Microtubule dynamics regulation contributes to endothelial morphogenesis

Abstract

Because little is known how microtubules contribute to cell migration in a physiological three-dimensional environment, we analyzed microtubule function and dynamics during in vitro angiogenesis in which endothelial cells form networks on a reconstituted basement membrane. Endothelial network formation resulted from distinct cell behaviors: matrix reorganization by myosin-mediated contractile forces, and active cell migration along reorganized, bundled matrix fibers. Inhibition of microtubule dynamics inhibited persistent cell migration, but not matrix reorganization. In addition, microtubule polymerization dynamics and CLASP2-binding to microtubules were spatially regulated to promote microtubule growth into endothelial cell protrusions along matrix tension tracks. We propose that microtubules counter-act contractile forces of the cortical actin cytoskeleton and are required to stabilize endothelial cell protrusions in a soft three-dimensional environment.

Microtubules in 3D cell motility

“…In fact, even low doses of different microtubule-targeting agents (MTAs), which were insufficient to block cell division, impaired matrix invasion by MDA-MB-231 cells (Tran et al, 2009). Microtubule requirement for pseudopod-based motility in 3D was also tested and confirmed by MTA-based approaches in endothelial cells (Lyle et al, 2012;Martins and Kolega, 2012;Pourroy et al, 2006). Nocodazole treatment also caused defects in elongation of invadopodia, which are small specialized protrusions observed in tumor cells that are thought to play a role in cancer invasion (Di Martino et al, 2016;Schoumacher et al, 2010).…”

The β3-integrin endothelial adhesome regulates microtubule dependent cell migration

“…Although no gross changes in cell microtubule arrays were observed, the increased bundling of microtubules in the β3HET and β3NULL ECs suggests they could be crosslinked by microtubule stabilising proteins. Microtubule bundles are known to form in ECs to assist with directional migration by counteracting actomyosin generated contractile forces; these bundles often target adhesion sites in EC protrusions (Lyle et al, 2012).…”

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

“…Over a time course of 10-12 hr, polarized wound-edge HUVECs migrate into the wound area and fill the wound, establishing a new confluent monolayer ( Figure 4A, t = 11 hr). As with HUVEC branching, current evidence suggests that polarization and migration is critically dependent upon the coordinated re-organization of the MT and actin cytoskeletons [43][44][45][46][47][48][49] . This is achieved, in part, through localized inhibition of MCAK-induced MT disassembly at the leading edge, but not at the trailing edge of wound-edge HUVECs 31 .…”

“…This is achieved, in part, through localized inhibition of MCAK-induced MT disassembly at the leading edge, but not at the trailing edge of wound-edge HUVECs 31 . The sensitivity of MT dynamics to wound-induced polarization is initiated by the Rac1 GTPase, which targets numerous downstream regulator proteins, including Aurora-A kinase that phosphorylates and inhibits MCAK at the leading edge, and other kinases that are capable of inhibiting or activating MAPs to locally modulate MT growth and shrinkage 18,31,36,48,[50][51][52][53] . Extensive experimental evidence supports the notion that coordination of leading edge protrusion and trailing edge contraction is governed through cycles of Rac1 activation to promote MT assembly at the leading edge and RhoA activation to acto-myosin contractility at the trailing edge, thereby driving directed motility 9,41,51,52,[54][55][56][57][58] .…”