2 research outputs found
Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells
Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules
and solutes between the blood and hepatocytes. The efficiency of this transport is realized via
transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with
the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The
cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a
hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent
phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of
several molecular inhibitors and by applying two high-resolution microscopy modalities: structured
illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise,
dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range
from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the
ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new
fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents.
We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration
diameter, while the inhibition of MLCK prevents the formation of new fenestrations
From fixed-dried to wet-fixed to live-comparative super-resolution microscopy of liver sinusoidal endothelial cell fenestrations
Fenestrations in liver sinusoidal endothelial cells
(LSEC) are transcellular nanopores of 50–350 nm diameter
that facilitate bidirectional transport of solutes and macromolecules between the bloodstream and the parenchyma of
the liver. Liver diseases, ageing, and various substances such
as nicotine or ethanol can negatively influence LSECs fenestrations and lead to defenestration. Over the years, the
diameter of fenestrations remained the main challenge for
imaging of LSEC in vitro. Several microscopy, or rather
nanoscopy, approaches have been used to quantify fenestrations in LSEC to assess the effect of drugs and, and toxins in
different biological models. All techniques have their limitations, and measurements of the “true” size of fenestrations
are hampered because of this. In this study, we approach the
comparison of different types of microscopy in a correlative
manner. We combine scanning electron microscopy (SEM)
with optical nanoscopy methods such as structured illumination microscopy (SIM) or stimulated emission depletion
(STED) microscopy. In addition, we combined atomic force
microscopy (AFM) with SEM and STED, all to better understand the previously reported differences between the reports
of fenestration dimensions. We conclude that sample dehydration alters fenestration diameters. Finally, we propose the
combination of AFM with conventional microscopy that allows for easy super-resolution observation of the cell dynamics with additional chemical information that can be
traced back for the whole experiment. Overall, by pairing the
various types of imaging techniques that provide topological
2D/3D/label-free/chemical information we get a deeper
insight into both limitations and strengths of each type microscopy when applied to fenestration analysis