Abstract
Exocytosis is a crucial cellular process involved in the release of neural transmitters or signaling hormones, and disposal of waste or toxic materials. The relationship between structural transition and temporal progression of this process is poorly understood, partly due to lack of adequate tools to resolve such dynamic structures at sufficient resolution in 3D. Exocytosis can be hijacked by some viruses, exemplified by the widely used model α-herpesvirus pseudorabies virus (PRV), which relies on exocytosis for trans-synaptic spread across neurons. Here, we have used cryo electron tomography (cryoET) to capture 199 events of PRV exocytosis from cultured hippocampal neurons. We established cumulative frequency analysis to estimate the relative duration of an exocytosis stage based on the frequency of observed viral particles at that stage. This analysis revealed that PRV exocytosis is biphasic, including a fast, “release phase” driven by fusion proteins and fused membranes, and a slow, “recovery phase” driven by flattening of curved membranes. The biphasic property of exocytosis discovered here appears to be conserved for membrane fusion during viral entry, and our approach of cumulative frequency analysis should have general utility for characterizing other membrane fusion events.
Highlights
Exocytosis is a cellular process by which cells use secretory vesicles to transport bulk materials onto the plasma membrane or out of the cell
These particles were interpreted as progeny viral particles in the process of exocytosis for the following reasons: First, enveloped viruses were observed inside cellular secretory vesicles (Supplementary Fig. S1a), but pseudorabies virus (PRV) is known to enter the neuronal cells by direct fusion with the plasma membrane and not by transcytosis or endocytosis[23,24]
Neurons on grids infected with PRV were frozen at 14–16 hpi, which is the time established by others as the ideal time to observe progeny PRV transport and exocytosis[15,25,26]
Summary
Exocytosis is a cellular process by which cells use secretory vesicles to transport bulk materials onto the plasma membrane or out of the cell. It occurs during release of neural transmitters and signaling hormones[1], disposal of waste or toxic materials[2], as well as repair of membrane wounds[3,4]. PRV, a neurotropic α-herpesvirus, hijacks neuronal exocytosis mechanism to accomplish trans-synaptic spread, and has been widely used as a self-amplifying “live” tracer for the study of brain circuitry[11,12,13].
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