Non-functional role of syntaxin 2 in insulin exocytosis by pancreatic β cells

This study was designed in order to examine the expression and functional role of syntaxin 2/epimorphin in pancreatic β cells. Northern blot analysis revealed that syntaxin 2 mRNA was able to be detected in mouse βTC3 cells, but not in isolated mouse islets. In agreement with this result, immunoblot analysis detected an appreciable amount of syntaxin 2 protein in βTC3 cells, but not in mouse islets. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 2 was little evident in islet cells of Langerhans, and somewhat predominant in exocrine tissues. In order to examine whether syntaxin 2 is anchored to cell surfaces in βTC3 cells, living cells were incubated with a monoclonal antibody against syntaxin 2 (MC-1). The antibody bound to their surfaces, indicating that syntaxin 2 was localized on cell surfaces. The addition of MC-1 to the culture medium of βTC3 cells did not affect insulin release under the presence or absence of 11 mM glucose, indicating that syntaxin 2 is not associated with insulin exocytosis. Thus, the expression of syntaxin 2 in islets of Langerhans is very low and the function of this protein is probably unrelated to the insulin exocytosis pathway. © 1997 John Wiley & Sons, Ltd.     

Inhibition of trichocyst exocytosis and calcium influx in Paramecium by amiloride and divalent cations

Summary— Regulated exocytosis of defensive secretory organelles, the trichocysts, as well as a transient Ca²⁺-influx can be induced in Paramecium by aminoethyldextran (Kerb?uf and Cohen, J Cell Biol (1990) 111, 2527). Knoll et al (Febs Lett (1992) 304, 265) reported that veratridine was also a secretagogue for Paramecium. Here we show that, like aminoethyldextran, veratridine induces a transient Ca²⁺-influx. Both aminoethyldextran-and veratridine-induced exocytosis and associated Ca²⁺-influx were: i) blocked in the nd12 thermosensitive mutant at the non-permissive temperature; and ii) inhibited by amiloride and four divalent cations, Ba²⁺, Mg²⁺, Sr²⁺ and Co²⁺. This suggests that, although of different chemical nature, aminoethyldextran and veratridine act through the same physiological pathway. In addition, the inhibitory doses are comparable to the ones found to inhibit a hyperpolarization-sensitive Ca²⁺-current described in Paramecium (Preston et al (1992) J Gen Physiol 100, 233). The possibility that the activation of this Ca²⁺-current by the secretagogue represents an early step in the regulation of trichocyst exocytosis is discussed.     

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.     

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.     

Yeast functional analysis: identification of two essential genes involved in ER to Golgi trafficking

We screened for genes potentially involved in the secretory and vacuolar pathways a collection of 61 yeast strains, each bearing an essential orphan gene regulated by the tetO₇-CYC1 promoter that can be down-regulated by doxycycline. After down-regulating the expression of these genes, we performed systematic Western blot analysis for markers of the secretory and vacuolar pathways that undergo post-translational modifications in their intracellular trafficking. Accumulation of protein precursors, revealed by Western immunoblot analysis, indicates defects in the secretory pathway or in associated biochemical modifications. After screening the whole collection, we identified two genes involved in ER to Golgi trafficking: RER2 , a cis -prenyl transferase, and USE1 , the function of which was unknown . We demonstrated that repression of USE1 also leads to BiP secretion, and therefore likely affects retrograde, in addition to anterograde, ER to Golgi trafficking. The collection also includes two essential genes involved in intracellular trafficking that were conveniently repressed without resulting growth or trafficking defects.     

Spatial proteomics of vesicular trafficking: coupling mass spectrometry and imaging approaches in membrane biology

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.     

Relation Between Cell Union and Nuclear Reorganization in Triplet Conjugants of Paramecium caudatum and Paramecium multimicronucleatum

