Actin‐spectrin scaffold supports open fenestrae in liver sinusoidal endothelial cells

Abstract

Fenestrae are open transmembrane pores that are a structural hallmark of healthy liver sinusoidal endothelial cells (LSECs). Their key role is the transport of solutes and macromolecular complexes between the sinusoidal lumen and the space of Disse. To date, the biochemical nature of the cytoskeleton elements that surround the fenestrae and sieve plates in LSECs remain largely elusive. Herein, we took advantage of the latest developments in atomic force imaging and super‐resolution fluorescence nanoscopy to define the organization of the supramolecular complex(es) that surround the fenestrae. Our data revealed that spectrin, together with actin, lines the inner cell membrane and provided direct structural support to the membrane‐bound pores. We conclusively demonstrated that diamide and iodoacetic acid (IAA) affect fenestrae number by destabilizing the LSEC actin‐spectrin scaffold. Furthermore, IAA induces rapid and repeatable switching between the open vs closed state of the fenestrae, indicating that the spectrin‐actin complex could play an important role in controlling the pore number. Our results suggest that spectrin functions as a key regulator in the structural preservation of the fenestrae, and as such, it might serve as a molecular target for altering transendothelial permeability.

Biophysical nanocharacterization of liver sinusoidal endothelial cells through atomic force microscopy

“…The nanometre-size transcellular pores were reported to respond to a variety of external stimuli becoming a great marker for the functional responsiveness of LSECs. Interestingly, there are only a few reports available, detailing the elastic properties of LSECs (Braet et al 1998b;Kus et al 2019;Zapotoczny et al 2019a). More than two decades ago, a first attempt was made to depict fenestrae in living LSECs.…”

“…Recently, we documented in detail the nanomechanical properties of this type of fenestrated sinusoidal endothelium exposed to various experimental conditions under live cell imaging conditions (Zapotoczny et al 2017a(Zapotoczny et al , 2019aRusaczonek et al 2019). Furthermore, significant progress was made in the detailed analysis on the organization of the cytoskeleton associated with fenestrae and sieve plates.…”

“…Using 4-D AFM, the fenestrae responses to a certain drug formulation can be readily assessed. Our method allows adding an agent (drug or toxin) to the medium and continues measurements in the same region (Zapotoczny et al 2017c(Zapotoczny et al , 2019a. This allowed us to observe the realtime response of the cell to an agent.…”

“…In a recent report, we showed that destabilization of spectrin-actin cytoskeleton forming FACR leads to rapid closing of fenestrae and also can cause fenestrae "blinking", i.e. a rapid switching between open and close state of fenestra (Zapotoczny et al 2019a). Those processes can only be analysed using live cell imaging techniques.…”

“…In particular, changes in the organization of LSEC-cytoskeleton were shown to alter the number and size of fenestrae, and thus the filtration efficiency of LSECs (Steffan et al 1987). Different elements of the cytoskeleton were reported to affect the porosity of LSECs, like actin, tubulin, myosin and spectrin (Steffan et al 1987;Braet et al 1996a;Gatmaitan et al 1996;Zapotoczny et al 2019a). Among others, the actin cytoskeleton has been studied widely in the last decades (Braet et al 1998b;Braet and Wisse 2002;Hunt et al 2019).…”