Stochastic Subcellular Organization of Dense-Core Vesicles Revealed by Point Pattern Analysis.

Dense-core vesicles (DCVs) are regulated secretory organelles found in many types of neurons. In neurons of the hippocampus, their cargo includes proteins that mediate several pivotal processes, including differentiation and synaptic plasticity. Motivated by interest in DCV distribution and its impact on cargo action, we have used fluorescence microscopy and statistical analysis to develop a quantitative model of the subcellular organization of DCVs in hippocampal neurons that are spontaneously active (their most prevalent state). We also have tested the functionally motivated hypothesis that these organelles are synaptically enriched. Variance-to-mean ratio, frequency distribution, and Moran's autocorrelation analyses reveal that DCV distribution along shafts, and within synapses, follows Poisson statistics, establishing that stochastically dictated organization sustains cargo function. Occupancy in boutons exceeds that at nearby extrasynaptic axonal sites by approximately threefold, revealing significant local presynaptic enrichment. Widespread stochastic organization is consistent with the emerging functional importance of synaptically and extrasynaptically localized DCVs. Presynaptic enrichment is consistent with the established importance of protecting presynaptic sites from depletion of DCV cargo. These results enhance understanding of the link between DCV organization and mechanisms of cargo action, and they reinforce the emerging theme that randomness is a prevalent aspect of synaptic organization and composition.

Super-resolution imaging of neuronal dense-core vesicles.

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization. Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

Extended-spectrum ß-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective.

The possible zoonotic spread of antimicrobial-resistant bacteria is controversial. This review discusses global molecular epidemiological data combining both analyses of the chromosomal background, using multilocus sequence typing (MLST), and analyses of plasmid (episomal) extended-spectrum ß-lactamase (ESBL)/AmpC genes in Escherichia coli present in humans and animals. For consideration of major epidemiological differences, animals were separated into livestock and companion animals. MLST revealed the existence of ESBL-producing isolates thoughout the E. coli population, with no obvious association with any ancestral EcoR group. A similar distribution of major ESBL/AmpC types was apparent only in human isolates, regardless of their geographical origin from Europe, Asia, or the Americas, whereas in animals this varied extensively between animal groups and across different geographical areas. In contrast to the diversity of episomal ESBL/AmpC types, isolates from human and animals mainly shared identical sequence types (STs), suggesting transmission or parallel micro-evolution. In conclusion, the opinion that animal ESBL-producing E. coli is a major source of human infections is oversimplified, and neglects a highly complex scenario.

Psychology graduate students' attitudes toward lesbians and gay men.

The extant literature concerning attitudes toward gay men and lesbians was reviewed, with a particular emphasis on the attitudes of mental health professionals. Seventy graduate students in a variety of psychology-related areas were surveyed about their attitudes toward lesbians and gay men. Findings from hierarchical multiple regression analyses indicated that after controlling for socially desirable responding, the only variable to predict negative attitudes toward gay men and lesbians was endorsement of traditional gender roles. The results of this investigation were placed within a theoretical framework and then implications for training future mental health practitioners were discussed.

Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging.

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning-related synaptic plasticity and potentiating N-methyl-D-aspartate (NMDA) receptor-dependent signaling. These findings suggest that tPA may localize to, and undergo activity-dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization-induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense-core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca(+2) concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse-pinch-linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA-type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory.

How neurosecretory vesicles release their cargo.

Neurons and related cell types often contain two major classes of neurosecretory vesicles, synaptic vesicles (SVs) and dense-core granules (DCGs), which store and release distinct cargo. SVs store and release classic neurotransmitters, which facilitate propagation of action potentials across the synaptic cleft, whereas DCGs transport, store, and release hormones, proteins, and neuropeptides, which facilitate neuronal survival, synaptic transmission, and learning. Over the past few years, there has been a major surge in our understanding of many of the key molecular mechanisms underlying cargo release from SVs and DCGs. This surge has been driven largely by the use of fluorescence microscopy (especially total internal reflection fluorescence microscopy) to visualize SVs or DCGs in living cells. This review highlights some of the recent insights into cargo release from neurosecretory vesicles provided by fluorescence microscopy, with emphasis on DCGs.

Mechanisms of transport and exocytosis of dense-core granules containing tissue plasminogen activator in developing hippocampal neurons.

