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Roles of cholesterol in vesicle fusion and motion.

ABSTRACT: Although it is well established that exocytosis of neurotransmitters and hormones is highly regulated by numerous secretory proteins, such as SNARE proteins, there is an increasing appreciation of the importance of the chemophysical properties and organization of membrane lipids to various aspects of the exocytotic program. Based on amperometric recordings by carbon fiber microelectrodes, we show that deprivation of membrane cholesterol by methyl-beta-cyclodextrin not only inhibited the extent of membrane depolarization-induced exocytosis, it also adversely affected the kinetics and quantal size of vesicle fusion in neuroendocrine PC12 cells. In addition, total internal fluorescence microscopy studies revealed that cholesterol depletion impaired vesicle docking and trafficking, which are believed to correlate with the dynamics of exocytosis. Furthermore, we found that free cholesterol is able to directly trigger vesicle fusion, albeit with less potency and slower kinetics as compared to membrane depolarization stimulation. These results underscore the versatile roles of cholesterol in facilitating exocytosis.

SUBMITTER: Zhang J

PROVIDER: S-EPMC2749761 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Roles of cholesterol in vesicle fusion and motion.

Zhang Jing J Xue Renhao R Ong Wei-Yi WY Chen Peng P

Biophysical journal 20090901 5

Although it is well established that exocytosis of neurotransmitters and hormones is highly regulated by numerous secretory proteins, such as SNARE proteins, there is an increasing appreciation of the importance of the chemophysical properties and organization of membrane lipids to various aspects of the exocytotic program. Based on amperometric recordings by carbon fiber microelectrodes, we show that deprivation of membrane cholesterol by methyl-beta-cyclodextrin not only inhibited the extent o ...[more]

PMID: 19720025

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Project description:Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Although cholesterol has been associated with the binding and entry of various enveloped viruses into cells, cholesterol's exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MβCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (VSV)-pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes.IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here, we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytium formation and virus-cell membrane fusion during viral entry. Furthermore, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.

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Roles of cholesterol in vesicle fusion and motion.

Publications

Roles of cholesterol in vesicle fusion and motion.

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