Essential role of germinal vesicle material in the meiotic cell cycle of Xenopus oocytes

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.

Omega images at the active zone may be endocytotic rather than exocytotic: Implications for the vesicle hypothesis of transmitter release

A Ca2+-dependent synaptic vesicle-recycling pathway emanating from the plasma membrane adjacent to the dense body at the active zone has been demonstrated by blocking pinch-off of recycling membrane by using the Drosophila mutant, shibire. Exposure of wild-type Drosophila synapses to low Ca2+/high Mg2+ saline is shown here to block this active zone recycling pathway at the stage in which invaginations of the plasma membrane develop adjacent to the dense body. These observations, in combination with our previous demonstration that exposure to high Ca2+ causes “docked” vesicles to accumulate in the identical location where active zone endocytosis occurs, suggest the possibility that a vesicle-recycling pathway emanating from the active zone may exist that is stimulated by exposure to elevated Ca2+, thereby causing an increase in vesicle recycling, and is suppressed by exposure to low Ca2+ saline, thereby blocking newly forming vesicles at the invagination stage. The presence of a Ca2+-dependent endocytotic pathway at the active zone opens up the following possibilities: (i) electron microscopic omega-shaped images (and their equivalent, freeze fracture dimples) observed at the active zone adjacent to the dense body could represent endocytotic images (newly forming vesicles) rather than exocytotic images; (ii) vesicles observed attached to the plasma membrane adjacent to the dense body could represent newly formed vesicles rather than vesicles “docked” for release of transmitter.

Opening of a monomer-monomer interface of the trimeric bacteriophage T4-coded GP45 sliding clamp is required for clamp loading onto DNA

The replication system of bacteriophage T4 uses a trimeric ring-shaped processivity clamp (gp45) to tether the replication polymerase (gp43) to the template-primer DNA. This ring is placed onto the DNA by an ATPase-driven clamp-loading complex (gp44/62) where it then transfers, in closed form, to the polymerase. It generally has been assumed that one of the functions of the loading machinery is to open the clamp to place it around the DNA. However, the mechanism by which this occurs has not been fully defined. In this study we design and characterize a double-mutant gp45 protein that contains pairs of cysteine residues located at each monomer-monomer interface of the trimeric clamp. This mutant protein is functionally equivalent to wild-type gp45. However, when all three monomer-monomer interfaces are tethered by covalent crosslinks formed (reversibly or irreversibly) between the cysteine pairs these closed clamps can no longer be loaded onto the DNA nor onto the polymerase, effectively eliminating processive strand-displacement DNA synthesis. Analysis of the individual steps of the clamp-loading process shows that the ATPase-dependent interactions between the clamp and the clamp loader that precede DNA binding are hyperstimulated by the covalently crosslinked ring, suggesting that binding of the closed ring induces a futile, ATP-driven, ring-opening cycle. These findings and others permit further characterization and ordering of the steps involved in the T4 clamp-loading process.

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway

A novel type I transmembrane protein of COPI-coated vesicles, p23, has been demonstrated to be localized mainly to the Golgi complex. This protein and p24, another member of the p24 family, have been shown to bind coatomer via their short cytoplasmic tails. Here we demonstrate that p23 continuously cycles through the early secretory pathway. The cytoplasmic tail of p23 is shown to act as a functional retrieval signal as it confers endoplasmic reticulum (ER) residence to a CD8–p23 fusion protein. This ER localization is, at least in part, a result of retrieval from post-ER compartments because CD8–p23 fusion proteins receive post-ER modifications. In contrast, the cytoplasmic tail of p24 has been shown not to retrieve a CD8–p24 fusion protein. The coatomer binding motifs FF and KK in the cytoplasmic tail of p23 are reported to influence the steady-state localization of the CD8–p23 fusion protein within the ER–Golgi recycling pathway. It appears that the steady-state Golgi localization of endogenous p23 is maintained by its lumenal domain, as a fusion protein with the lumenal domain of CD8, and the membrane span as well as the cytoplasmic tail of p23 is no longer detected in the Golgi.

Amyloid β peptide alters intracellular vesicle trafficking and cholesterol homeostasis

Amyloid β peptide (Aβ) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Aβ induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Aβ and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Aβ decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Aβ may be relevant to the chronic neurodegeneration observed in AD.

Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP

The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307–319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+ cellular vaccines in which defined T cell epitopes are continuously synthesized.

Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons

Tetanus neurotoxin causes the spastic paralysis of tetanus by blocking neurotransmitter release at inhibitory synapses of the spinal cord. This is due to the penetration of the toxin inside the neuronal cytosol where it cleaves specifically VAMP/synaptobrevin, an essential component of the neuroexocytosis apparatus. Here we show that tetanus neurotoxin is internalized inside the lumen of small synaptic vesicles following the process of vesicle reuptake. Vesicle acidification is essential for the toxin translocation in the cytosol, which results in the proteolytic cleavage of VAMP/synaptobrevin and block of exocytosis.

Gi regulation of secretory vesicle swelling examined by atomic force microscopy

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the α subunit of a heterotrimeric GTP binding protein Gαi3 and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15–25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that Gαi3 protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A)

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein common to all synaptic and endocrine vesicles. Unlike many proteins involved in synaptic exocytosis, SV2 has no homolog in yeast, indicating that it performs a function unique to secretion in higher eukaryotes. Although the structure and protein interactions of SV2 suggest multiple possible functions, its role in synaptic events remains unknown. To explore the function of SV2 in an in vivo context, we generated mice that do not express the primary SV2 isoform, SV2A, by using targeted gene disruption. Animals homozygous for the SV2A gene disruption appear normal at birth. However, they fail to grow, experience severe seizures, and die within 3 weeks, suggesting multiple neural and endocrine deficits. Electrophysiological studies of spontaneous inhibitory neurotransmission in the CA3 region of the hippocampus revealed that loss of SV2A leads to a reduction in action potential-dependent γ-aminobutyric acid (GABA)ergic neurotransmission. In contrast, action potential-independent neurotransmission was normal. Analyses of synapse ultrastructure suggest that altered neurotransmission is not caused by changes in synapse density or morphology. These findings demonstrate that SV2A is an essential protein and implicate it in the control of exocytosis.

Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey

Zinc transporter-3 (ZnT-3), a member of a growing family of mammalian zinc transporters, is expressed in regions of the brain that are rich in histochemically reactive zinc (as revealed by the Timm’s stain), including entorhinal cortex, amygdala, and hippocampus. ZnT-3 protein is most abundant in the zinc-enriched mossy fibers that project from the dentate granule cells to hilar and CA3 pyramidal neurons. We show here by electron microscopy that ZnT-3 decorates the membranes of all clear, small, round synaptic vesicles (SVs) in the mossy fiber boutons of both mouse and monkey. Furthermore, up to 60–80% of these SVs contain Timm’s-stainable zinc. The coincidence of ZnT-3 on the membranes of SVs that accumulate zinc, and its homology with known zinc transporters, suggest that ZnT-3 is responsible for the transport of zinc into SVs, and hence for the ability of these neurons to release zinc upon excitation.

The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Proteolytic Processing and Ca2+-binding Activity of Dense-Core Vesicle Polypeptides in Tetrahymena

Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles.

Immunoisolation and Characterization of a Subdomain of the Endoplasmic Reticulum That Concentrates Proteins Involved in COPII Vesicle Biogenesis

Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from both rough ER and Golgi. We hypothesized that this compartment corresponds to hypertrophied ER exit sites that have expanded in response to overexpression of E1. In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation. Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present. By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes. These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites. Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.

A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.