A regulated secretory pathway in cultured hippocampal astrocytes.

Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40-50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca(2+), a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca(2+)](i) transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo [Ca(2+)](i) increase upon specific stimuli is present in cultured hippocampal astrocytes.     

Trafficking of astrocytic vesicles in hippocampal slices

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.     

Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses.

The protein brain-derived neurotrophic factor (BDNF) has been postulated to be a retrograde or paracrine synaptic messenger in long-term potentiation and other forms of activity-dependent synaptic plasticity. Although crucial for this concept, direct evidence for the activity-dependent synaptic release of BDNF is lacking. Here we investigate secretion of BDNF labelled with green fluorescent protein (BDNF-GFP) by monitoring the changes in fluorescence intensity of dendritic BDNF-GFP vesicles at glutamatergic synaptic junctions of living hippocampal neurons. We show that high-frequency activation of glutamatergic synapses triggers the release of BDNF-GFP from synaptically localized secretory granules. This release depends on activation of postsynaptic ionotropic glutamate receptors and on postsynaptic Ca(2+) influx. Release of BDNF-GFP is also observed from extrasynaptic dendritic vesicle clusters, suggesting that a possible spatial restriction of BDNF release to specific synaptic sites can only occur if the postsynaptic depolarization remains local. These results support the concept of BDNF being a synaptic messenger of activity-dependent synaptic plasticity, which is released from postsynaptic neurons.     

Intravesicular calcium release mediates the motion and exocytosis of secretory organelles: a study with adrenal chromaffin cells

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.     

Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy: distinct behavior of granule motion in biphasic insulin release.

To study insulin exocytosis by monitoring the single insulin secretory granule motion, evanescent wave microscopy was used to quantitatively analyze the final stage of insulin exocytosis with biphasic release. Green fluorescent protein-tagged insulin transfected in MIN6 beta cells was packed in insulin secretory granules, which appeared to preferentially dock to the plasma membrane. Upon fusion evoked by secretagogues, evanescent wave microscopy revealed that fluorescence of green fluorescent protein-tagged insulin brightened, spread (within 300 ms), and then vanished. Under KCl stimulation, which represents the 1st phase of release, the successive fusion events were seen mostly from previously docked granules for the first minute, followed by the recruitment of new granules to the plasmalemmal docking sites. Stimulation with glucose, in contrast, caused the fusion events from previously docked granules for the first 120 s, thereafter a continuous fusion (2nd phase of release) was observed over 10 min mostly from newly recruited granules that progressively accumulated on the plasma membrane. Thus, our data revealed the distinct behavior of the insulin granule motion during the 1st and 2nd phase of release.     

Protein discharge from immature secretory granules displays both regulated and constitutive characteristics.

At physiological glucose concentrations, isolated pancreatic islets release a minor portion of their newly synthesized insulin and precursors in a phase of secretion which is largely complete by 4 h of chase. Discharge during this period can be amplified by secretagogues, yet is not abolished by conditions which fully suppress regulated release from dense core secretory granules. The ability to stimulate the secretion and the biochemical profile of released proinsulin-related peptides indicate that secretion during this period originates from immature granules. The stoichiometry of release of labeled C-peptide:insulin during this phase is 1:1 at high glucose concentrations. However, at physiologic or low concentrations, C-peptide is released in molar excess of insulin as if the exocytotic vesicles carrying this secretion were budding from a post-Golgi compartment in which the lumen was composed of condensing insulin and soluble C-peptide. These findings can be explained by a model for regulated secretory protein traffic in which direct exocytosis of young granules is stimulated by higher glucose concentrations and vesicle budding from immature granules occurs at lower concentrations. Thus, insulin targeting from immature granules exhibits both regulated and constitutive-like characteristics.     

The secretory response through electric stimulation of differentiated PC12 rat pheochromocytoma cells transfected with neuropeptide Y fused with enhanced green fluorescent protein

Exocytosis in pheochromocytoma cells was induced by electric stimulation. To chase the movement of vesicles by electric stimulation, dense-core secretory vesicles were visualized by expression of the fusion protein between neuropeptide Y and enhanced green fluorescent protein (EGFP) in these differentiated PC12 rat pheochromocytoma cells. When the cells were stimulated with constant voltage potential at –300 mV, the movement of dense-core secretory vesicles could be regulated.     

