Hindered submicron mobility and long-term storage of presynaptic dense-core granules revealed by single-particle tracking.

Dense-core granules (DCGs) are organelles found in neuroendocrine cells and neurons that house, transport, and release a number of important peptides and proteins. In neurons, DCG cargo can include the secreted neuromodulatory proteins tissue plasminogen activator (tPA) and/or brain-derived neurotrophic factor (BDNF), which play a key role in modulating synaptic efficacy in the hippocampus. This function has spurred interest in DCGs that localize to synaptic contacts between hippocampal neurons, and several studies recently have established that DCGs localize to, and undergo regulated exocytosis from, postsynaptic sites. To complement this work, we have studied presynaptically localized DCGs in hippocampal neurons, which are much more poorly understood than their postsynaptic analogs. Moreover, to enhance relevance, we visualized DCGs via fluorescence labeling of exogenous and endogenous tPA and BDNF. Using single-particle tracking, we determined trajectories of more than 150 presynaptically localized DCGs. These trajectories reveal that mobility of DCGs in presynaptic boutons is highly hindered and that storage is long-lived. We also computed mean-squared displacement curves, which can be used to elucidate mechanisms of transport. Over shorter time windows, most curves are linear, demonstrating that DCG transport in boutons is driven predominantly by diffusion. The remaining curves plateau with time, consistent with motion constrained by a submicron-sized corral. These results have relevance to recent models of presynaptic organization and to recent hypotheses about DCG cargo function. The results also provide estimates for transit times to the presynaptic plasma membrane that are consistent with measured times for onset of neurotrophin release from synaptically localized DCGs.

Efficient copackaging and cotransport yields postsynaptic colocalization of neuromodulators associated with synaptic plasticity.

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy.

Real-time imaging of the dynamics of secretory granules in growth cones.

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.

Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator/green fluorescent protein hybrid.

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

A dominant repressor of cyclic adenosine 3',5'-monophosphate (cAMP)-regulated enhancer-binding protein activity inhibits the cAMP-mediated induction of the somatostatin promoter in vivo.

The transactivation of genes through the cAMP-regulated enhancer (CRE) is proposed to occur by the binding and phosphorylation of the transcription factor CREB (CRE-binding protein). Originally believed to be a single protein, more than 10 different CREB proteins have been cloned. The contributions of each of these factors to gene regulation have yet to be determined unambiguously. We have isolated a CREB cDNA that contains a mutation of a single amino acid in the DNA-binding domain. In gel shift assays, this mutant, designated KCREB, is unable to bind to the somatostatin (SS) CRE. In addition, KCREB acts as a dominant repressor of the wild-type factor, blocking the ability of wild-type CREB to bind to the CRE when present as a KCREB:CREB heterodimer. The KCREB mutant also acts as a dominant repressor in vivo, completely blocking the ability of wild-type CREB to mediate induction by protein kinase-A of a SS CRE reporter gene in F9 teratocarcinoma cells. We have used this mutant to analyze the participation of CREB in the induction of the SS promoter in CA-77 cells, a medullary thyroid carcinoma cell line that produces high levels of SS. Although KCREB can block a portion of the cAMP induction of the SS promoter in CA-77 cells, approximately 45% of the induction remains insensitive to the mutant. These data support the paradigm that CREB is involved in the cAMP induction of SS in vivo. Furthermore, the inability of KCREB to completely block cAMP-mediated SS expression in CA-77 cells suggests that additional factors may contribute to the cAMP regulation of CRE function.

all-trans-Retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.

all-trans-Retinal was previously shown to stimulate high levels of superoxide release by guinea pig neutrophils. When the cells, previously labeled with [3H]inositol, are treated with all-trans-retinal, they exhibit a decrease in the levels of [3H]inositol phospholipids and an increase in the accumulation of [3H]inositol phosphates. The maximal accumulation of inositol phosphates and the optimal rate of superoxide release occurred together at approximately 7 min after stimulation. The levels of [3H]inositol phosphates accumulated were comparable to those observed when the cells were stimulated with a chemotactic peptide. In direct measurements, using concentrations that stimulate intact cells maximally, all-trans-retinal was found not to inhibit protein kinase C from the cytosol of neutrophils significantly. This contrasts with the situation with this kinase obtained from other sources. These observations represent additional effects of vitamin A on cells.

The role of exoenzyme S in infections with Pseudomonas aeruginosa.

To define the contribution of exoenzyme S to the pathogenesis of infections with Pseudomonas aeruginosa, we have compared the ability of an exoenzyme S-deficient mutant, 388 exs1::Tn1, and that of its exoenzyme S-producing parent to colonize and disseminate in burned mice infected with this organism. Both the exoenzyme S-deficient mutant and the parent strain proliferated in burned skin, but only the parent strain was able to effectively disseminate to blood and other tissues. The reduced ability of the mutant to disseminate was not due to alterations in serum sensitivity, lipopolysaccharide composition, or motility. The exoenzyme S-deficient mutant was able to disseminate in the presence of the exoenzyme S-producing parent. Antibody to purified exoenzyme S was able to greatly reduce dissemination of the exoenzyme S-producing parent strain but did not prevent colonization in the burned skin. These data suggest that exoenzyme S does not contribute to the initial colonization but does contribute to the establishment of disseminated infection.

Defective triggering of polymorphonuclear leukocyte oxidative metabolism by Legionella pneumophila toxin.

Preincubation of human polymorphonuclear leukocytes (PMNLs) with Legionella pneumophila toxin impaired activation of the superoxide-generating complex induced by latex particles and by the Ca++ ionophore A23187. The toxin had no effect, however, on activation of the complex induced by phorbol myristate acetate (PMA), concanavalin A, valinomycin, or bromolasalocid. The toxin prevented PMNL plasma membrane depolarization induced by A23187 but failed to influence the membrane depolarization induced by PMA. These observations indicate that Legionella pneumophila toxin selectively impairs activation of the phagocyte superoxide-generating complex without affecting the functional integrity of components of the complex.

Effect of oxygen-dependent antimicrobial systems on Legionella pneumophila.

Legionella pneumophila was susceptible to the antimicrobial action of oxygen metabolites generated by both the myeloperoxidase-H(2)O(2)-halide and the xanthine oxidase systems.

The effects of Legionella pneumophila toxin on oxidative processes and bacterial killing of human polymorphonuclear leukocytes.

The effect of Legionella pneumophila toxin on selected functions of human polymorphonuclear leukocytes was investigated. Amounts of L. pneumophila toxin that had no effect on leukocyte viability or phagocytosis significantly decreased hexose monophosphate shunt activity and O2 consumption during phagocytosis and bacterial iodination and killing in a dose-dependent fashion. The mechanism of action of this toxin appears to be unique among bacterial products thus far studied.

Early electrochemical events in lectin-lymphocyte interaction. I. Mechanism of lectin-mediated changes in the electrophoretic mobility of lymphocytes.

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.