The structural-functional hallmark of liver sinusoidal endothelial cells (LSEC) is the presence of fenestrae (or fenestrations) grouped in sieve plates. Fenestrae are transcellular pores in 50-300 nm supported by a (sub)membranous cytoskeletal lattice. Their function covers the bidirectional transport of solutes and macromolecules between the blood vessels and hepatocytes. Therefore, any alterations in the number and/or size of fenestrae may impair liver homeostasis. Isolated LSECs in culture lose their characteristic phenotype in 1-3 days, hindering the research of fenestrae. For nearly five decades, the hunt has been open to depict fenestrae under live and relevant physiological conditions (Braet and Wisse 2017). Over the last decades, beautiful research was made on fenestrated LSECs using electron microscopy. Fixed at a certain time, previously drug-treated LSECs were investigated using scanning and transmission electron microscopy. Recently, with the development of optical nanoscopy also fluorescence-based techniques revealed actin-built net forming fenestrae. Moreover, research using correlative atomic force microscopy (AFM) and direct stochastic optical reconstruction microscopy (dSTROM) indicate that spectrin-actin cytoskeleton is responsible for open fenestrae (Zapotoczny et al. 2019a). However, only with the development of atomic force microscopy (AFM) based techniques, it became possible to track the dynamic structure of fenestrae in living cells in time (Zapotoczny et al. 2019b). By performing multiple force-distance curves in millisecond time, the mode named Quantitative Imaging (QI) allowed scanning the topography of LSECs with unprecedented resolution, minimizing the lateral forces. AFM remains a unique methodology to investigate real-time alterations in LSECs for a prolonged time.
In our current research, here at the Department of Biophysical Microstructures, we focus on elasticity driving the number of fenestrae in isolated murine LSECs. Having the complex information about the course of the force curves collected in each pixel point of the image, we can calculate nanomechanical properties (e.g. Young modulus) distribution over the image. It allowed us to investigate the crosstalk between the fenestrated morphology, polymerization of the actin cytoskeleton, and elasticity of LSECs (Zapotoczny et al. 2020). We believe that by understanding the changes in the cytoskeleton of LSECs we could answer the questions about why LSECs lose their fenestrated morphology so fast in culture. Our research is conducted in close collaboration with prof. Stefan Chlopicki from Jagiellonian Centre for Experimental Therapeutics (JCET). Together, in 2020 we have started the project Sonata 15 “From the structure of fenestrations in live Liver Sinusoidal Endothelial Cells to real-time in vitro pharmacology”.
- F. Braet, E. Wisse – Gentle palpating liver sinusoidal endothelial cells reveals the dynamic behavior and formation of fenestrae – a new window for biomedical research – Hepatology 67 (2017)1–36.
- B. Zapotoczny, F. Braet, E. Kus, et al. – Actin‐Spectrin Scaffold Supports Open Fenestrae in Liver Sinusoidal Endothelial Cells – Traffic 20 (2019a) 932-942.
- B. Zapotoczny, F. Braet, E. Wisse, et al. – Biophysical nanocharacterization of liver sinusoidal endothelial cells through atomic force microscopy – Biophys Rev 12 (2020) 625–636.
- B. Zapotoczny, K. Szafranska, E. Kus, et al. – Tracking Fenestrae Dynamics in Live Murine Liver Sinusoidal Endothelial Cells – Hepatology 69 (2019b) 876–888.