Neurotransmitters are released from nerve terminals and neuroendocrine cells by calcium-dependent exocytosis of vesicles. Before fusion, vesicles are docked to the plasma membrane and rendered release competent through a process called priming. Electrophysiological methods such as membrane capacitance measurements and carbon fiber amperometry accurately measure the fusion step of exocytosis with high time resolution but provide only indirect information about priming and docking. Total internal reflection fluorescence microscopy (TIRFM) enables the real-time visualization of vesicles, near the plasma membrane, as they undergo changes from one molecular state to the other. We devised a new method to analyze the mobility of vesicles, which not only allowed us to classify the movement of vesicles in three different categories but also to monitor dynamic changes in the mobility of vesicles over time. We selectively enhanced priming by treating bovine chromaffin cells with phorbol myristate acetate (PMA) or by overexpressing Munc13-1 (mammalian Unc) and analyzed the mobility of large dense-core vesicles. We demonstrate that nearly immobile vesicles represent primed vesicles because the pool of vesicles displaying this type of mobility was significantly increased after PMA treatment and Munc13-1 overexpression and decreased during tetanus toxin expression. Moreover, we showed that the movement of docked but unprimed vesicles is restricted to a confined region of ϳ220 nm diameter. Finally, a small third population of undocked vesicles showed a directed and probably active type of mobility. For the first time, we can thus distinguish the molecular state of vesicles in TIRFM by their mobility.
Calcium-activator protein for secretion (CAPS) is a cytosolic protein that associates with large dense-core vesicles and is involved in their secretion. Mammals express two CAPS isoforms, which share a similar domain structure including a Munc13 homology domain that is believed to be involved in the priming of secretory vesicles. A variety of studies designed to perturb CAPS function indicate that CAPS is involved in the secretion of large dense-core vesicles, but where in the secretory pathway CAPS acts is still under debate. Mice in which one allele of the CAPS-1 gene is deleted exhibit a deficit in catecholamine secretion from chromaffin cells. We have examined catecholamine secretion from chromaffin cells in which both CAPS genes were deleted and show that the deletion of both CAPS isoforms causes a strong reduction in the pool of rapidly releasable chromaffin granules and of sustained release during ongoing stimulation. We conclude that CAPS is required for the adequate refilling and/or maintenance of a rapidly releasable granule pool.
The calcium-dependent activator proteins for secretion, CAPS1 and CAPS2, facilitate syntaxin opening during synaptic vesicle priming.
Single sodium channel currents were studied in mouse neuroblastoma cells. Channel conductance (gamma) was 11.6 +/- 3.4 pS at 6-8 degrees C. Unitary current amplitudes and channel activity increased with increasing temperature. The Arrhenius plot of the conductance was linear between 5 and 35 degrees C with an activation enthalpy of 27.1 kJ/mole (Q10 = 1.28). Amplitude distributions were fitted by the sum of two Gaussian functions indicating the presence of two different single channel amplitudes: smaller i1 and larger i2. The relative probability of appearance of i1, which has a shorter mean open time, was higher during the early phase of depolarization (t less than 16 ms). The open time histograms, inactivating phase of macroscopic currents and delay time histograms were fitted by the sum of two exponentials. The distinct kinetic and steady-state parameters reflect two open states of sodium channels.
Total internal reflection fluorescence microscopy (TIRF-Microscopy) allows the observation of individual secretory vesicles in real-time during exocytosis. In contrast to electrophysiological methods, such as membrane capacitance recording or carbon fiber amperometry, TIRF-Microscopy also enables the observation of vesicles as they reside close to the plasma membrane prior to fusion. However, TIRF-Microscopy is limited to the visualization of vesicles that are located near the membrane attached to the glass coverslip on which the cell grows. This has raised concerns as to whether exocytosis measured with TIRF-Microscopy is comparable to global secretion of the cell measured with membrane capacitance recording. Here we address this concern by combining TIRF-Microscopy and membrane capacitance recording to quantify exocytosis from adrenal chromaffin cells. We found that secretion measured with TIRF-Microscopy is representative of the overall secretion of the cells, thereby validating for the first time the TIRF method as a measure of secretion. Furthermore, the combination of these two techniques provides a new tool for investigating the molecular mechanism of synaptic transmission with combined electrophysiological and imaging techniques.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.