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Role of actin-based contraction and scaffolding in hepatocyte polarization, generation of liver-specific microarchitecture and liver tissue functioning

Final Report Summary - LIVER (Role of actin-based contraction and scaffolding in hepatocyte polarization, generation of liver-specific microarchitecture and liver tissue functioning)

Liver function depends on not only the presence of specific cells in the tissue, but also on their characteristic arrangement into a unique microarchitecture. One of the crucial requirements for this microarchitecture is the establishment of correct cell polarity.

Generation of cell polarity involves the establishment of distinct cellular membrane domains – apical and basolateral, separated by tight junctions (TJs) and adherens junctions (AJs). Hepatocytes exhibit an apicobasal polarity that is more complex than the one in polarized columnar epithelial cells, e.g. the cells lining the kidney or the bile duct, which display a “simple” apico-basal polarization state (see schematic in Figure 1). The apical membranes of hepatocytes, however, form a three-dimensional (3D) narrow belt between adjacent cells, which collectively give rise to the bile canalicular (BC) network, an essential component for bile secretion and overall liver function.

Interestingly, during the development of the liver tissue, both hepatocytes (with hepatocytic polarity) and cholangiocytes (cells lining the bile duct acquiring the simple apico-basal polarity) share a common precursor cell called hepatoblast. This implies that the molecular machinery to generate the two different types of polarity is fundamentally the same, and that changes in activation of specific key pathways to inner or outer cues leads to the development of the distinct polarization morphology. Therefore, the primary aim of this project was to understand the molecular principles underlying the polarization of hepatocytes, and how the molecular machinery forming the simple versus hepatic apico-basal polarity is regulated to give rise to distinct morphological outcomes. Since actin filaments are enriched at the cell cortex underneath the apical plasma membrane in polarized cells, the project focused on the role of the actin machinery in shaping the apical region of polarized hepatocytes and the generation of the BC.

First, we investigated the mechanical properties of the apical surface, hypothesizing that the narrow condensed apical surface of the hepatocyte could result from stronger myosin contractility, and by inhibiting the ability of myosin to contract we could relax and open up the apical surface to mimic the characteristic simple apico-basal polarity. Even though most of the inhibitors affecting the myosin-dependent contractility led to the enlargement of the apical lumen in polarized hepatocytes, none of them resulted in changing the type of polarization to simple apico-basal polarity. Moreover, laser ablation of actin filaments underneath the apical surface did not induce apical surface opening. Thus, mechanical tension does not play a role in determining/switching the type of polarity between hepatocytic and simple apico-basal polarity.

To identify the key molecular pathways involved in the shaping of the one-of-a-kind hepatocytic apical domain, we performed RNA deep sequencing on our samples. In collaboration with the sequencing facility at CRTD (Center for Regenerative Therapies Dresden), and our in-house bioinformatics experts, we performed RNA deep sequencing on samples reflecting either (i) unpolarized, (ii) simple apico-basally polarized or (iii) hepatocytically polarized cells. This dataset proved to be invaluable to the progress of this project in terms of highlighting pathways of interest for further studies, as well as corroborating information from the studies performed in parallel on cells.

In parallel with the RNA sequencing and bioinformatics analysis, we hypothesized that visualizing the actin filaments and their network underneath the apical surface could be essential in assisting in narrowing down the possible mechanism by which the apical surface is shaped and draw connections between the microscopic and RNA sequencing data. Structured illumination microscopy (SIM) was chosen due to the best imaging outcome. Detailed image analysis, in collaboration with Dr. F. Jug and B. Lombardot, revealed that the actin filaments in hepatocytic polarity are longer, more interconnected, and display a different orientation with more branched Y-shaped structures than in simple apico-basal polarity.

From the SIM data analysis, and in corroboration with the RNA deep sequencing data, we hypothesized that actin severing and capping pathways and their distinct regulation could be responsible for the formation of distinct polarity states. This hypothesis is being tested concurrently with over-expression techniques in polarized hepatoblasts, and siRNA knock-down in MDCK cells and cholangiocytes. If successful, the next steps will follow with testing on developing liver models, such as over-expression in adult mice with 70% hepatectomy, a technique fully developed in our lab at MPI-CBG by a colleague (Dr. K. Meyer).