CYTOSKELETON STUDIES

POLARITY STUDIES

Multicellular reorganization is intimately coupled to the dynamics of the actin cytoskeleton through integrin-mediated cell adhesion and cadherin-mediated cell-cell interactions. Hepatocytes seeded on Primaria dishes initially form monolayers where they develop an extensive network of stress fibers throughout the cytoplasm (Fig. 1). The stress fibers are linked to the cell-substratum contacts via focal adhesion complexes. These contacts act as cell tethers to the substratum. In the early stage of spheroid formation, adjacent cells form monolayer patches and establish intercellular contacts. Cells within a monolayer patch pull each other together to form multi-layer patches and eventually aggregates. Treatment with cytochalasin D, which disorganizes microfilament organization, hinders the self-assembly process (Fig. 2). Thus, the initial stage of hepatocyte spheroid self-assembly can be postulated as mechanistically directed in part by the development of stress fibers and the establishment of cell-cell contacts.

The organization of actin filaments undergoes significant changes as the spheroid self-assembly progresses. The distributed stress fiber network in cell patches gradually localize along the cell cortex as spheroids are formed (Fig. 3). The cortical microfilament configuration favors the compaction of hepatocyte aggregates, as actomyosin filaments are considered contractile elements. This is also observed in many epithelial tissues in vivo, where bundles of cortical microfilaments generate and transmit tension orchestrating a series of morphological changes. Parallel to in vivo tissue, hepatocyte aggregates undergo compaction resulting in spheroids with a smooth external surface. Conceivably, the compaction is expected to be hindered if the microfilament lattice is disrupted. Indeed, at low concentrations of cytochalasin D, the hepatocytes form loose aggregates but do not form compacted spheroids. At higher cytochalasin D concentrations, the formation of aggregates is completely inhibited. Thus, the results suggest that the integrity of the microfilament lattice is critical for hepatocyte spheroid self-assembly.