super-resolution-imaging-of-neuronal-dense-core-vesicles

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 ...

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.

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

Kehr-Modell von Dynamin Aktivit&#xE4;t. Visualisierung der Kopplung zwischen Exozytose und Endozytose unter einer abklingenden Welle Mikroskop mit gr&#xFC;n fluoreszierenden Proteine.

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.

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

Neuronalen Calcium-Sensor-1 bindet an sekretorischen Organellen und Funktionen in basale und stimulierte Exozytose in PC12-Zellen.

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.

Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies.

Retraction. Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies.

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.

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

Mechanismen der Transport-und Exozytose von Dense-Core-Granulaten, die Gewebe-Plasminogen-Aktivator bei der Entwicklung von Neuronen im Hippocampus.

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.

How neurosecretory vesicles release their cargo.

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.

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

Tissue Plasminogen Activator (tPA) hat in einer Vielzahl von wichtigen Zellfunktionen, einschlie&szlig;lich synaptische Plastizit&auml;t im Zusammenhang mit lernen und potentiating N-Methyl-D-Aspartat (NMDA)-Rezeptor-abh&auml;ngige Signalisierung verwickelt. Diese Forschungsergebnisse legen nahe, dass tPA lokalisieren zu kann, und leistungsabh&auml;ngige Sekretion von Synapsen zu unterziehen; schl&uuml;ssige Daten unterst&uuml;tzen diese Hypothesen sind jedoch schwer fassbaren geblieben. Um diese Fragen zu kl&auml;ren, untersucht wir die Verteilung, die Dynamik und die Depolarisierung induziert Sekretion von tPA in Neuronen, mit fluoreszierende Chim&auml;ren der tPA. Wir fanden, dass tPA im dichten Kern Granulat (DCGs) befindet, die Datenverkehr postsynaptisches dendritische Dornen und Stacheln, &uuml;ber einen l&auml;ngeren Zeitraum bleiben kann. Wir fanden auch die Depolarisierung induziert durch hohe Kalium Ebenen eine langsame, teilweise exocytotic Freisetzung von tPA aus DCGs in Stacheln hervorruft, die extrazellul&auml;re Ca(+2) Konzentrationen abh&auml;ngig ist. Dieser langsame, teilweise Version veranschaulicht die Exozytose erfolgt &uuml;ber einen Mechanismus, wie z. B. Sicherung-Prise-verweilen, die partielle Freigabe und Wiederverwendung von DCG-Ladung erm&ouml;glicht und schl&auml;gt einen Mechanismus, der Neuronen berufen k&ouml;nnen, um zu vermeiden, abbauende tPA bei aktiven Synapsen. Unsere Ergebnisse zeigen auch die Freisetzung von tPA an einem Standort, der Interaktion mit NMDA-Typ Glutamatrezeptoren erleichtert, und sie bieten direkte Best&auml;tigung der grundlegenden Hypothesen &uuml;ber die tPA-Lokalisierung und Ver&ouml;ffentlichung, die auf seinen Neuromodulatory Funktionen, z. B. in lernen und Ged&auml;chtnis zu tragen.

T&auml;tigkeit-abh&auml;ngige Freisetzung von Tissue Plasminogen Activator aus dendritischen Dornen von Neuronen, die von Leben-Zelle Darstellung offenbart.

Article	Year
Sweeping model of dynamin activity. Visualization of coupling between exocytosis and endocytosis under an evanescent wave microscope with green fluorescent proteins. The Journal of biological chemistry\| PubMed ID: 11856729	2002
Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells. Journal of cell science\| PubMed ID: 12006624	2002
Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies. Nature biotechnology\| PubMed ID: 14608366	2003
Retraction. Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies. Nature biotechnology\| PubMed ID: 15122294	2004
Mechanisms of transport and exocytosis of dense-core granules containing tissue plasminogen activator in developing hippocampal neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 15788766	2005
How neurosecretory vesicles release their cargo. The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry\| PubMed ID: 16514013	2006
Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging. Journal of neurobiology\| PubMed ID: 16555239	2006

Bethe A. Scalettar

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