The role of microtubules on cell secretory processes, membrane traffic, and organelle and mRNA transport has been studied extensively. However, their mechanical role on tissue formation is less well understood. To determine the role of microtubules on hepatocyte spheroid formation, cells were cultured in the presence of microtubule-disorganizing drugs such as taxol and nocodazole. Taxol causes much of the free tubulin to assemble into microtubules creating disordered microtubule networks while nocodazole disrupts the microtubules by promoting their depolymerization. The spheroid self-assembly in the presence of these drugs proceeds as in normal cultures, i.e. in the absence of any cytoskeleton-disrupting drug. Incubation with taxol or nocodazole compromised the microtubule network but did not affect the morphology of spheroids. In order to visualize the microtubule network, hepatocytes are transfected with a plasmid encoding the co-expression of GFP and a-tubulin. Observation is done under a confocal microscope. The delivery of the GFP-a-tubulin plasmid and its subsequent expression in the cultured cells enables the visualization of the delicate microtubule structure in its natural intracellular environment. In this way, the risk of generating artifacts due to specific antibody raising and fixation of the cells as in immunofluoresence microscopy techniques is eliminated. A continuous microtubule network is clearly displayed by spread hepatocytes in control cultures as indicated by the characteristic GFP fluorescence (Fig. 1). Conversely, spread hepatocytes treated with taxol or nocodazole exhibited no organized microtubules.

Figure 1. Microtubule network of spread hepatocytes in control cultures and after incubation with taxol [click on each fluorescence image for a series of sections through the spheroid (QuickTime movies)].

Hepatocyte microtubules were also visualized in five-day spheroids. Confocal microscopic images of cells collected at different depths within mature spheroids revealed a fluorescent filamentous structure in the absence of drugs whereas individual microtubules were not seen in the cells of spheroids incubated with taxol or nocodazole. Instead, a few bright spots of fluoresence probably corresponding to aggregates of altered microtubules, commonly observed in taxol, were visible in the perinuclear regions and elsewhere in the cytoplasm.We found that the cell spreading and patch formation is not affected by treatment with microtubule-disorganizing drugs. In the presence of taxol or nocodazole, hepatocytes preserve their planar shape as well as their contacts with neighboring cells and the substratum and proceed to form aggregates. Despite the lack of organized microtubules as revealed by confocal microscopy, the resulting cell clusters underwent compaction, giving rise to spheroids with indistinguishable cell-cell boundaries on the exterior. Therefore, unlike in the case of actin filaments, microtubule integrity is not imperative for efficient hepatocyte spheroid self-assembly.

Figure 2. Microtubule network in hepatocytes within spheroids: in control cultures and after incubation with taxol [click on each fluorescence image for a series of sections through the spheroid (QuickTime movies)].