Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis

Abstract

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging

“…All cells were cultured and maintained at 37°C in a 5% CO 2 environment. Gene transduction using lentivirus or adenovirus gene transfer was performed on HUVEC cultures as described [7, 10]. …”

“…The pFUGW vector was sufficiently used to mediate gene transduction into the endothelial cells [7, 10, 13, 16, 17]. The pFUGW vector is highly sufficient compared to an earlier used lentivirus vector that exhibited low gene transduction efficacy [18], as this vector apparently lacks the cis -acting element, the central polypurine tract, that is important for transduction of nonproliferating cell types [19].…”

“…Excitation and emission wave length was adjusted accordingly. Laser scanning microscopy (LSM) was performed as described elsewhere [10]. Briefly, confocal LSM was carried out using a Zeiss Elyra/LSM 780 microscope.…”

“…A highly dynamic actin-driven structure, called junction-associated intermittent lamellipodia (JAIL) was identified, which leads directly to the formation of new VE-cadherin-mediated cell adhesion sites. JAIL appear constitutively and thereby ensure the integrity of endothelial cell contacts, but are also responsible for coordination of cell contact dynamics during cell migration and angiogenesis [7, 10]. It has also been demonstrated that the relative VE-cadherin concentration (Rel-VEcad-C), which is defined by the ratio between the total VE-cadherin amount at a given cell boundary length divided by the respective length, is reduced when cells lengthen (e.g., after application of VEGF) [10].…”

“…JAIL appear constitutively and thereby ensure the integrity of endothelial cell contacts, but are also responsible for coordination of cell contact dynamics during cell migration and angiogenesis [7, 10]. It has also been demonstrated that the relative VE-cadherin concentration (Rel-VEcad-C), which is defined by the ratio between the total VE-cadherin amount at a given cell boundary length divided by the respective length, is reduced when cells lengthen (e.g., after application of VEGF) [10]. As a consequence of the decreased Rel-VEcad-C, actin-driven and Arp2/3 complex and WAVE/WASP family protein-controlled JAIL were formed, which in turn established new VE-cadherin adhesion sites via the intermediate formation of VE-cadherin plaques exactly at JAIL sites.…”