Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes.
Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.
Mukherjee
Soumi
S
Centre for Cellular and Molecular Biology, Hyderabad, India.
Kalipatnapu
Shanti
S
Pucadyil
Thomas J
TJ
Chattopadhyay
Amitabha
A
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure*; D002784:Cholesterol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology*; D066298:In Vitro Techniques; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism
C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D005456:Fluorescent Dyes; D010714:Phosphatidylethanolamines; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan