Biomedical subjects

Helmut Plattner

Publications and source records attributed to Helmut Plattner.

29 records · Page 2Linked to original sources

PubMed2004

Acidocalcisomes of Phytomonas françai possess distinct morphological characteristics and contain iron.

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites, and recently found in other unicellular eukaryotes. The aim of this study was to identify the presence of acidocalcisomes in the plant trypanosomatid Phytomonas françai. Electron-dense organelles of P. françai were shown to contain large amounts of oxygen, sodium, magnesium, phosphorus, potassium, calcium, iron, and zinc as determined by X-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation or by elemental mapping. The presence of iron is not common in other acidocalcisomes. In situ, but not when purified, these organelles showed an elongated shape differing from previously described acidocalcisomes. However, these organelles also possessed a vacuolar H+-pyrophosphatase (V-H+-PPase) as determined by biochemical methods and by immunofluorescence microscopy using antibodies against the enzyme. Together, these results suggest that the electron-dense organelles of P. françai are homologous to the acidocalcisomes described in other trypanosomatids, although with distinct morphology and elemental content.

Animals↗

PubMed2004

Immunolocalization of actin in Paramecium cells.

We have selected a conserved immunogenic region from several actin genes of Paramecium, recently cloned in our laboratory, to prepare antibodies for Western blots and immunolocalization. According to cell fractionation analysis, most actin is structurebound. Immunofluorescence shows signal enriched in the cell cortex, notably around ciliary basal bodies (identified by anti-centrin antibodies), as well as around the oral cavity, at the cytoproct and in association with vacuoles (phagosomes) up to several mum in size. Subtle strands run throughout the cell body. Postembedding immunogold labeling/EM analysis shows that actin in the cell cortex emanates, together with the infraciliary lattice, from basal bodies to around trichocyst tips. Label was also enriched around vacuoles and vesicles of different size including "discoidal" vesicles that serve the formation of new phagosomes. By all methods used, we show actin in cilia. Although none of the structurally well-defined filament systems in Paramecium are exclusively formed by actin, actin does display some ordered, though not very conspicuous, arrays throughout the cell. F-actin may somehow serve vesicle trafficking and as a cytoplasmic scaffold. This is particularly supported by the postembedding/EM labeling analysis we used, which would hardly allow for any large-scale redistribution during preparation.

Actins↗

PubMed2003

Dense-core secretory vesicle docking and exocytotic membrane fusion in Paramecium cells.

Work with Paramecium has contributed to the actual understanding of certain aspects of exocytosis regulation, including membrane fusion. The system is faster and more synchronous than any other dense-core vesicle system described and its highly regular design facilitates correlation of functional and ultrastructural (freeze-fracture) features. From early times on, several crucial aspects of exocytosis regulation have been found in Paramecium cells, e.g. genetically controlled microdomains (with distinct ultrastructure) for organelle docking and membrane fusion, involvement of calmodulin in establishing such microdomains, priming by ATP, occurrence of focal fusion with active participation of integral and peripheral proteins, decay of a population of integral proteins ("rosettes", mandatory for fusion capacity) into subunits and their lateral dispersal during fusion, etc. The size of rosette particles and their dispersal upon focal fusion would be directly compatible with proteolipid V(0) subunits of a V-ATPase, much better than the size predicted for oligomeric SNARE pins (SCAMPs are unknown from Paramecium at this time). However, there are some restrictions for a straightforward interpretation of ultrastructural results. The rather pointed, nipple-like tip of the trichocyst membrane could accommodate only one (or very few) potential V(0) counterpart(s), while the overlaying domain of the cell membrane contains numerous rosette particles. Particle size is compatible with V(0), but larger than that assumed for the SNARE complexes. When membrane fusion is induced in the presence of antibodies against cell surface components, focal fusion is seen to occur with dispersing rosette particles but without dispersal of their subunits and without pore expansion. Clearly, this is required for completing fusion and pore expansion. After cloning SNARE and V(0) components in Paramecium (with increasing details becoming rapidly available), we may soon be able to address the question more directly, whether any of these components or some new ones to be detected, serve exocytotic and/or any other membrane fusions in Paramecium.

Animals↗

PubMed2003

Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells.

