation of fusion pore geometry and kinetics of endo- and exocytotic vesicles, indicating that both share a common dynamin-regulated structural intermediate, the fusion pore.

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.

Actin Polymerization Does Not Provide Direct Mechanical Forces for Vesicle Fission during Clathrin-Mediated Endocytosis

Actin polymerization is important for vesicle fission during clathrin-mediated endocytosis (CME), and it has been proposed that actin polymerization may promote vesicle fission during CME by providing direct mechanical forces. However, there is no direct evidence in support of this hypothesis. In the present study, the role of actin polymerization in vesicle fission was tested by analyzing the kinetics of the endocytic tubular membrane neck (the fission-pore) with cell-attached capacitance measurements to detect CME of single vesicles in a millisecond time resolution in mouse chromaffin cells. Inhibition in dynamin GTPase activity increased the fission-pore conductance (Gp), supporting the mechanical role of dynamin GTPase in vesicle fission. However, disruptions in actin polymerization did not alter the fission-pore conductance Gp, thus arguing against the force-generating role of actin polymerization in vesicle fission during CME. Similar to disruptions of actin polymerization, cholesterol depletion results in an increase in the fission-pore duration, indicating a role for cholesterol-dependent membrane reorganization in vesicle fission. Further experiments suggested that actin polymerization and cholesterol might function in vesicle fission during CME in the same pathway. Our results thus support a model in which actin polymerization promotes vesicle fission during CME by inducing cholesterol-dependent membrane reorganization.

Synaptotagmin 1 Is Necessary for the Ca2+Dependence of Clathrin-Mediated Endocytosis

The role of Ca²⁺ in synaptic vesicle endocytosis remains uncertain due to the diversity in various preparations where several forms of endocytosis may contribute variably in different conditions. Although recent studies have demonstrated that Ca²⁺ is important for clathrin-mediated endocytosis (CME), the mechanistic role of Ca²⁺ in CME remains to be elucidated. By monitoring CME of single vesicles in mouse chromaffin cells with cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate that the dynamics of vesicle fission during CME is Ca²⁺ dependent but becomes Ca²⁺ independent in synaptotagmin 1 (Syt1) knock-out cells. Our results thus suggest that Syt1 is necessary for the Ca²⁺ dependence of CME.

Hormonal inhibition of endocytosis: novel roles for noradrenaline and G protein Gz

The modulation of endocytosis following exocytosis by noradrenaline (NA), a physiological inhibitor of insulin secretion, was investigated in INS 832/13 cells using patch-clamp capacitance measurements. Endocytosis was inhibited by NA in a pertussis toxin-insensitive manner. Dialysing a synthetic peptide mimicking the C-terminus of the α-subunit of G(z) into the cells blocked the inhibition of endocytosis by NA. Cell-attached capacitance measurements indicated that inhibition by NA was due to a decreased number of endocytotic events without a change in vesicle size. Analysis of fission pore closure kinetics revealed two distinct fission modes, with NA selectively inhibiting the rapid fission pore closure events. Comparison of the actions of NA and deltamethrin, a calcineurin antagonist and potent inhibitor of endocytosis, demonstrated that they inhibit endocytosis by different mechanisms. These findings establish novel actions for NA and G(z) in insulin-secreting cells and possibly other cell types.

