A role for microtubules in endothelial cell protrusion in three-dimensional matrices.

Most cells reside in vivo in a three-dimensional (3D) environment surrounded by extracellular matrix and other neighbouring cells, conditions that are different from those found by cells cultured in vitro on two-dimensional (2D) substrata. Cell morphology and behaviour are very different under these two different conditions, but the structural basis for these differences is still not understood, especially the role of microtubules (MTs). To address this issue, we studied the early spreading behaviour of bovine aortic endothelial cells (BAECs) cultured in 3D collagen matrices and on 2D substrata, in the presence of MT-disrupting drugs. We found that depolymerisation of MTs greatly reduces the ability of BAECs to form large and stable protrusions inside 3D collagen matrices, an effect that is less pronounced when the cells are cultured on 2D substrata. Colcemid-treated BAECs inside 3D matrices begin assembling protrusions and pull on the matrix, but they fail to extend those protrusions deep into the matrix. It has been previously reported that MT disruption affects Rho signalling which may result in increased cell rigidity and adhesiveness to 2D matrices. Accordingly, we demonstrate that colcemid treatment indeed leads to activation of Rho-kinase (ROCK) targets, which in turn results in activation of regulatory myosin light chains, and that blocking of ROCK mitigates some of the effects of MT disruption in cell spreading in 3D. Our results show that MT depolymerisation is particularly disruptive when cells interact with pliable 3D matrices, suggesting a role for MTs and the Rho pathway in the fine-tuning of contractile and adhesive forces necessary to sustain cell motility in vivo.