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 ...

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.

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
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
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

Janis E. Lochner

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