Morphology and force probing of primary murine liver sinusoidal endothelial cells

Abstract

Liver sinusoidal endothelial cells (LSECs) represent unique type of endothelial cells featured by their characteristic morphology, ie, lack of a basement membrane and presence of fenestrations-transmembrane pores acting as a dynamic filter between the vascular space and the liver parenchyma. Delicate structure of LSECs membrane combined with a submicron size of fenestrations hinders their visualization in live cells. In this work, we apply atomic force microscopy contact mode to characterize fenestrations in LSECs. We reveal the structure of fenestrations in live LSECs. Moreover, we show that the high-resolution imaging of fenestrations is possible for the glutaraldehyde-fixed LSECs. Finally, thorough information about the morphology of LSECs including great contrast in visualization of sieve plates and fenestrations is provided using Force Modulation mode. We show also the ability to precisely localize the cell nuclei in fixed LSECs. It can be helpful for more precise description of nanomechanical properties of cell nuclei using atomic force microscopy. Presented methodology combining high-quality imaging of fixed cells with an additional nanomechanical information of both live and fixed LSECs provides a unique approach to study LSECs morphology and nanomechanics that could foster understanding of the role of LSECs in maintaining liver homeostasis.

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

“…Our recent work demonstrated that even better insight into the dynamics of the fenestra structure could be achieved using multimodal AFM imaging. Dynamically tracking the detailed structure and dynamics of the fenestrae in vitro for the first time in living (ie, nonfixed) LSECs, enabled us to obtain time‐dependent high‐resolution images of the actin cytoskeleton that supports the fenestrae, confirming the previously reported fenestrae‐associated cytoskeletal structures . We showed that some FACRs are closed, that is, filled with lipid membrane, while others are open .…”

“…Because of the decreased stiffness (ie, relatively low Young's modulus) of living LSECs after CB treatment (see Figure ), we were unable to trace FACRs in real time . To further investigate the interconnection between actin and spectrin, we performed high‐resolution AFM imaging on fixed LSECs.…”

Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed

“…A virtual issue appeared in the Journal of Molecular Recognition in 2017 . Nine research articles composed this special issue . At the end of the conference, it was announced that the next one will be organized in Kraków, in 2017.…”

Gentle palpating liver sinusoidal endothelial cells reveals the dynamic behavior and formation of fenestrae: A new window for biomedical research

“…Although significant progress has been made on the structural and functional aspects of fenestrae since their original description in liver sinusoidal endothelial cells (LSECs), dynamic observations of life cells on a time‐lapse basis have been lacking until now. Over the past months, major breakthroughs on fenestra biology by Zapotoczny and colleagues have been reported by employing atomic force microscopy (AFM), providing liver sinusoidal cell biologists with the first glimpse of the increase versus decrease in fenestrae number under live‐cell imaging conditions in vitro (Fig. ).…”

“…In conclusion, this novel experimental AFM design reported by Zapotoczny et al should be considered as a future benchmark to study intact LSEC fenestra dynamics. One can foresee, when combined with multiarray AFM cantilevers and correlated imaging approaches, that high‐throughput screening of various bioactive substances will be in reach shortly. In support, the team of Zapotoczny succeeded in establishing a workflow that enables the automated quantification of fenestra size and number in a statistically rigorous manner .…”