Formation of spheroid structures in a human colon carcinoma cell line involves a complex series of intercellular...

The structural remodelling of tissues that occurs in vivo during animal morphogenesis can often prove difficult to study. Here we investigate the organizational processes of the LIM 1863 colon carcinoma cell line as it transforms from a single-cell stage into multicellular spherical structures called 'organoids'. The organoids can be dissociated into a viable single-cell suspension when cultured in calcium-depleted medium, and then induced to reform the organoid structure by the readdition of calcium. Previous studies have shown that initial cell attachment under these conditions is characterized by a novel mechanism of cell engulfment termed 'clutching'. This investigation reveals the subsequent appearance of junctional complexes between groups of 'clutched' cells prior to lumen formation, and the ultimate 'declutching' of entrapped cells as a means of cell rearrangement. Intact actin filaments but not microtubules were required for the initial clutching events, while inhibition of microtubule polymerization resulted in aberrant apical protein polarization, but did not affect the development of a luminal space within the spheroids. Single cells exhibited pools of intracellular microvilli contained in vacuolar apical compartments, which were resistant to the effects of cytoskeleton-disrupting drugs. However, these structures did not seem to be responsible for the swift development of the luminal surface observed in these cells. Two other cell lines, MDCK and DU 4475, were found to exhibit similar clutching conformations when induced to form three-dimensional structures, suggesting that this may be a widespread mechanism of cell rearrangement that reflects the process of organ morphogenesis in vivo.