Norepinephrine Inhibits Endocytosis In Insulin-secreting Cells

Norepinephrine (NE) is a well-known physiological inhibitor of insulin secretion in pancreatic β cells. We investigated modulation of exocytosis and endocytosis by NE in INS 832/13 cells using whole-cell capacitance measurements. Exocytosis was stimulated by depolarizing pulses from −70mV to +10mV of variable duration and was followed by compensatory endocytosis. Inhibition of Ca2+-evoked exocytosis by NE was overcome by increasing Ca2+ influx, either by increasing the depolarizing pulse duration (up to 500ms) or by increasing the extracellular Ca2+ concentration up to 10mM. When stimulated by a short train of 500ms pulses in the presence of NE (5μM), robust exocytosis was observed but endocytosis was markedly inhibited. The NE inhibition of endocytosis was abolished by the α2-adrenergic receptor antagonist yohimbine (10μM) and was not affected by PTX-treatment (150ng/ml), demonstrating that NE inhibition of endocytosis is mediated via the α2-adrenergic receptor and not via Gi and/or Go proteins. When a synthetic peptide that mimicked the last 13 c-terminal amino acids of the Gzα subunit was dialyzed into the cells via the whole-cell patch pipette, NE inhibition of endocytosis was fully blocked, suggesting that Gz may be mediating the inhibition. Single vesicle recordings by cell attached capacitance measurements indicate that inhibition of endocytosis by NE is due to a decreased number of endocytic events without a significant change in endocytic vesicle size. Further analysis of fission pore kinetics revealed that NE selectively inhibited the rapid fission events. Our findings establish a novel action for NE and suggest the possibility that NE may modulate endocytosis in the central nervous system and elsewhere.

A Role For Protein Phosphorylation In Fusion Pore Opening And Transmitter Release

Activity-dependent regulation of protein kinase A (PKA) and protein kinase C (PKC), is essential for modulation of neurosecretion. To study the role of protein phosphorylation in the dynamics of the fusion pore opening and transmitter release, carbon fiber amperometry and cell-attached capacitance measurements were performed in bovine chromaffin cells. In cells treated with the PKC inhibitor, bisindolylmaleimide I (100nM, 10Ki), release frequency was significantly reduced to 5.0±1.2events/min (from control 17.1±2.0events/min). Amperometric spikes also showed increased half width of 22.3±2.0ms (from 11.5±0.9ms control) and smaller amperometric spike amplitude of 21.9±3.7pA (from 56.2±9.4pA control). In the cells treated with PKA inhibitor, KT 5720 (500nM, 10Ki), the duration of foot signals of amperometric events was prolonged to 14.4±1.6ms (from 9.0±1.0 ms control), consistent with prolonged fusion pore duration in cell-attached capacitance measurements of 27.7±6.8ms (from 11.8±1.4ms control). In contrast to PKC inhibitor, KT 5720 did not affect the release frequency.These results indicate that PKC affects the rate of fusion pore formation and release after full fusion but not early fusion pore expansion, while PKA specifically affects the expansion of the early fusion pore as well as release after full fusion.View Large Image | View Hi-Res Image | Download PowerPoint Slide

F-Actin and Myosin II Accelerate Catecholamine Release from Chromaffin Granules

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Cell-attached capacitance measurements at release sites of a central nerve terminal

Cell-attached capacitance measurements at release sites of a central nerve terminal

Regulated Exocytosis and Kiss-and-Run of Synaptic-Like Microvesicles in INS-1 and Primary Rat β-Cells

We have applied cell-attached capacitance measurements to investigate whether synaptic-like microvesicles (SLMVs) undergo regulated exocytosis in insulinoma and primary pancreatic beta-cells. SLMV and large dense-core vesicle (LDCV) exocytosis was increased 1.6- and 2.4-fold upon stimulation with 10 mmol/l glucose in INS-1 cells. Exocytosis of both types of vesicles was coupled to Ca(2+) entry through l-type channels. Thirty percent of SLMV exocytosis in INS-1 and rat beta-cells was associated with transient capacitance increases consistent with kiss-and-run. Elevation of intracellular cAMP (5 micromol/l forskolin) increased SLMV exocytosis 1.6-fold and lengthened the duration of kiss-and-run events in rat beta-cells. Experiments using isolated inside-out patches of INS-1 cells revealed that the readily releasable pool (RRP) of SLMVs preferentially undergoes kiss-and-run exocytosis (67%), is proportionally larger than the LDCV RRP, and is depleted more quickly upon Ca(2+) stimulation. We conclude that SLMVs undergo glucose-regulated exocytosis and are capable of high turnover. Following kiss-and-run exocytosis, the SLMV RRP may be reloaded with gamma-aminobutyric acid and undergo several cycles of exo- and endocytosis. Our observations support a role for beta-cell SLMVs in a synaptic-like function of rapid intra-islet signaling.

The exocytotic event in chromaffin cells revealed by patch amperometry.

In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at <3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, generating a 'foot' signals which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane.