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M Heidecker

Publications and source records attributed to M Heidecker.

10 recordsLinked to original sources
PubMed2003

Impact of hypoosmotic challenges on spongy architecture of the cytoplasm of the giant marine alga Valonia utricularis.

The ultrastructure of the several micrometers thick cytoplasmic layer of the giant marine alga Valonia utricularis displays characteristics which are apparently linked with the capability of this alga to regulate turgor pressure. Transmission and scanning electron microscopy of cells prefixed in different ways, including a protocol that allows prefixation of the alga in a turgescent state, revealed a highly dendritic network of cytoplasmic strands connecting and enveloping the chloroplasts and the nuclei. Innumerable vacuolar entities are embedded in the network, giving the cytoplasm a spongy appearance. Vacuolar perfusion of turgor-pressure-clamped cells with prefixation solution containing tannic acid presented evidence that these vacuolar entities together with the huge central vacuole form a large unstirred continuum. In contrast to the tonoplast, the plasmalemma followed smoothly the lining of the cell wall, even at the numerous cell wall ingrowths. Sucrose, but not polyethylene glycol 6000, induced chloroplast clustering. Acute hypoosmotic treatment (established by reduction of external NaCl or by replacement of part of the external NaCl by equivalent osmotic concentrations of sucrose or polyethylene glycol 6000) resulted in a local relocation of the chloroplasts and cytoplasm towards the central vacuole. This effect did not occur when the relatively low reflection coefficients of these two osmolytes were taken into account. The increase in spacing between the spongy cytoplasm and the plasmalemma by chloroplast relocation (viewed by confocal laser scanning microscopy) was associated with a speckled appearance of the affected surface area under the light microscope. As indicated by electron microscopy, hypoosmotically induced chloroplast relocation resulted from disproportionate swelling of the vacuolar entities located close to the plasmalemma. The cytoskeleton in the cytoplasm and the mucopolysaccharide network in the central vacuole apparently resisted swelling of these compartments. This finding has the important consequence that relevant hydrostatic pressure gradients can be built up throughout the entire multifolded vacuolar space. This gradient could represent the trigger for turgor pressure regulation which is manifested electrically first in the tonoplast.

Chlorophyta↗
PubMed2003

Structural peculiarities dominate the turgor pressure response of the marine alga Valonia utricularis upon osmotic challenges.

The pressure response of (plant) cells to osmotic challenges depends on the reflection coefficient, sigma, of osmotically active solutes; it is less than predicted by the van't Hoff equation if sigma < 1. In Valonia utricularis, sigma is significantly reduced by internal (and, to a lesser extent, by external) unstirred layers, protecting the cytoplasm against vacuolar osmotic fluctuations. As shown by scanning and transmission electron microscopy, diffusion-restricted spaces are formed by innumerable small vacuoles that are interconnected with each other and with the central vacuole. They are embedded in networks of cytoplasmic strands connecting and encircling the organelles. Unstirred layers are also created in the central vacuole by an extensive network of acid mucopolysaccharide filaments (visualized by alcian blue staining). Mucopolysaccharides apparently also affect steady-state turgor by reducing the water activity. When the effective vacuolar osmotic pressure was adjusted to that of the bath by perfusion with an artificial vacuolar sap (AVS), an "offset turgor pressure" of 17 +/- 5 kPa was recorded. Consistent with the ultrastructural data, sigma values less than unity were calculated from the pressure response upon vacuolar addition of KCl or sucrose by perfusion (sigma(iKCl) = 0.63 +/- 0.13; sigma(isuc) = 0.58 +/- 0.17). Dilution of AVS yielded slightly higher sigma(iKCl) values (0.73 +/- 0.35). External addition to the artificial sea water (ASW) indicated that sigma(e) > sigma(i) for these osmotica. However, even in this case, sigma(esuc) (0.86 +/- 0.09) and sigma(ePEG) (0.58 +/- 0.08) were significantly less than sigma(eNaCl) (0.94 +/- 0.05) and sigma(eKCl) (0.91 +/- 0.13), presumably due to unstirred layers within the 4 micro m thick cell wall. Consistent with the low sigma values, a partial replacement of NaCl by osmotically equivalent amounts of sucrose (ASW(suc)), PEG and dextran, respectively, as well as replacement of Cl(-) by the large anion MES(-) induced an 'anomalous' hyposmotic turgor pressure response followed by the usual backregulation of pressure. After a 2-day preincubation in ASW(suc), significantly lower sigma(e) values were obtained both hyperosmotically (sigma(eNaCl) = 0.78 +/- 0.14; sigma(esuc) = 0.72 +/- 0.15) and hyposmotically (sigma(eNaCl) = 0.70 +/- 0.17; sigma(esuc) = 0.63 +/- 0.09), probably due to long-term effects on membrane structure to be elucidated yet. The freshwater alga Chara corallina lacked these apparently closely related structural and biophysical features of Valonia.