An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites

mRNA transport and local translation in the neuronal dendrite is implicated in the induction of synaptic plasticity. Recently, we cloned an RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1), that is suggested to be important for the stabilization of mRNA. We report here that SYNCRIP is a component of mRNA granules in rat hippocampal neurons. SYNCRIP was mainly found at cell bodies, but punctate expression patterns in the proximal dendrite were also seen. Time-lapse analysis in living neurons revealed that the granules labeled with fluorescent protein-tagged SYNCRIP were transported bi-directionally within the dendrite at approximately 0.05 microm/s. Treatment of neurons with nocodazole significantly inhibited the movement of green fluorescent protein-SYNCRIP-positive granules, indicating that the transport of SYNCRIP-containing granules is dependent on microtubules. The distribution of SYNCRIP-containing granules overlapped with that of dendritic RNAs and elongation factor 1alpha. SYNCRIP was also found to be co-transported with green fluorescent protein-tagged human staufen1 and the 3'-untranslated region of inositol 1,4,5-trisphosphate receptor type 1 mRNA. These results suggest that SYNCRIP is transported within the dendrite as a component of mRNA granules and raise the possibility that mRNA turnover in mRNA granules and the regulation of local protein synthesis in neuronal dendrites may involve SYNCRIP.     

Nearly neutral secretory vesicles in Drosophila nerve terminals

The acidity of mammalian secretory vesicles drives concentration and processing of their contents. Here, pH-sensitive green fluorescent protein (GFP) variants show that the > or =30-fold (H+) difference between secretory vesicles (pH < or = 5.7) and the cytoplasm (pH = 7.2) in mammalian cells is not present in peptidergic and small synaptic vesicles of the Drosophila neuromuscular junction. First, we find that fluorescence from Topaz-tagged atrial natriuretic factor, a peptidergic vesicle pH indicator, is only modestly affected by collapsing the H+ gradient in type III synaptic boutons. Quantitation shows that peptidergic vesicles are nearly neutral (pH = 6.74 +/- 0.05), even when temperature is elevated. Furthermore, small synaptic vesicles in glutamatergic synaptic boutons, studied with synaptophluorin, are as alkaline as peptidergic vesicles. Finally, yellow fluorescent protein measurements show that cytoplasmic pH is only slightly different than in mammals (pH = 7.4). Thus, the marked acidity of mammalian secretory vesicles is not conserved in evolution, and a modest vesicular H+ gradient is sufficient for supporting neurotransmission.     

Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells

The molecular mechanisms underlying "kiss and run" or "cavicapture" exocytosis of dense core secretory vesicles are presently unclear. Although dynamin-1 has previously been implicated in the recapture process in neurons, the recruitment of this fission protein to a single exocytosing vesicle has not been examined in real time during peptide release from pancreatic beta-cells. Imaged simultaneously in clonal insulin-secreting cells by dual color total internal reflection fluorescence microscopy, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y and green fluorescent protein (GFP)-tagged synaptotagmin-1 or synaptobrevin-2 rapidly diffused from sites of exocytosis, whereas the vesicle membrane protein phogrin and tissue plasminogen activator (tPA) were retained, consistent with fusion pore closure. Vesicle recovery frequently involved the recruitment of enhanced GFP-tagged dynamin-1, and GTPase-defective dynamin-1(K44E) increased the dwell time of tPA-mRFP at the plasma membrane. By contrast, recruitment of GFP chimeras of clathrin, epsin, and amphiphysin was not observed. Expression of dynamin-1(K535A), mutated in the pleckstrin homology domain, caused the apparent full fusion of vesicles, as reported by the additional release of tPA-mRFP (15-nm diameter) and enhanced GFP-tagged phogrin. We conclude that re-uptake of vesicles after peptide release by cavicapture corresponds to a novel form of endocytosis in which dynamin-1 stabilizes and eventually closes the fusion pore, with no requirement for "classical" endocytosis for retreat from the plasma membrane.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Use of targetable gfp-tagged neuropeptide for visualizing neuropeptide release following execution of a behavior

