High-resolution Membrane Capacitance Measurements Research Articles

Vesicle cholesterol controls exocytotic fusion pore

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

Vesicle Cholesterol Controls Exocytotic Fusion Pore

In lysosomal storage diseases (LSD) cholesterol accumulates in vesicles, which may affect vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes evokes vesicle hormonal secretion. A new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In fibroblasts lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

How goblet cells respond to dry eye: adaptive and pathological roles of voltage-gated calcium channels and P2X7 purinoceptors.

Dry eye is a common sight-impairing, painful disorder characterized by disruption of the preocular tear film, whose integrity is required for ~70% of the eye's refractive power. A universal feature of clinical dry eye is hyperosmolarity of the tears resulting from their accelerated evaporation due to dysfunction of tear- and oil-producing ocular glands. A key adaptive response to dryness/hyperosmolarity is release of tear-stabilizing mucin by conjunctival goblet cells. Yet the mechanisms mediating this response to hyperosmolarity remain poorly understood. In this study of freshly excised rat conjunctiva, perforated-patch recordings revealed that during sustained hyperosmolarity, the development of a nonspecific cation (NSC) conductance depolarizes the goblet cells to a near-optimal voltage for the tonic activation of their voltage-gated calcium channels (VGCCs). In turn, as demonstrated by high-resolution membrane capacitance measurements, VGCC activation boosts the exocytotic response of conjunctival goblet cells to neural input. However, over time, VGCC activation also increases the vulnerability of these cells to the lethality of hyperosmolarity. Viability assays further revealed that hyperosmotic-induced goblet cell death is critically dependent on P2X7 receptor channels. Similar to the yin-yang impact of VGCCs on goblet cell physiology and pathobiology, P2X7 activation not only compromises goblet cell viability but also enhances exocytotic activity. Thus, the NSC/VGCC and P2X7 purinoceptor pathways are components of a previously unappreciated high-gain/high-risk adaptive strategy to combat ocular dryness. These pathways boost release of tear-stabilizing mucin at the risk of jeopardizing the viability of the conjunctival goblet cells, whose loss is a histopathological hallmark of irreversible mucin-deficient dry eye.

A Mechanism for Exocytotic Arrest by the Complexin C-Terminus

Hindering premature vesicular fusion is key to build up a molecularly primed ready releasable pool. Complexin-II (CpxII), a 16 KD cytosolic protein, inhibits non-synchronous vesicle fusion at release sites, and by that accumulates a ready releasable pool of primed vesicles that are released in synchrony upon immediate elevation of intracellular calcium. Functionally, CpxII is equipped with four independent yet synergetic domains. A central helix mediates the CpxII-SNARE (N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors) complex interaction, while an accessory helix is important for the stability of the central helix and for reducing premature fusion. The upstream N-terminal domain facilitates exocytosis, whereas the downstream C-terminal domain (CTD) inhibits premature fusion of vesicles. The molecular mechanisms how the CTD blocks asynchronous release are still unclear. With the help of high resolution membrane capacitance measurements coupled with calcium imaging, carbon fiber amperometry, and biochemical assays, we show that infusion of CpxII far CTD -derived peptide into mouse chromaffin cells is able to boost the synchronous release by reducing the premature vesicle fusion. A similar phenotype is also observed by over-expressing the full length WT protein. Using in vitro binding assays, we show that the CTD interacts with SNARE proteins. Moreover, we found that the CTD peptide lowers the rate of SDS resistant SNARE complex formation. We also show that the CTD shares a high degree of similarity to the SNARE protein SNAP25-SN1 domain. Furthermore chimeras of CpxII:SNAP25-SN1 fully restore CpxII function, and even “superclamp” tonic secretion. Collectively, these results provide evidence for a clamping mechanism in which the CTD of CpxII prevents the spontaneous assembly of SNARE complexes, enabling the build-up of a ready releasable pool for synchronised Ca2+-triggered exocytosis.

Role of ion channels in the functional response of conjunctival goblet cells to dry eye.

Optimal vision requires an ocular surface with a stable tear film whose many critical tasks include providing >70% of the eye's refractive power. However, for millions, tear film instability produces uncomfortable sight-impairing dry eye. Despite the multitude of etiologies for dry eye, a universal hallmark is hyperosmolarity of the tear film. Presently, knowledge of how the ocular surface responds to hyperosmolarity remains incomplete with little understood about the role of ion channels. This bioelectric analysis focused on conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to dry eye. In freshly excised rat conjunctiva, perforated-patch recordings demonstrated that a ≥10% rise in osmolarity triggers goblet cells to rapidly generate a ~15-mV hyperpolarization due to the oxidant-dependent activation of ATP-sensitive K+ (KATP) channels. High-resolution membrane capacitance measurements used to monitor exocytosis revealed that this hyperpolarization results in an approximately fourfold boost in exocytotic activity evoked by cholinergic input, which in vivo occurs via a neural reflex and depends chiefly on calcium influxing down its electro-gradient. We discovered that this adaptive response is transient. During 30-80 min of hyperosmolarity, development of a depolarizing nonspecific cation conductance fully counterbalances the KATP-driven hyperpolarization and thereby eliminates the exocytotic boost. We conclude that hyperosmotic-induced hyperpolarization is a previously unappreciated mechanism by which goblet cells respond to transient ocular dryness. Loss of this voltage increase during long-term dryness/hyperosmolarity may account for the clinical conundrum that goblet cells in chronically dry eyes can remain filled with mucin even though the tear film is hyperosmotic and mucin-deficient.

The uptake, retention and clearance of drug-loaded dendrimer nanoparticles in astrocytes - electrophysiological quantification.

Nanoparticle-based drug delivery systems may impose risks to patients due to potential toxicity associated with a lack of clearance from cells or prolonged carrier-cell retention. This work evaluates vesicular cell uptake, retention and the possible transfer of endocytosed methylprednisolone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (NPs) into secretory vesicles of rat cultured astrocytes. The cells were incubated with NPs and unitary vesicle fusions/fissions with the plasma membrane were monitored employing high-resolution membrane capacitance measurements. In the NP-treated cells the frequency of unitary exocytotic events was significantly increased. The presence of NPs also induces an increase in the size of exocytotic vesicles interacting with the plasma membrane, which exhibit transient fusion with prolonged fusion pore dwell-time. Live-cell confocal imaging revealed that once NPs internalize into endocytotic compartments they remain in the cell for 7 days, although a significant proportion of these merge with secretory vesicles destined for exocytosis. Co-localization studies show the route of clearance of NPs from cells via the exocytotic pathway. These findings bring new insight into the understanding of the intracellular trafficking and biological interactions of drug-loaded dendrimer NPs targeting astrocytes.

High-resolution membrane capacitance measurements for studying endocytosis and exocytosis in yeast.

Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell-attached configuration of the patch-clamp techniques we were able to resolve the elementary processes of endo- and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2-1 fF which translates to vesicle diameters of 90-200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo- and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.

Molecular Mechanisms of Endocytosis and Exocytosis in Yeast Studied by High-Resolution Membrane Capacitance Measurements

Exo- and endocytosis are crucial in numerous physiological processes in eukaryotic cells, such as polar growth, cell division, motility or signaling. Endo- as well as exocytotic events result in small changes of the cell surface area, which can be resolved in electrophysiological recordings in the form of discrete steps in membrane capacitance. By applying high resolution recordings with yeast protoplasts, we were able to resolve discrete changes in membrane capacitance mainly in the range 0.2-1.2fF, corresponding to fusion and fission of single vesicles of 80-250nm. Analysis of exo- and endocytosis in the yeast sec6-4 mutant, which is characterized by a temperature dependent block of exocytosis, revealed low fission/fusion activity at room temperature. Incubation at the restrictive temperature of 37°C for 3h and shifting to the permissive temperature of 25°C resulted in a transient increase in endo-/exocytotic activity of up to 10-fold. This study demonstrates that patch clamp capacitance measurements in yeast provide a novel in vivo tool to examine the molecular mechanisms of endo-/exocytosis in this model organism. The recordings gain information in real time on the geometry of endo-/exocytotic vesicles, on the kinetics of the events and their dependence on signaling cascades and key proteins.

High-resolution membrane capacitance measurements for the study of exocytosis and endocytosis

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (C(m)), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution C(m) measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk C(m) changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of C(m) with attofarad resolution and allows characterization of fusion pore properties. Although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. After initial hardware setup and culture preparation, several C(m) measurements can be performed daily.

P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells

Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.