Cell Membrane↗
PubMed2003

Gating of Cl- currents in protoplasts from the marine alga Valonia utricularis depends on the transmembrane Cl- gradient and is affected by enzymatic cell wall degradation.

The electrical properties of protoplasts of the turgor pressure-regulating giant marine alga Valonia utricularis were investigated by using the patch-clamp technique. In the whole-cell configuration, large inward currents were elicited by negative-going voltage pulses. The time-dependent component was predominantly carried by Cl-, as revealed by 'tail current' analysis. When experiments were performed on protoplasts directly after mechanical release from the 'mother cell', small outward currents were additionally observed at membrane voltages more positive than ECl-. These outward currents disappeared to a large extent after treatment of the protoplasts with a mixture of cell wall-degrading enzymes. Plots of the chord conductance versus the clamped membrane voltage revealed that enzymatic treatment affected the gating properties. By fitting Boltzmann distributions to the data, a midpoint potential of + 5 +/- 5 mV (n = 7) was obtained in symmetrical Cl- solutions for mechanically released protoplasts. In contrast, protoplasts treated additionally with enzymes exhibited a midpoint potential of -13 +/- 5 mV (n = 8). By varying the external and internal Cl- concentration, gating was also shown to depend on the Cl- gradient across the plasmalemma both in enzymatically treated and untreated protoplasts. Plotting of the midpoint potential against the Nernst potential of Cl- rendered a slope less than 1 (0.70 and 0.64, respectively) indicating that gating did not strictly depend on the electrochemical Cl- gradient. The voltage- and Cl--dependence as well as inhibition experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) suggested that the Cl- conductance of the membrane is dominated by the Valonia Anion Channel 1 (VAC1) described by Heidecker, M., Wegner, L.H., Zimmermann, U. 1999: A patch-clamp study of ion channels in proto-plasts prepared from the marine alga Valonia utricularis. J. Membrane Biol. 172:235-247. The relevance of the findings for membrane potential control and turgor regulation in V. utricularis as well as the general implications of the data for electrophysiological work on protoplasts (that are usually obtained by enzymatic digestion of plant tissue) are discussed.

Cells, Cultured↗
PubMed1999

A patch-clamp study of ion channels in protoplasts prepared from the marine alga Valonia utricularis.

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant "mother cells" (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123-134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl(-) channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K(+) channel (VKC1, Valonia K(+) Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl(-) media, VAC1 was most frequently observed and had a single channel conductance of 36 +/- 7 pS (n = 4) in the outside-out and 33 +/- 5 pS (n = 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 +/- 4 mV (n = 4, outside-out) and 9 +/- 7 mV (n = 10, cell-attached) close to the Nernst potential of Cl(-) and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl(-) media (bath/pipette; E(Cl(-)) -20 +/- 7 mV (n = 4); Nernst potential -28 mV). Consistent with a selectivity for Cl(-), VAC1 was inhibited by 100 micronM DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl(-) conditions yielded a midpoint potential of -12 +/- 5 mV (n = 4, outside-out) and -3 +/- 6 mV (n = 9, cell-attached) and corresponding apparent minimum gating charges of 15 +/- 3 (n = 4) and 18 +/- 5 (n = 9). The midpoint potential shifted to more negative values in the presence of a Cl(-) gradient. VAC2 was activated by voltages more negative than E(Cl(-)) and was always observed together with VAC1, but less frequently. It showed a "flickering" gating. The single channel conductance was 99 +/- 10 pS (n = 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 +/- 5 pS (n = 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 +/- 27 pS (n = 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl(-) conductance of the plasmalemma of the turgescent "mother cells" and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein.