Dense-core granules (DCGs) are organelles found in specialized secretory cells, including neuroendocrine cells and neurons. Neuronal DCGs facilitate many critical processes, including the transport and secretion of proteins involved in learning, and yet their transport and exocytosis are poorly understood. We have used wide-field and total internal reflection fluorescence microscopy, in conjunction with transport theory, to visualize the transport and exocytosis of DCGs containing a tissue plasminogen activator-green fluorescent protein hybrid in cell bodies, neurites, and growth cones of developing hippocampal neurons and to quantify the roles that diffusion, directed motion, and immobility play in these processes. Our results demonstrate that shorter-ranged transport of DCGs near sites of exocytosis in hippocampal neurons and neuroendocrine cells differs markedly. Specifically, the immobile fraction of DCGs within growth cones and near the plasma membrane of hippocampal neurons is small and relatively unaltered by actin disruption, unlike in neuroendocrine cells. Moreover, transport of DCGs in these domains of hippocampal neurons is unusually heterogeneous, being significantly rapid and directed as well as slow and diffusive. Our results also demonstrate that exocytosis is preceded by substantial movement and heterogeneous transport; this movement may facilitate delivery of DCG cargo in hippocampal neurons, given the relatively low abundance of neuronal DCGs. In addition, the extensive mobility of DCGs in hippocampal neurons argues strongly against the hypothesis that cortical actin is a major barrier to membrane-proximal DCGs in these cells. Instead, our results suggest that extended release of DCG cargo from hippocampal neurons arises from heterogeneity in DCG mobility.

Same-sex couple perceptions of family support: a consensual qualitative study.

Few studies have examined the family context in which same-sex couples negotiate their lives and relationships. Consensual qualitative research methods (Hill, Thompson, & Williams, 1997) were used to analyze 14 same-sex couples' conversations about family support. Couples perceived that their families are positively supportive, non-supportive, or ambivalent in their support. These perceptions led to positive or negative emotional reactions in the couple members and to specific coping. The majority of couples perceived that family support (or lack of support) had an effect on the quality of their couple relationship. No general or typical response strategies to lack of family of origin support emerged, suggesting a lack of models or norms for same-sex couples. The implications for psychotherapeutic interventions with same-sex couples are discussed.

Correlates of high-risk sexual behavior among young men who have sex with men.

Data from a sample (n = 154) of young men who have sex with men (YMSM), aged 13-21, residing in five mid-sized cities in the midwestern United States were used to test the hypothesis that personality variables (impulsive decision making, sensation seeking, anxiety/depression, internalized homophobia) contribute uniquely to the prediction of unprotected anal sex beyond what is accounted for by other high-risk behaviors (i.e., substance use and number of sex partners) and partner status (having a dating partner). Results of hierarchical regression analyses indicated that number of reported anal sex partners and partner status were positively associated with self-reported frequency of unprotected anal sex. Beyond these factors, impulsive decision making significantly contributed to the predictive model. These findings indicate that designing effective prevention programming for YMSM should take into consideration relationship status and the impulsiveness of sexual decision making among members of the target group.

Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies.

The difficulty of deriving binding ligands to targets identified by genomic sequencing has led to a bottleneck in genomic research. By inserting diverse antibody binding loops into four of the exposed loops at one end of green fluorescent protein (GFP), we have mimicked the natural antibody binding footprint to create robust binding ligands that combine the advantages of antibodies (high affinity and specificity) with those of GFP (intrinsic fluorescence, high stability, expression and solubility). These 'fluorobodies' have been used effectively in enzyme-linked immunosorbent assays (ELISAs), flow cytometry, immuno-fluorescence, arrays and gel shift assays, and show affinities as high as antibodies. Furthermore, the intrinsic fluorescence of fluorobodies correlates with binding activity, allowing the rapid determination of functionality, concentration and affinity. These properties render them especially suitable for the high-throughput genomic scale selections required in proteomics, as well as in diagnostics, target validation and drug development.

Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells.

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue, frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.

Sweeping model of dynamin activity. Visualization of coupling between exocytosis and endocytosis under an evanescent wave microscope with green fluorescent proteins.

