Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

Abstract

The compliance and dimensionality of the ECM regulate distinct changes in microtubule growth speed and growth persistence.

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

“…HUVECs cultured on fibronectin coated glass substrates ( Figure 3A), or on stiff 2-dimensional polyacrylamide ECMs (55 kPa) typically display a round or elongated morphology with few distinct branched protrusions ( Figure 3B). However, when cultured on very soft 2-dimensional polyacrylamide ECMs (0.7 kPa), HUVECs display a highly branched morphology that is known to result from compliance-induced downregulation of myosin-II contractility 4,5,31 ( Figure 3C). Thus, HUVECs regulate their cellular morphology through ECM mechanosensing, and this is accomplished through localized modulation of MT dynamics and actomyosin contractility.…”

“…Track overlays are color-coded and color distinctions can be determined by parameters entered by the user. Commonly, these distinctions use the global mean of MT growth speed and MT growth lifetime 5,31 and thereby split the data into four groups: slow and short-lived (red), slow and long-lived (yellow), fast and short-lived (green), and fast and long-lived MT growth (blue; Figure 2G). PlusTipTrackermediated tracking of EB3-labeled MT growth dynamics also enables user-defined Regions of Interest (ROIs).…”

“…Moreover, continuous, rapid imaging of fluorescently-labeled EB3 on the second time scale leads to photobleaching of the EB3 signal. Attempts to overcome this issue by conducting short imaging series of EB3 (1-2 min acquisition series) every 30 minutes have been successful, but could only be accomplished in a small number of ECs that had optimal expression of EB3 and that did not display adverse effects to repeated laser illumination 5 .…”

“…For example, soluble signaling molecules such as VEGF induce actin polymerization and MT growth toward the VEGF cue in endothelial cell types 2,[82][83][84][85][86][87] . Additionally, in response to physical interactions with the ECM (for example, compliance and dimensionality mechanosensing), HUVECs modulate their MT dynamics by locally regulating MAPs to promote MT growth within elongating branched protrusions, and this serves to guide HUVEC migration in the direction of the cue 5,31,88 . Thus, polarization in response to extracellular signaling cues highlights the need for experimental evaluation of localized changes in cytoskeletal dynamics.…”

“…In order for EC branches to form, myosin-II must be locally downregulated, resulting in localized membrane protrusions 4 . At the same time and in the same location within the cell, MT growth dynamics become modified resulting in slow and persistent MT growth to promote branch elongation and stability 5 . This coordinated cytoskeletal regulation allows ECs to polarize their morphology to facilitate directional migration.…”