Vangl2-Dependent Plasma Membrane Protrusion Behaviors Require an Intact Fibronectin Extracellular Matrix

Abstract

Vang-Like 2 (Vangl2), a key player in the planar cell polarity (PCP) pathway, regulates collective and directed cell migration in zebrafish gastrulae. vangl2 mutant zebrafish embryos exhibit a strong convergence and extension phenotype, characterized by a shortened and broadened body axis. To date, Vangl2’s role in developing plasma membrane protrusions, including lamellipodia-like large protrusions and filopodia, is unclear. Previous works claim loss of Vangl2 results in decreased fibronectin extracellular matrix (ECM), while loss of Glypican4, a PCP protein thought to be working in opposition to Vangl2, causes increased fibronectin ECM. We aim to establish the role of Vangl2 and fibronectin ECM in membrane-protrusive activity. We report vangl2 mutant cells produce excess unpolarized membrane protrusions, while glypican4 mutant cells do not. Our data indicate non-canonical Wnt/Glypican4 signaling inhibits membrane protrusion polarization but minimally affects cell trajectory directness and protrusion quantity. Further, Vangl2 and its binding partner, Prickle1a, regulate membrane protrusion quantity, polarization, and productivity. Protein localization confocal imaging studies indicate Vangl2 accumulates in newly developing large membrane protrusions. Similar to vangl2 mutant cells, fibronectin morphant cells exhibit loss of cell trajectory directness, increased numbers of membrane protrusions, and decreased large protrusion polarity. Additionally, Vangl2 fails to effectively localize to the cell surface in fibronectin morphant embryos. Injecting fibronectin mRNA into vangl2 mutant embryos rescues membrane protrusion and cell trajectory defects but does not correct cellular length-width ratios, mediolateral cell alignment, or the embryonic convergence and extension phenotype. Our work establishes cell-matrix interactions are integral for Vangl2-dependent membrane protrusion activities in migrant zebrafish gastrula cells.