BACKGROUND: The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a 'leading edge' of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum. RESULTS: Purified GFP-actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10-20% of the total actin. Microfilaments containing GFP-actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP-actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP-actin and phalloidin fluorescence, but there were distinct sites in which GFP-actin was more prominent. CONCLUSIONS: Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

Actin Cytoskeleton↗
PubMed1997

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.

Actins↗
PubMed1996

Light-directed generation of the actin-activated ATPase activity of caged heavy meromyosin.

An understanding of the molecular mechanism of muscle contraction will require a complete description of the kinetics of the myosin motor in vitro and in vivo. To this end chemical relaxation studies employing light-directed generation of ATP from caged ATP have provided detailed kinetic information in muscle fibers. A more direct approach would be to trigger the actin-activated ATPase activity from a caged myosin, i.e., myosin whose activity is blocked upon derivatization with a photolabile protection group. Herein we report that a new type of caged reagent can be used to prepare a caged heavy meromyosin by modification of critical thiol groups, i.e., a chemically modified motor without activity that can be reactivated at will using a pulse of near-ultraviolet light. Heavy meromyosin modified at Cys-707 with the thiol reactive reagent 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene does not exhibit an actin-activated ATPase activity and may be viewed as a caged protein. Absorption spectroscopy showed that the thioether bond linking the cage group to Cys-707 is cleaved following irradiation (340-400 nm) via a transient aci-nitro intermediate which has an absorption maximum at 440 nm and decays with a rate constant of 45.6 s(-1). The in vitro motility assay showed that caged heavy meromyosin cannot generate the force necessary to move actin filaments although following irradiation of the image field with a 30 ms pulse of 340-400 nm light the caged group was removed with the concomitant movement of most filaments at a velocity of 0.5-2 micron/s compared to 3-4 micron/s for unmodified HMM. The specificity and simplicity of labeling myosin with the caged reagent should prove useful in studies of muscle contraction in vivo.

Actins↗
PubMed1995

Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin.

Distance relationships between phalloidin binding sites on F-actin have been investigated using fluorescence resonance energy transfer (FRET) techniques in solution and on single F-actin filaments bound to heavy meromyosin (HMM). Filaments saturated with an equimolar concentration of fluoresceinisothiocyanatophalloidin (FITC-ph) as the donor and tetramethylrhodamineisothiocyanatophalloidin (TRITC-ph) as the acceptor and control filaments saturated with either FITC-ph or TRITC-ph were characterized by absorption and fluorescence spectroscopy and the in vitro motility assay. Fluorescence excitation polarization spectroscopy showed hetero-FRET occurred within colabeled filaments, whereas homo-FRET was observed in control filaments. The distance measured between adjacent phalloidin binding sites using randomly labeled FITC-ph and TRITC-ph was 37.2 A using steady-state spectral analysis and 36.9 A using time-resolved spectroscopy with a radial coordinate of 14.5 A. Measurements of the distance between fluorescent phalloidin groups using the atomic model of F-actin [Lorenz, M., Popp, D., & Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836] suggest transfer occurs between adjacent phalloidin molecules on opposite actin helices (39 A), rather than between adjacent phalloidins along the same actin helix (55.4 A). A quantitative fluorescence microscope technique was described that measures the proximity of adjacent FITC-ph and TRITC-ph on single filaments immobilized on HMM. Here a distance of 36.2 A was calculated which was unchanged during ATP-dependent sliding of F-actin on HMM. Spatially resolved FRET measurements are being used to observe the effects of actin binding proteins on the structural properties along the length of single actin filaments.

Actins↗
PubMed1994

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found. Through the coupling of SBMC to streptavidin,a plethora of biotin-based tracer molecules are available for immunocytochemical studies.

Animals↗