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

For nearly five decades the hunt has been on to depict fenestrae under live and relevant physiological conditions. Although significant progress has beenmade 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. 1). The relevance of those observations is paramount as the processes of hyperfenestration and defenestration are implemented in numerous human diseases such as fibrosis, hepatitis, steatosis, aging, and drug-induced liver failure. Detailed studies have shown that fenestrae grouped in sieve plates act as a dynamic filter, the liver sieve, which filters solutes and particles that are exchanged between the liver sinusoids and the space of Disse. Adverse changes to the bidirectional nature of the liver sieve hamper normal liver function. Previous reports on fenestra structure and changes in number and diameter were mainly restricted to “static” electron microscopic observations in situ or in vitro on chemically or physically fixed LSECs. Despite the power of electron microscopy to resolve fine structure, this imaging approach excludes the disclosure of dynamic subcellular detail at high temporal resolution. As such, outstanding questions concerning fenestra size changes, formation, and disappearance have been largely unanswered. Since the inception of the Nobel Prize–winning AFM in 1986, this microscopic tool, which uses a fine tip mounted on a flexible cantilever to probe the sample at ambient conditions, has been used to study various structure–function relationships. AFM has been applied to a diverse set of liver cells, enabling the exploration of different aspects in liver health and disease. The article published in the August issue of Scientific Reports by Zapotoczny et al., however, is a game changer as it allowed the team to depict fenestrae under artifact-free live-cell imaging conditions at an unsurpassed timeresolving resolution of 1 millisecond per pixel, enabling the depiction of a single sieve plate in a time window as short as 30 seconds per image frame. Taking advantage of the latest advances in force spectroscopy-based AFM imaging, the authors elegantly illustrated the rapid rise of newly born fenestrae under the influence of the actin-disrupting drug cytochalasin B. Conversely, in another experimental set, the authors captured the rapid decline in the number of fenestrae when exposed to the adenosine triphosphate-depleting agent antimycin A. Those data elaborated earlier electron microscopic observations by filling in missing pieces of the “image” puzzle. The dawn of fenestra-forming centers and the closure of fenestrae could be revealed. In addition, and maybe more importantly, the AFM images informed the scientist about the fast turnover and short life span of fenestrae under different experimental conditions. Notable is the relevance of the data as LSECs were subjected to minimum sample manipulation steps that would otherwise adversely affect cell viability and intact surface topology, underpinning once more the importance of the data. In line, this innovative AFM imaging mode allowed the team to image live LSECs at minimum interaction forces (i.e., 100-300 pN), turning around previous attempts to dynamically study sieve plates and fenestra regulation (reviewed in Braet et al.) 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. Such large cell-screening assays at unprecedented speed and resolution will enable full documentation of the morphological and biochemical properties of LSEC fenestrae and, as