Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Stabilization of MyoD by direct binding to p57(Kip2).

Recent data have demonstrated the role of Cdk1- and Cdk2-dependent phosphorylation of MyoD(Ser200) in the regulation of MyoD activity and protein turnover. In the present study, we show that in presence of p57(Kip2), MyoD(Ala200), a MyoD mutant that cannot be phosphorylated by cyclin-Cdk complexes, displayed activity 2-5-fold higher than of MyoD(Ala200) alone in transactivation of muscle-specific genes myosin heavy chain, creatine kinase, and myosin light chain 1. Furthermore, p57(Kip2) increases the levels of MyoD(Ala200) in cotransfected cells. This result implies that p57(Kip2) may regulate MyoD through a process distinct from its function as a cyclin-dependent kinase inhibitors. We report that overexpression of p57(Kip2) increased the half-life of MyoD(Ala200). This increased half-life of MyoD involves a physical interaction between MyoD and p57(Kip2) but not with p16(Ink4a), as shown by cross-immunoprecipitation not only on overexpressed proteins from transfected cells, but also on endogenous MyoD and p57(Kip2) from C2C12 myogenic cells. Mutational and functional analyses of the two proteins show that the NH(2) domain of p57(Kip2) associates with basic region in the basic helix-loop-helix domain of MyoD. Competition/association assays and site-directed mutagenesis of the NH(2) terminus of p57(Kip2) identified the intermediate alpha-helix domain, located between the Cdk and the cyclin binding sites, as essential for MyoD interaction. These data show that the alpha-helix domain of p57(Kip2), which is conserved in the Cip/Kip proteins, is implicated in protein-protein interaction and confers a specific regulatory mechanism, outside of their Cdk-inhibitory activity, by which the p57(Kip2) family members positively act on myogenic differentiation.

Dimerization of the amino terminal domain of p57Kip2 inhibits cyclin D1-cdk4 kinase activity.

Previous studies have led to the proposal that a single molecule of Cki can associate with the cyclin/Cdk complex to repress its activity. On the other hand, multiple inhibitor molecules are required to inhibit Cdks. In the present work, by using differently tagged p57Kip2 proteins we demonstrate that p57Kip2 can bind to itself in vitro and in vivo. Mutational deletion analysis showed that the NH2 terminal domain of p57Kip2 is necessary and sufficient to dimerization. Using an in vitro competition/association assay, we demonstrate that cyclin D1 alone, Cdk4 alone and/or cyclin D1/Cdk4 complexes do not compete for the p57Kip2 homodimers formation. However, a mutation in the alpha-helix domain of p57Kip2 (R33L) strongly reduced homodimer formation but did not modify interaction with cyclin D1-Cdk4 complexes. Also, increasing amounts of p57Kip2 lead in vivo to a significant augmentation in the level of p57Kip2 homodimerization associated with cyclin D1-Cdk4 complexes and to a marked inhibition of the cyclin D1-Cdk4 kinase activity. Altogether, these data suggest a model whereby p57Kip2 associates with itself by using the NH2 domain to form a homodimeric species which interacts with and inhibits the cyclin D1-Cdk4 complexes.

p57(Kip2) stabilizes the MyoD protein by inhibiting cyclin E-Cdk2 kinase activity in growing myoblasts.

We show that expression of p57(Kip2), a potent tight-binding inhibitor of several G(1) cyclin-cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation. We examined the effect of p57(Kip2) on the activity of the transcription factor MyoD. In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57(Kip2), p21(Cip1), and p27(Kip1) but not p16(Ink4a) induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57(Kip2). Forced expression of p57(Kip2) correlated with hypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mutant arrested cells at the G(1) phase transition and induced hypophosphorylation of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57(Kip2). In addition, the NH2 domain of p57(Kip2) necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts. Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57(Kip2) could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.