Atomic Force Microscopy Reveals the Dynamic Morphology of Fenestrations in Live Liver Sinusoidal Endothelial Cells

Abstract

Here, we report an atomic force microscopy (AFM)-based imaging method for resolving the fine nanostructures (e.g., fenestrations) in the membranes of live primary murine liver sinusoidal endothelial cells (LSECs). From data on topographical and nanomechanical properties of the selected cell areas collected within 1 min, we traced the dynamic rearrangement of the cell actin cytoskeleton connected with the formation or closing of cell fenestrations, both in non-stimulated LSECs as well as in response to cytochalasin B and antimycin A. In conclusion, AFM-based imaging permitted the near real-time measurements of dynamic changes in fenestrations in live LSECs.

Tracking Fenestrae Dynamics in Live Murine Liver Sinusoidal Endothelial Cells

“…Initially, the FFCs in a variety of forms (floating, flattening, spiraling) were identified using electron microscopy . In our previous work using QI AFM, we demonstrated that after application of cytochalasin B, a compound that disturbs actin polymerization, expansion of newly formed sieve plates occurred in the direction of FFC migration. The application of jasplakinolide, an actin‐modulating agent promoting actin polymerization, resulted in the appearance of many discrete islands of nonfenestrated areas within the sieve plates of live LSECs.…”

“…Unless otherwise stated, LSECs were measured in 25 mM HEPES buffered medium. All images were acquired in force versus distance (FD)–based imaging mode (QI; JPK Instruments) according to a method described elsewhere . In brief, a single FD curve is performed in every pixel point of the image and then translated into images of topography and stiffness.…”

“…Topographical images were translated from the selected trigger force up to the maximum loading force used in the acquired FD curves. Linear fit to the selected area of the FD curve was used to calculate stiffness . An image is always acquired from the bottom to the top, and image acquisition time varies from tens of seconds to several minutes, dependent on the point‐to‐point resolution and scanning area size.…”

“…, , , and and Supporting Animations S1 and S5, where the most detailed structure of fenestrae‐associated structures measured for a prolonged time was necessary. The loading force used varied from 100 to 400 pN and was adjusted to obtain a clear image of fenestrae and cytoskeleton fibers at 80% of loading force . The obtained images of topography and stiffness were analyzed using JPK Data Processing Software.…”

“…Over the years there was a pursuit to visualize the ultrastructure of LSECs revealing the nature of fenestrae. Last year our team reported that the quantitative imaging (QI) mode of atomic force microscopy (AFM) allowed the relatively fast and high‐resolution dynamic imaging of fenestrae in cultured LSECs . Moreover, high‐resolution three‐dimensional (3‐D) imaging of fenestrae on fixed LSECs using superresolution fluorescence microscopy recently became possible, indicating the future potential to investigate live LSECs under relevant physiological conditions .…”