Hematopoietic cells have long been defined as round, nonpolar cells that show uniform distribution of cell surface-associated molecules. However, recent analyses of the immunological synapse and the importance of lipid microdomains in signaling have shed new light on the aspect of lymphocyte polarization during the activation processes, but none of the molecules implicated so far in either the activation process or the microdomain residency are known to have a preferential localization in nonactivated cells. Chemical crosslinking and fluorescence resonance energy transfer methods have allowed the visualization of certain glycosylphosphatidylinositol-anchored proteins in lipid rafts but so far no microdomain resident protein has been shown to exist as visible stable platforms in the membrane. We report here that two lipid microdomain resident proteins, flotillins/reggies, form preassembled platforms in hematopoietic cells. These platforms recruit signaling molecules upon activation through lipid rafts. The preassembled platforms significantly differ from the canonical cholesterol-dependent "lipid rafts," as they are resistant to cholesterol-disrupting agents. Most evidence for the functional relevance of microdomains in living cells remains indirect. Using laser scanning confocal microscopy, we show that these proteins exist as stable, microscopically patent domains localizing asymmetrically to one pole of the cell. We present evidence that the asymmetric concentration of these microdomain resident proteins is built up during cytokinesis.

Antigens, CD↗

PubMed2003

Molecular aspects of membrane trafficking in paramecium.

Results achieved in the molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum --> Golgi --> lysosomes or secretory vesicles; endo- and phagosomes --> lysosomes/digesting vacuoles), but also of some unique features, e.g. and elaborate phagocytic route with the cytoproct and membrane recycling to the cytopharynx, as well as the osmoregulatory system with multiple membrane fusion sites. Exocytosis sites for trichocysts (dense-core secretory vesicles), parasomal sacs (coated pits), and terminal cisternae (early endosomes) display additional regularly arranged predetermined fusion/fission sites, which now can be discussed on a molecular basis. Considering the regular, repetitive arrangements of membrane components, availability of mutants for complementation studies, sensitivity to gene silencing, and so on, Paramecium continues to be a valuable model system for analyzing membrane interactions. This review intends to set a new baseline for ongoing work along these lines.

Animals↗

PubMed2003

Molecular identification of a calcium-inhibited catalytic subunit of casein kinase type 2 from Paramecium tetraurelia.

We have previously described the occurrence in Paramecium of a casein kinase (CK) activity (EC 2.7.1.37) with some unusual properties, including inhibition by Ca(2+) (R. Kissmehl, T. Treptau, K. Hauser, and H. Plattner, FEBS Lett. 402:227-235, 1995). We now have cloned four genes, PtCK2alpha1 to PtCK2alpha4, all of which encode the catalytic alpha subunit of type 2 CK (CK2) with calculated molecular masses ranging from 38.9 to 39.4 kDa and pI values ranging from 8.8 to 9.0. They can be classified into two groups, which differ from each other by 28% on the nucleotide level and by 18% on the derived amino acid level. One of them, PtCK2alpha3, has been expressed in Escherichia coli and characterized in vitro. As we also have observed with the isolated CK, the recombinant protein preferentially phosphorylates casein but also phosphorylates some Paramecium-specific substrates, including the exocytosis-sensitive phosphoprotein pp63/parafusin. Characteristically, Ca(2+) inhibits the phosphorylation at elevated concentrations occurring during stimulation of a cell. Reconstitution with a recombinant form of the regulatory subunit from Xenopus laevis, XlCK2beta, confirms Ca(2+) sensitivity also under conditions of autophosphorylation. This is unusual for CK2 but correlates with the presence of two EF-hand calcium-binding motifs, one of which is located in the N-terminal segment essential for constitutive activity, as well as with an aberrant composition of normally basic domains recognizing acidic substrate domains. Immunogold localization reveals a considerable enrichment in the outermost cell cortex layers, excluding cilia. We discuss a potential role of this Ca(2+)-inhibited PtCK2alpha species in a late step of signal transduction.

Amino Acid Sequence↗

PubMed2002

NSF regulates membrane traffic along multiple pathways in Paramecium.

