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

SummaryAngiogenesis is the fundamental process by which new blood vessels form from pre-existing vasculature. It plays a critical role in the formation of the vasculature during development and is triggered in response to tissue hypoxia in adult organisms. 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. Here, we show that FMNL3 interacts with Cdc42 and RhoJ, two Rho family GTPases known to be required for lumen formation. FMNL3 and RhoJ are concentrated at the early apical surface, or AMIS, and regulate the formation of radiating actin cables from this site. In diverse biological systems, formins mediate polarized trafficking through the generation of similar actin filaments tracks. We show that FMNL3 and RhoJ are required for polarized trafficking of podocalyxin to the early apical surface—an important event in vascular lumenogenesis

The Formin FMNL3 is a Cytoskeletal Regulator of Angiogenesis

The 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

Identification of FMNL3 as a novel regulator of the cytoskeleton in angiogenesis

Sprouting angiogenesis is the formation of new blood vessels from the pre-existing vasculature. Endothelial cells in quiescent blood vessels form a highly organised array with tight cell-cell junctions and uniform polarity. When pro-angiogenic signals initiate angiogenesis, endothelial, cells must become invasive and migrate into the extracellular matrix that surrounds the blood vessels. Sprouting endothelial cells become highly elongated, and this involves dramatic changes in cell shape and polarity. The mechanisms controlling angiogenic morphogenesis are poorly characterised. Cell shape and polarity are orchestrated by the cytoskeleton. The aim of this study was to characterise the cytoskeleton as endothelial cells convert to an angiogenic morphology, and identify novel regulators of this process. An in vitro model of angiogenesis was used to study endothelial cell elongation, in which endothelial cells form capillary-like tubes. It was found that this process was accompanied by the reorganisation of the microtubule network to form a parallel array along the length of the endothelial cells. These microtubules accumulated a modified form of the tubulin monomer, which indicates microtubule stabilisation. Stabilised microtubules were not found in quiescent endothelial cells, and so were associated with the initiation or maintenance of endothelial cell elongation. The formin family of cytoskeletal regulators have been shown to cause similar stabilisation of the microtubule network, in addition to modulating actin dynamics. A screen of the form in family of proteins in the in vitro model demonstrated that the novel formin FMNL3 is critical for endothelial cell elongation. Loss of FMNL3 caused a dramatic decrease in endothelial cell tube formation and resulted in disorganisation of the microtubule cytoskeleton. This study describes the identification of FMNL3 as a novel cytoskeletal regulator and its characterisation in endothelial cells.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The formin FMNL3 is a cytoskeletal regulator of angiogenesis

The 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