Mechanisms of Transport and Exocytosis of Dense-Core Granules Containing Tissue Plasminogen Activator in Developing Hippocampal Neurons

Abstract

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.

Plasminogen and its fragments in rat brain: a plausible role for astrocytes in angiostatin generation

“…For years, Pg has been thought to be primarily a liver-derived protein [25], then the question is how widely Pg may be expressed in other organs, including the brain. Currently, there is an ample body of literature regarding the involvement of Pg/ Pm system in CNS development, normal neural functioning as well as neurologic disorders [1,2,5]. Our data expand evidences that rat brain cells are able to produce Pg and are in accordance with the previous reports indicating that Pg is expressed by specific populations of hippocampal, cortical and hypothalamic neurons in murine brain [13,26].…”

“…Pm generated on the cell surface modulates the astrocytic cytoskeleton and downstream signaling in astrocytes via the Rho/Rho kinase (ROCK) pathway [3]. It has been established that Pg/Pm system participates in synaptic plasticity, neurite outgrowth, and migration of granule cells during cerebellum development [4,5]. Though the main proteins of this system have been shown to be closely involved in regulation of many processes in central nervous system (CNS), of equal interest is the inquiry of functional significance of proteolytic fragments of Pg/Pm, referred to as angiostatins, in the CNS.…”

Matrix-Dependent Local Retention of Secretory Vesicle Cargo in Cortical Neurons

“…Whether these mechanisms also exist in neurons is unclear. Neuronal exocytosis may differ from neuroendocrine cells (Silverman et al, 2005).…”

Imaging of evoked dense-core-vesicle exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion events

“…By contrast, recently developed imaging techniques have allowed the tracking of individual vesicles and the visualization of single SV exocytic events in neurons (Aravanis et al, 2003;Gandhi and Stevens, 2003;Murthy et al, 1997;Ryan et al, 1997;Zenisek et al, 2000) and DCV exocytic events in endocrine cells (Steyer et al, 1997;Takahashi et al, 2002;Taraska et al, 2003). DCV transport in central neurons has also been visually tracked by fluorescence microscopy (Shakiryanova et al, 2006;Silverman et al, 2005), but the exocytosis of peptidergic DCVs has not been studied by single-vesicle imaging.…”