N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells. In the ciliated protozoon Paramecium, complex but well-defined transport routes exist, constitutive and regulated exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through contractile vacuoles, that can all be studied independently at the whole cell level. To unravel the role of NSF and of the SNARE machinery in this complex traffic, we looked for NSF genes in Paramecium, starting from a partial sequence found in a pilot random sequencing project. We found two very similar genes, PtNSF1 and PtNSF2, which both seem to be expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band. PtNSF gene silencing results in decreasing phagocytotic activity, while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly attached at the cell membrane, persists. Ultrastructural analysis of silenced cells shows deformation or disappearance of structures involved in membrane traffic. Aggregates of numerous small, smooth vesicles intermingled with branches of ER occur in the cytoplasm and are most intensely labeled with anti-NSF Ab-gold. Furthermore, elongated vesicles of approximately 30 nm diameter can be seen attached at cortical calcium storage compartments, the alveolar sacs, whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some low frequency fusion events was visualized in non-silenced cells by immuno-fluorescence, after cautious permeabilization in the presence of ATP-gamma-S and NEM. Our data document that PtNSF is involved in distinct pathways of vesicle traffic in Paramecium and that actual sensitivity to silencing is widely different, apparently dependent on the turnover of membrane-to-membrane attachment formation.

Amino Acid Sequence↗

PubMed2002

Crystal structure analysis of the exocytosis-sensitive phosphoprotein, pp63/parafusin (phosphoglucomutase), from Paramecium reveals significant conformational variability.

During exocytosis of dense-core secretory vesicles (trichocysts) in Paramecium, the protein pp63/parafusin (pp63/pf) is transiently dephosphorylated. We report here the structures of two crystal forms of one isoform of this protein which has a high degree of homology with rabbit phosphoglucomutase, whose structure has been reported. As expected, both proteins possess highly similar structures, showing the same four domains forming two lobes with an active-site crevice in between. The two X-ray structures that we report here were determined after crystallization in the presence of sulfate and tartrate, and show the lobes arranged as a closed and an open conformation, respectively. While both conformations possess a bound divalent cation, only the closed (sulfate-bound) conformation shows bound sulfate ions in the "phosphate-transfer site" near the catalytic serine residue and in the "phosphate-binding site". Comparison with the open form shows that the latter dianion is placed in the centre of three arginine residues, one contributed by subunit II and two by subunit IV, suggesting that it causes a contraction of the arginine triangle, which establishes the observed conformational closure of the lobes. It is therefore likely that the closed conformation forms only when a phosphoryl group is bound to the phosphate-binding site. The previously published structure of rabbit phosphoglucomutase is intermediate between these two conformers. Several of the known reversible phosphorylation sites of pp63/pf-1 are at positions critical for transition between the conformations and for binding of the ligands and thus give hints as to possible roles of pp63/pf-1 in the course of exocytosis.

Adenosine Triphosphate↗

PubMed2002

My favorite cell--Paramecium.

A Paramecium cell has a stereotypically patterned surface, with regularly arranged cilia, dense-core secretory vesicles and subplasmalemmal calcium stores. Less strikingly, there is also a patterning of molecules; for instance, some ion channels are restricted to certain regions of the cell surface. This design may explain very effective and selective responses, such as that to Ca(2+) upon stimulation. It enables the cell to respond to a Ca(2+) signal precisely secretion (exocytosis) or by changing its ciliary activity. These responses depend on the location and/or type of signal, even though these two target structures co-exist side-by-side, and normally only limited overlap occurs between the different functions. Furthermore, the patterning of exocytotic sites and the possibility of synchronous exocytosis induction in the sub-second time range have considerably facilitated analyses, and thus led to new concepts of exocytotic membrane fusion. It has been possible to dissect complicated events like overlapping Ca(2+) fluxes produced from external sources and from internal stores. Since molecular genetic approaches have become available for Paramecium, many different gene products have been identified only some of which are known from "higher" eukaryotes. Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation.

Animals↗

PubMed2002

N-ethylmaleimide-sensitive factor is required to organize functional exocytotic microdomains in paramecium.

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the "rosettes" of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.

Animals↗

PubMedDate not supplied

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells.

Ca2+ signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the < or =10(3) dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Ca2+-dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-localised, and characterised as phosphoglucomutase, the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo, followed by MALDI analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial Ser/Thr residues. Although present also in exo(-) mutants, pp63/pf is selectively de-phosphorylated only in exo(+) strains during synchronous exocytosis (80 ms) and re-phosphorylated within approximately 20 s, i.e., the time required to re-establish [Ca2+] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider Ca2+/calmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Ca2+-activated guanylate cyclase. Another kinase, casein kinase 2, is inhibited by Ca2+ and, hence, activated with some delay in parallel to decreasing [Ca2+] after exocytosis. In total, several Ca2+-sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo(-) mutants) and NADH formation, with effects on Ca2+ signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca2+ dynamics during synchronous exo- and endocytosis.

Animals↗