Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.
Fenestrations are pores in liver sinusoidal endothelial cells that filter substrates and debris between the blood and hepatocytes. Fenestrations have significant roles in aging and the regulation of lipoproteins. However their small size (<200 nm) has prohibited any functional analysis by light microscopy. We employed structured illumination light microscopy to observe fenestrations in isolated rat liver sinusoidal endothelial cells with great clarity and spatial resolution. With this method, the three-dimensional structure of fenestrations (diameter 123+/-24 nm) and sieve plates was elucidated and it was shown that fenestrations occur in areas of abrupt cytoplasmic thinning (165+/-54 nm vs. 292+/-103 nm in non-fenestrated regions, P<0.0001). Sieve plates were not preferentially co-localized with fluorescently labeled F-actin stress fibers and endothelial nitric oxide synthase but appeared to occur in primarily attenuated non-raft regions of the cell membrane. Labyrinthine structures were not seen and all fenestrations were short cylindrical pores. In conclusion, three-dimensional structured illumination microscopy has enabled the unlimited power of fluorescent immunostaining and co-localization to reveal new structural and functional information about fenestrations and sieve plates.
Cogger
Victoria C
VC
Centre for Education and Research on Ageing and the ANZAC Research Institute, Sydney Medical School, University of Sydney and Concord RG Hospital, Sydney 2139, Australia. victoria.cogger@sydney.edu.au
McNerney
Gregory P
GP
Nyunt
Tun
T
DeLeve
Laurie D
LD
McCourt
Peter
P
Smedsrød
Bård
B
Le Couteur
David G
DG
Huser
Thomas R
TR
D000199:Actins; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D008853:Microscopy; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000199:Actins; D052250:Nitric Oxide Synthase Type III