Previous work has shown that a transgene consisting of a fusion between the rat atrial natriuretic peptide and a green fluorescent protein reporter (ANF-gfp) is processed, localized, and released, as would be an endogenous neuropeptide when it is expressed in the nervous system of Drosophila melanogaster using the GAL4/UAS expression system. Here we have tested the utility of this targetable transgene for detecting neuropeptide release following the execution of a peptide-controlled behavior. For the behavior we used ecdysis, the behavior expressed by insects to shed their old cuticle at the end of the molt. We found that larval ecdysis was accompanied by a readily detectable reduction in gfp fluorescence from relevant secretory cells in the periphery and peptidergic neurons in the CNS. We also found that expression of the ANF-gfp products did not have detrimental effects on larval ecdysis or adult circadian rhythmicity, when the transgene was expressed in peptidergic cells that are known to control these behaviors. Finally, we used a broadly expressed GAL4 driver to show that the UAS-ANF-gfp transgene could be used to identify axons that show a reduction in gfp fluorescence following the expression of ecdysis behavior. These findings, coupled with the availability of an increasing number of strains bearing different GAL4 drivers, suggest that this transgene will be a useful tool for identifying peptidergic neurons and secretory cells (and, eventually, their secretory product) that release their peptide content during the occurrence, in the intact animal, of a developmental, physiological or behavioral process of interest.     

Imaging secretory vesicles by fluorescent protein insertion in propeptide rather than mature secreted peptide

We combined confocal and live-cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose-regulated insulin vesicle secretion. The 'Ins-C-GFP' reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins-C-GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins-C-GFP colocalized GFP with insulin at vesicle dense cores by immuno-electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C-GFP detected by anti-GFP Western blots, and of insulin detected by anti-insulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY^®TR, was applied to islets expressing Ins-C-GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides.     

Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster.

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.     

Use of targetable gfp‐tagged neuropeptide for visualizing neuropeptide release following execution of a behavior

Previous work has shown that a transgene consisting of a fusion between the rat atrial natriuretic peptide and a green fluorescent protein reporter (ANF‐gfp) is processed, localized, and released, as would be an endogenous neuropeptide when it is expressed in the nervous system of Drosophila melanogaster using the GAL4/UAS expression system. Here we have tested the utility of this targetable transgene for detecting neuropeptide release following the execution of a peptide‐controlled behavior. For the behavior we used ecdysis, the behavior expressed by insects to shed their old cuticle at the end of the molt. We found that larval ecdysis was accompanied by a readily detectable reduction in gfp fluorescence from relevant secretory cells in the periphery and peptidergic neurons in the CNS. We also found that expression of the ANF‐gfp products did not have detrimental effects on larval ecdysis or adult circadian rhythmicity, when the transgene was expressed in peptidergic cells that are known to control these behaviors. Finally, we used a broadly expressed GAL4 driver to show that the UAS‐ANF‐gfp transgene could be used to identify axons that show a reduction in gfp fluorescence following the expression of ecdysis behavior. These findings, coupled with the availability of an increasing number of strains bearing different GAL4 drivers, suggest that this transgene will be a useful tool for identifying peptidergic neurons and secretory cells (and, eventually, their secretory product) that release their peptide content during the occurrence, in the intact animal, of a developmental, physiological or behavioral process of interest. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 181–191, 2004     

A Highlights from MBoC Selection: Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein

Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO–tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K⁺] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.     

Synaptotagmin VII is targeted to dense-core vesicles and regulates their Ca2+ -dependent exocytosis in PC12 cells

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.     

Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104

We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)-tagged IDA-1. IDA-1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA-1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron-dense objects in the neuronal processes. The post-translational processing of IDA-1::GFP in transgenic worms was dependent on the neuropeptide proprotein convertase EGL-3, indicating that the protein was efficiently targeted to the peptidergic secretory pathway. Time-lapse epifluorescence microscopy of IDA-1::GFP revealed that DCVs moved in a saltatory and bidirectional manner. DCV velocity profiles exhibited multiple distinct peaks, suggesting the participation of multiple molecular motors with distinct properties. Differences between velocity profiles for axonal and dendritic processes furthermore suggested a polarized distribution of the molecular transport machinery. Study of a number of candidate mutants identified the kinesin UNC-104 (KIF1A) as the microtubule motor that is specifically responsible for anterograde axonal transport of DCVs at velocities of 1.6 microm/s-2.7 microm/s.     

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.     

Secretory granules: and the last shall be first..

By tagging secretory granules with the fluorescent protein dsRed-E5, which changes its emission from green to red over time, Duncan et al. analysed the age-dependent distribution of secretory vesicles within chromaffin cells. This elegant study illustrates as never before how age is a critical factor that segregates granules with respect to their localization and mobility and the probability of them undergoing exocytosis in response to different stimuli.