The novel formin FMNL3 is a cytoskeletal regulator of angiogenesis.

Abstract

SummaryThe process of angiogenesis requires endothelial cells (ECs) to undergo profound changes in shape and polarity. Although this must involve remodelling of the EC cytoskeleton, little is known about this process or the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of ECs actively undergoing angiogenic morphogenesis. We found that elongation of ECs during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified formin-like 3 (FMNL3; also known as FRL2) as a crucial regulator of EC elongation during angiogenesis. We showed that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for this alignment during angiogenic morphogenesis. FMNL3 was highly expressed in the ECs of zebrafish during development and embryos that were depleted for FMNL3 showed profound defects in developmental angiogenesis that were rescued by expression of the human gene. We conclude that FMNL3 is a new regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent ECs into their elongated angiogenic forms.

Reverse Genetic Screening Reveals Poor Correlation between Morpholino-Induced and Mutant Phenotypes in Zebrafish

“…Most strikingly, we failed to observe defects in embryos bearing mutations in genes previously shown to be required for ISV formation using MOs (Table 1; Table S3). For example, individual MO knockdowns of fmnl3 and pdgfrb cause a block in ISV sprouting (Hetheridge et al, 2012; Wiens et al, 2010). However, embryos mutant for fmnl3 um150 or pdgfrb um148 display ISV growth that is indistinguishable from wild type siblings (Figure 2A, E, F).…”

The Formin FMNL3 Controls Early Apical Specification in Endothelial Cells by Regulating the Polarized Trafficking of Podocalyxin

“…This process requires complex and coordinated regulation of the endothelial cell cytoskeleton to control cell shape and polarity. In our previous work, we showed that the cytoskeletal regulator FMNL3/ FRL2 controls the alignment of stabilized microtubules during polarized endothelial cell elongation and that depletion of FMNL3 retards elongation of the intersegmental vessels in zebrafish [1]. Recent work has shown that FMNL3 is also needed for vascular lumen formation [2], a critical element of the formation of functional vessels.…”

“…ECs first undergo polarized elongation in the direction of the proangiogenic signal to become tip cells, which then guide the migration of the following stalk cells [12]. ECs depleted of FMNL3 show inhibition of this polarized elongation [1,13]. As the vessel matures, adjacent stalk cells undergo apical-basolateral polarization perpendicular to this axis to begin the process of lumen formation [14,15].…”

Formins as effector proteins of Rho GTPases

“…FMNL3 in contrast regulates endothelial cell elongation during angiogenic morphogenesis by microtubule alignment 96 and seems to be involved in cell motility 97 . Both, FMNL2 and FMNL3 are predominantly associated with the plasma membrane and this localization depends on their N-terminal myristoylation (Table 2).…”