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M Lindau

Publications and source records attributed to M Lindau.

At least 37 records · Page 2Linked to original sources
PubMed1995

Regulation of granule size in human and horse eosinophils by number of fusion events among unit granules.

1. We have investigated the granule size distributions in human and horse eosinophils by time-resolved patch-clamp capacitance measurements. 2. During exocytosis of single granules the electrical capacitance of the plasma membrane increases in discrete steps. The steps in horse cells are about six times larger than those in human cells in accordance with the difference in granule size. 3. In both species a multimodal capacitance step size distribution is observed with a first peak at 6-7 fF corresponding to granules with a diameter of about 450-500 nm and a surface area of about 0.7 microns2, which we call the unit granule. The other peaks in the distributions correspond to multiples of the surface area of these units. 4. These results show that the larger granules are formed by fusion of several unit granules and the final size of mature granules is determined by the number of units allowed to fuse with each other. Whereas in human eosinophils most granules consist of one or two units, most granules of horse eosinophils are formed by fusion of seven to fifteen units. 5. The intracellular fusion events associated with vesicular traffic are believed to occur constitutively. In contrast, our results indicate that a cellular mechanism exists which regulates the size of the mature granules by determining the number of units allowed to fuse with each other. In view of our recent report that granule-granule fusion can be activated by GTP gamma S, this regulation may possibly involve GTP-binding proteins.

Animals↗
PubMed1995

Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion.

Several proteins involved in exocytosis have been identified recently, but it is still completely unclear which molecules perform the fusion event itself. Although in viral fusion the fusion proteins are known, even there the molecular mechanism remains controversial. Investigation of single fusion events by electrophysiological techniques together with fluorimetric measurements have now provided some insight into the properties of the first aqueous connection, the fusion pore. This pore has an initial size similar to an ion channel and allows movement of lipids only after it has substantially expanded, indicating that it is initially not a purely lipidic structure, but incorporates lipids when it expands. Although neurotransmitter release may occur through narrow transient fusion pores, the fusion pore of synaptic vesicles probably expands vey rapidly, making it unlikely that secretion is performed by rapid exo/endocytosis without full fusion under normal conditions. Recent recordings from small membrane patches have made it possible to resolve fusion events from vesicles as small as synaptic vesicles. Future experiments using excised patches may provide an approach to identify the molecular machinery of exocytotic membrane fusion.

Animals↗
PubMed1995

Influence of conductance changes on patch clamp capacitance measurements using a lock-in amplifier and limitations of the phase tracking technique.

We characterized the influence of conductance changes on whole-cell patch clamp capacitance measurements with a lock-in amplifier and the limitations of the phase-tracking method by numerical computer simulations, error formulas, and experimental tests. At correct phase setting, the artifacts in the capacitance measurement due to activation of linear conductances are small. The cross talk into the capacitance trace is well approximately by the second-order term in the Taylor expansion of the admittance. In the case of nonlinear current-voltage relationships, the measured conductance corresponds to the slope conductance in the range of the sine wave amplitude, and the cross talk into the capacitance trace corresponds to the second-order effect of the slope conductance. The finite gating kinetics of voltage-dependent channels generate phase-shifted currents. These lead to major artifacts in the capacitance measurements when the angular frequency of the sine wave is close to the kinetic rate constant of the channel. However, when the channel kinetics are sufficiently slow, or sufficiently fast, the cross talk is still close to the second-order effect of the measured conductance. The effects of activation of voltage-dependent currents on the capacitance measurements may be estimated, provided a detailed characterization of the kinetics and voltage dependence is available. A phase error of the lock-in amplifier of a few degrees leads to significant projections. The phase-tracking method can be used to keep the phase aligned only during periods of low membrane conductance. However, nonideal properties of the equivalent circuit, in particular the fast capacitance between the pipette and the bath solutions, may lead to large phase errors when the phase-tracking method is used, depending on the electrical properties of the cell. In this article we provide practical values, setting the range where possible artifacts are below defined limits. For proper evaluation of capacitance measurements, the capacitance and conductance traces should always be displayed together.

Animals↗
PubMed1995

The exocytotic fusion pore of small granules has a conductance similar to an ion channel.

We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex.

Cytoplasmic Granules↗
PubMed1994

Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.

An increase in free Ca2+ triggers exocytosis in pituitary nerve terminals leading to an increase in membrane area and membrane capacitance. When Ca2+ is increased by step depolarization, an instantaneous capacitance increase during the first 80 ms is followed by a slow increase extending over several seconds. We measured capacitance changes associated with exocytosis and endocytosis in single pituitary nerve terminals internally perfused with high Ca2+. At 50 microM Ca2+ the capacitance increased by up to 2%/s, similar to the slow phase observed during depolarization. Our results indicate that at the site of fusion very high Ca2+ is required. Following exocytosis, large downward capacitance steps were measured, reflecting endocytosis of large vacuoles. These events were not abrupt but reflected a gradual decrease of fission pore conductance from 8 nS to < 40 pS during 500 ms, revealing the dynamics of individual fission pore closures. Above 300 pS, narrowing of the endocytotic fission pore was approximately 10 times slower than the previously reported expansion of the exocytotic fusion pore. The transition between 300 pS and 0 pS took approximately 200 ms, whereas it has been reported that the exocytotic fusion pore measured in mast cells opens from 0 to 280 pS in < 100 microseconds. The time course of closing of the fission pore may be explained by an exponential decrease in pore diameter occurring at a constant rate.

Animals↗
PubMed1994

Compound exocytosis and cumulative degranulation by eosinophils and their role in parasite killing.

The killing of metazoan parasitic larvae by eosinophils occurs following cell adhesion and the secretion o f their cytotoxic proteins onto the surface o f these targets. In eosinophils, as in mast cells and neutrophils, stimulus-secretion coupling is mediated by GTP-binding proteins. In this article, Susanne Scepek Redwon Moqbel and Manfred Lindou summarize recent results indicating that the granule-fusion events activated by GTP-binding proteins lead to compound exocytosis and cumulative fusion. They propose that these exocytotic processes, following contact with opsonized larvae, may direct secretion to a restricted space defined by the site o f contact Such a focused release may be essential for effective targeting in parasite killing, thus preventing uncontrolled random diffusion of the secreted cytotoxic proteins with the possible undesirable consequences of damage to intact host tissue.

Journal Article↗
PubMed1994

Chloride channels in mast cells: block by DIDS and role in exocytosis.

In rat peritoneal mast cells, we have investigated the influence of the chloride transport blocker 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and the extracellular chloride concentration on the chloride current induced by intracellular application of cyclic AMP (cAMP) and on hexosaminidase secretion from intact cells stimulated with compound 48/80. The inhibition of the Cl-current by extracellular DIDS is voltage and time dependent. Upon depolarization from -10 to +70 mV, the outward current diminishes with millisecond kinetics. The size of the steady state current and the time constant of the decrease both decrease with increasing DIDS concentrations. The steady state current at +70 mV is blocked by DIDS with an IC50 of 2.3 microM. The number of open channels at -10 mV is reduced with an IC50 of 22 microM. The electrophysiological and pharmacological properties of this current are most similar to those of the Cl- current in T lymphocytes activated by osmotic stress (Lewis, R. S., P. E. Ross, and M. D. Cahalan. 1993. Journal of General Physiology. 101:801-826). Extracellular DIDS also inhibits exocytosis. At optimal stimulation with 10 micrograms/ml compound 48/80 secretion is inhibited with an IC50 = 50 microM and a Hill coefficient n = 10. At half optimal stimulation with 1 microgram/ml inhibition occurs with an IC50 = 10 microM and n = 1. Substitution of extracellular chloride by glutamate has only very small effects on secretion stimulated with 10 micrograms/ml compound 48/80. We conclude that activation of the chloride current in mast cells is not essential for stimulation of exocytosis but may enhance secretion at suboptimal stimulation. Alternatively, the channel may play a role in volume regulation following degranulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Focal exocytosis by eosinophils--compound exocytosis and cumulative fusion.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.

Animals↗
PubMed1993

The calcium signal in human neutrophils and its relation to exocytosis investigated by patch-clamp capacitance and Fura-2 measurements.

Intracellular calcium ([Ca2+]i) and exocytosis of human neutrophils were investigated with patch-clamp capacitance and Fura-2 fluorescence measurements. Intracellular application of GTP gamma S induces a calcium transient and exocytosis. The onset of degranulation occurs at the time where the maximal [Ca2+]i is reached. Despite the close correlation in time, buffering [Ca2+]i at the resting level or at approximately 2 microM leaves the extent and the time course of degranulation unchanged. The decay of the calcium transient is due to diffusional equilibration between the cytosol and the pipette volume. GTP gamma S activates no cellular mechanisms for Ca2+ reuptake or extrusion. The endogenous calcium buffer capacity can be estimated to be as low as that of approximately 90 microM Fura-2. Stimulation with fMLP also induces degranulation and a calcium transient. The decay of fMLP-induced calcium transients is much faster than that of GTP gamma S-induced transients and is independent of diffusion indicating that fMLP also induces rapid reuptake or extrusion of Ca2+. Degranulation but not the calcium transient requires the presence of intracellular GTP. Different signalling pathways appear to be involved in GTP gamma S- and fMLP-stimulated calcium signals. The intracellular calcium release is not an essential signal to initiate exocytosis in neutrophils.

