Date Published: September 24, 2012
Publisher: Public Library of Science
Author(s): Dmitri Svistounov, Alessandra Warren, Gregory P. McNerney, Dylan M. Owen, Dusan Zencak, Svetlana N. Zykova, Harry Crane, Thomas Huser, Ronald J. Quinn, Bård Smedsrød, David G. Le Couteur, Victoria C. Cogger, Bernadette Allinquant. http://doi.org/10.1371/journal.pone.0046134
Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.
Liver sinusoidal endothelial cells (LSECs) act as a filter between the lumen of the hepatic sinusoid and the surrounding hepatocytes. A major role of the LSEC is to minimize any barrier for the bi-directional transfer of small or soluble substrates between blood and the extracellular space of Disse, while excluding larger circulating particles such as blood cells, platelets and chylomicrons. This physiological role is achieved by the presence of numerous transcellular pores in LSECs called fenestrations. Fenestrations are approximately 50–150 nm in diameter and most are aggregated into groups of 10–100, so-called liver sieve plates . The diameter and number of fenestrations are altered by various liver diseases, diabetes mellitus and old age and are influenced by cytokines and hormones . Alteration in the size and number of fenestrations influences the hepatic trafficking of lipoproteins , clearance of pharmaceutical agents , liver regeneration  and interactions between lymphocytes and hepatocytes .
Fenestrations and rafts are both cell membrane structures that are below the limit of resolution of light microscopy . The morphology of fenestrations has been studied primarily using electron microscopy while that of rafts has been studied using fluorescence microscopy . 3D-SIM is a super-resolution fluorescence microscopy technique that provides the opportunity to simultaneously study both membrane rafts and fenestrations and their distribution in isolated LSECs. 3D-SIM provides high resolution images of fenestrations and associated structures, such as the cellular cytoskeleton . Here, we also applied 3D-SIM to visualize membrane rafts. Using the fluorescent raft stain, Bodipy FL C5 ganglioside GM1, membrane rafts were found to be aggregated preferentially in the perinuclear region of LSECs, with a more diffuse distribution in the peripheral cytoplasmic extensions, and were generally thicker than the surrounding cell membrane. This pattern of distribution of rafts was confirmed using TIRFM with two raft stains, Bodipy FL C5 ganglioside GM1 and NBD-cholesterol. With 3D-SIM, a few clustered rafts sections were also apparent in the peripheral regions of the cells. These were about 1–2 µm in diameter and some had a raised perimeter, consistent with predictions based on line tension . As reported previously , 3D-SIM revealed that fenestrations are clustered in groups of 10–100 fenestrations called liver sieve plates that occupy 5–10% of the entire cell membrane. Moreover, the SIM images revealed that there is a distinct inverse distribution between liver sieve plates and membrane rafts. On the basis of this observation, we investigated whether manipulating membrane rafts had any effect on fenestrations.