SYNOPSIS Triplet conjugants of Paramecium caudatum which appeared naturally in mating mixtures and those of Paramecium multimicronucleatum which were produced by conjugation-inducing chemicals were isolated. Triplet conjugants lasting for more than 3 h were stained to examine macronuclear events. In P. caudatum , only 2 triplets among 182 (1%) contained macronuclear fragmentation in all 3 members. The most frequently occurring triplets (79%) were those producing 1 cell without and 2 cells with macronuclear fragments. There were also triplets (17%) producing 1 cell with, and 2 without macronuclear fragments, and some (3%) with 3 cells that contained no fragments. The length of persistence of the triplet was not responsible for the occurrence of macronuclear fragmentation in the 3rd cell of the triplet. In P. multimicronucleatum , the same 4 classes of triplets occurred, but the most frequently occurring class was that consisting of 3 cells (91%) with macronuclear fragments. Induction of nearly 100% of triplets with 3 such cells was possible by isolating the triplets' from a culture which was treated chemically at about 24 h after the last feeding. Treatment with chemicals in starved cultures resulted in triplets with incompletely fragmented or nonfragmented macronuclei. Further, in P. multimicronucleatum , chemicallyinduced triplets involving only holdfast pairs to which the 3rd cells were uniting often produced 3 cells with fragmented macronuclei.     

Regulation of SNAREs by tomosyn and ROCK: implication in extension and retraction of neurites

Extension of neurites requires the SNARE-dependent fusion of plasmalemmal precursor vesicles with the plasma membrane of growth cones. Here, we show that tomosyn localizes at the palm of growth cones and inhibits the fusion of the vesicles there, thus promoting transport of the vesicles to the plasma membrane of the leading edges of growth cones. Tomosyn localizes because ROCK activated by Rho small G protein phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the SNARE complex. In retraction of neurites, tomosyn localizes all over the edges of the neurites and inhibits fusion of the vesicles with the plasma membrane. Thus, tomosyn demarcates the plasma membrane by binding to syntaxin-1 phosphorylated by ROCK, and thereby regulates extension and retraction of neurites.     

Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.     

Expression of the green fluorescent protein in Paramecium tetraurelia

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.     

Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.     

Acute manipulation and real-time visualization of membrane trafficking and exocytosis in Drosophila

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Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA⁺ or cnrB⁺) of the donor 9–24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells. In unstable transformation, injected macronucleoplasm is distributed only to one of the daughter cells at every division until about the fifth to seventh fission after injection and then begins to assort equally to daughter cells. The cell cycle stage at injection may influence the mode of transformation. Interspecific microinjection of macronucleoplasm from P. multimicronucleatum and P. tetraurelia to P. caudatum. resulted in the expression of foreign genes in P. caudatum. In one case, injection of macronucleoplasm of P. tetraurelia produced a stable transformant indicating replication of foreign macronucleoplasm in P. caudatum. This work reveals the mode of transformation by injected macronucleoplasm and shows the possibility of transformation among Paramecium species, which is significant in the study of the conservation of gene products and the mechanism of gene expression in different species. © 1992 Wiley-Liss, Inc.     

Molecular biology of the genes for immobilization antigens in Paramecium

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition to known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51C and 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria , a freshwater protozoan, typically harbors hundreds of symbiotic algae ( Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria . Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross-breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria , F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.     

Differential contribution of syntaxin 1 and SNAP-25 to secretion in noradrenergic and adrenergic chromaffin cells

We used botulinum neurotoxins (BoNT) to examine whether differences in the secretory activity of noradrenergic and adrenergic chromaffin cells are related to differences in the exocytotic machinery of these two types of bovine adrenal medulla cells. Cleavage of syntaxin and SNAP-25 by BoNT/C1 decreased in a dose-dependent way the release of both noradrenaline and adrenaline, but noradrenaline release was more sensitive to BoNT/C1. Cleavage of SNAP-25 by BoNT/A also had a larger inhibitory effect on noradrenaline release than on adrenaline release. Neither BoNT/C1 nor BoNT/A affected the intracellular Ca2+ responses induced by K+-depolarisation, and the extent of the inhibition of K+-evoked catecholamine release by selective blockers of voltage-gated Ca2+ channels was not affected by BoNT/C1. Therefore, our data do not support the hypothesis of a regulatory effect of syntaxin or SNAP-25 on the activity of Ca2+ channels. The lower sensitivity of adrenaline release to BoNT was not due to a reduced ability of the toxins to enter or to cleave their protein targets in adrenergic cells, since immunoblot analysis showed the cleavage of a larger fraction of syntaxin 1A in adrenergic cells, as compared to the cleavage in noradrenergic cells. The immunoblot analysis also showed larger amounts of syntaxin 1A in noradrenergic chromaffin cells than in adrenergic cells. Thus, in spite of a greater cleavage of syntaxin 1A in adrenergic cells by BoNT/C1, adrenaline release was less sensitive to BoNT/C1, suggesting that the release process in noradrenergic cells might be more dependent on syntaxin 1A and SNAP-25, as compared to adrenergic cells.