Calcium↗
PubMed1993

Individual differences in vowel production.

It is often assumed that a relatively small set of articulatory features are universally used in language sound systems. This paper presents a study which tests this assumption. The data are x-ray microbeam pellet trajectories during the production of the vowels of American English by five speakers. Speakers were consistent with themselves from one production of a word to the next, but the articulatory patterns manifested by this homogeneous group were speaker specific. Striking individual differences were found in speaking rate, the production of the tense/lax distinction of English, and in global patterns of articulation. In terms of a task-dynamic model of speech production, these differences suggested that the speakers used different gestural target and stiffness values, and employed different patterns of interarticulator coordination to produce the vowels of American English. The data thus suggest that, at some level of speech motor control, speech production tasks are specified in terms of acoustic output rather than spatiotemporal targets or gestures.

Female↗
PubMed1993

The membrane fusion events in degranulating guinea pig eosinophils.

We have investigated the granule fusion events associated with exocytosis in degranulating peritoneal guinea pig eosinophils by time-resolved patch-clamp capacitance measurements using the phase detector technique. Intracellular stimulation with micromolar calcium and GTP gamma S induces a 2- to 3-fold capacitance increase. The main phase of the capacitance increase occurs after a delay of 2-7 minutes and is composed of well-resolved capacitance steps. The number of steps is very close to the number of crystalloid granules contained in a resting cell and the step size distribution with a peak at 9 fF is in excellent agreement with the granule size distribution determined by electron microscopy. The individual granules thus fuse sequentially with the plasma membrane. The stepwise capacitance increase is frequently preceded by an apparently continuous capacitance increase which consists of steps smaller than 4 fF, indicating exocytosis of small vesicles as distinct from crystalloid-containing granules. In some cases the time course of the opening of individual fusion pores could be recorded, and this revealed metastable conductance states below 300 pS but random fluctuations at higher conductance levels. This behaviour suggests that the small fusion pore might be a protein structure similar to an ion channel, which becomes a continuously variable lipid pore at higher conductances. In some cells a significant capacitance decrease was observed which is apparently continuous, suggesting a process of membrane uptake by endocytosis of small vesicles.

Animals↗
PubMed1992

Depolarization, intracellular calcium and exocytosis in single vertebrate nerve endings.

We have investigated the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve terminals. The change of [Ca2+]i and vasopressin release were measured with a time resolution of less than 1 s in response to K(+)-induced depolarization. Exocytosis was also monitored in the whole-terminal patch-clamp configuration by time resolved capacitance measurements while [Ca2+]i was simultaneously followed by fura-2 fluorescence measurements. In intact as well as patch-clamped nerve terminals sustained depolarization leads to a sustained rise of [Ca2+]i. The rate of vasopressin release from intact nerve terminals rises in parallel with [Ca2+]i but then declines rapidly towards basal (t1/2 approximately 15 s) despite the maintained high [Ca2+]i indicating that only a limited number of exocytotic vesicles can be released. We demonstrate that in nerve terminals exocytosis can be followed during step depolarization by capacitance measurements. The capacitance increase starts instantaneously whereas [Ca2+]i rises with a half time of several hundred milliseconds. An instantaneous steep capacitance increase is followed by a slow increase with a slope of 25-50 fF/s indicating the sequential fusion of predocked and cytoplasmic vesicles. During depolarization the capacitance slope declines to zero with a similar time course as the vasopressin release indicating a decrease in exocytotic activity. Depolarization per se in the absence of a sufficient rise of [Ca2+]i does not induce exocytosis but elevation of [Ca2+]i in the absence of depolarization is as effective as in its presence. The experiments suggest that a rapid rise of [Ca2+]i in a narrow region beneath the plasma membrane induces a burst of exocytotic activity preceding the elevation of bulk [Ca2+]i in the whole nerve terminal.

Animals↗
PubMed1992

Sodium, calcium and exocytosis: confessions of calcified scientists.

Stimulated exocytotic secretion from nerve endings is initiated by an increase in intracellular free calcium concentration. We summarize here our latest findings regarding the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve endings. In addition, we present surprising findings for a regulatory role of intracellular Na+ on exocytosis.

Animals↗
PubMed1990

Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils.

The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.

Animals↗