Vesicle recycling through exocytosis and endocytosis is mediated by a coordinated cascade of protein-protein interactions. Previously, exocytosis and endocytosis were studied separately so that the coupling between them was understood only indirectly. We focused on the coupling of these processes by observing the secretory vesicle marker synaptobrevin and the endocytotic vesicle marker dynamin I tagged with green and red fluorescent proteins under an evanescent wave microscope in pheochromocytoma cells. In control cells, many synaptobrevin-expressing vesicles were found as fluorescent spots near the plasma membrane. Upon electrical stimulation, many of these vesicles showed an exocytotic response as a transient increase in fluorescence intensity followed by their disappearance. In contrast, fluorescent dynamin appeared as clusters increasing slowly in number upon stimulation. The clusters of fluorescent dynamin moved around beneath the plasma membrane for a significant distance. Simultaneous observations of green fluorescent dynamin and red fluorescent synaptobrevin indicated that more than 70% of the exocytotic responses of synaptobrevin had no immediate dynamin counterpart at the same site. From these findings it was concluded that dynamin-mediated recycling is not directly coupled to exocytosis but rather completed by a scanning movement of dynamin for the sites of invaginating membrane destined to endocytosis.

Autocatalytic polysialylation of polysialyltransferase-1.

Polysialic acid (PSA) is a specific and highly regulated post-translational modification of the neural cell adhesion molecule NCAM. Synthesis of PSA depends on the activity of a single enzyme, the polysialyltransferase-1 (PST-1), recently cloned from three mammalian species. The present study was carried out to investigate the catalytic mechanism of PST-1. Using a newly developed in vitro assay system, we demonstrate autopolysialylation for PST-1. The synthesis of PSA chains, which involved N-glycosylation sites, occurred immediately after contact with the activated sugar donor CMP-Neu5Ac. In contrast to the polysialylation of NCAM, where terminal sialylation in either the alpha2,3 or alpha2,6 position is required, the autopolysialylation could be started in the asialo-PST-1 isolated from CHO cells of the Lec2 complementation group. Pre-formed PSA chains were not transferred to NCAM. Nevertheless, the autocatalytic step is likely to be a prerequisite for enzymatic activity, since agalacto-PST-1 isolated from Lec8 cells was functionally inactive. Our data describe a novel route of autocatalytic maturation of a glycosyltransferase and thereby provide a new basis for studies aimed at elucidating and influencing the catalytic functions of PST-1.

Polysialylation of NCAM by a single enzyme.

The addition of poly-alpha2,8-N-acetylneuraminic acid (polysialic acid; PSA) to the neural cell adhesion molecule NCAM plays a crucial role in neural development [1-3], neural regeneration [4], and plastic processes in the vertebrate brain associated with neurite outgrowth [5], axonal pathfinding [6], and learning and memory [7,-9]. PSA levels are decreased in people affected by schizophrenia [10], and PSA has been identified as a specific marker for some neuroendocrine and lymphoblastoid tumours [11-13]; expression of PSA on the surface of these tumour cells modulates their metastatic potential [11-13]. Studies aimed at understanding PSA biosynthesis and the dynamics of its production have largely been promoted by the cloning of polysialyltransferases (PST-1 in hamster; PST in human and mouse) [14-16]. However, the number of enzymes involved in the biosynthesis of PSA has not been identified. Using incompletely glycosylated NCAM variants and soluble recombinant glycosyltransferases, we reconstituted the site at which PST-1 acts to polysialylate NCAM in vitro. The data presented here clearly demonstrate that polysialylation of NCAM is catalyzed by a single enzyme, PST-1, and that terminal sialylation of the N-glycan core is sufficient to generate the PSA acceptor site. Our results also show that PST-1 can act on core structures with the terminal sialic acid connected to galactose via an alpha2,3 or alpha2,6 linkage.

Expression cloning of the Golgi CMP-sialic acid transporter.

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE.

Homopolymeric alpha-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.

Molecular characterization of eukaryotic polysialyltransferase-1.

Polysialic acid (PSA) is a dynamically regulated product of post-translational modification of the neural cell adhesion molecule, NCAM. Presence of the large anionic carbohydrate modulates NCAM binding properties and, by increasing the intercellular space, influences interactions between other cell surface molecules. PSA expression underlies cell type- and developmental-specific alterations and correlates with stages of cellular motility. In the adult, PSA becomes restricted to regions of permanent neural plasticity and regenerating neural and muscle tissues. Recent data implicate its important function in spatial learning and memory, and in tumour biology. Here we describe the molecular characterization of polysialyltransferase-1, the key enzyme of eukaryotic PSA synthesis. In reconstitution experiments, the newly cloned enzyme induces PSA synthesis in all NCAM-expressing cell lines. Our data therefore represent convincing evidence that the polycondensation of alpha-2,8-linked sialic acids in mammals is the result of a single enzymatic activity and provide a new basis for studying the functional role of PSA in neuro- and tumour biology.