A potential Rho GEF and Rac GAP for coupled Rac and Rho cycles during mesenchymal-to-epithelial-like transitions

Abstract

The leading edge-to-cadherin contact transitions that occur during metazoan developmental processes and disease states require fine coordination of Rac and Rho pathways. Recently the elmo-mbc complex, a Rac GEF and RhoGAP19D, a Rho GAP were identified as key, conserved regulators that link Rac and Rho during these transitions. The corresponding Rho GEF and Rac GAP remain hidden amongst the large family of GEF and GAP proteins. Identification of these regulators is essential to understand GTPase coordination during these transitions. Here we find two candidates based on the mammalian literature and use RNAi to explore the fly ortholog effects on the dorsal closure epidermis. RhoGEF64C and RhoGAP92B are strong contenders to couple Rac and Rho during mesenchymal-to-epithelial-like transitions.

Keywords: Net1; Rac; Rho; RhoGAP19D; RhoGAP92B; RhoGEF64C; dorsal closure; elmo-dock; elmo-mbc; mesenchymal-to-epithelial transition.

Figure 1.

(A) Current model of Rho regulation during MET-like transitions (B) Schematic of pros and cons of Mammalian (italics) and Drosophila (bold italics) studies (C) Rho regulator orthologies for Mammals (italics) and Drosophila (bold italics). Orthologies were identified using simple BlastP (NCBI) searches between mouse/human and Drosophila (D) Montages of maximal intensity Z-stack projections of embryos, which express endogenous DE-cadherin-GFP and ectodermal Gal4-driven UAS-Lifeact-RFP with indicated UAS-RNAi constructs. Scale bar = 25 µm. (E) Single focal plane of an epidermal leading edge in en2.4-GAL4 UAS-LifeAct-RFP embryos of indicated UAS-RNAi constructs. Scale bar = 5 µm. (F) Quantification of the number of lamellipodia detected per length of engrailed positive leading edge from still images in indicated embryo backgrounds

Figure 2.

(A) GFP-channel kymographs generated from (1 frame/min) movies of embryos that express endogenous DE-cadherin-GFP and ectodermal Gal4-driven UAS-Lifeact-RFP with indicated UAS-RNAi constructs. Schematic shows region of kymograph selected. Brightest signal indicates cadherin signal at epidermal leading edges that convergence over time. The closure speeds calculated for each condition. (B) Maximal intensity Z-stack projection of DE-cadherin-GFP at epidermal seams in indicated embryos. Scale bar = 5 µm. Yellow arrow indicates newest cadherin contact. Yellow dashed lines show regions with constricted cells. (C) Schematic that indicates cadherin dimensions measured in the zone of epidermal cadherin contact formation. Box plot of measurements of indicated cadherin dimensions for wt and depleted cells. Measurements pooled from 3–5 embryos and include anterior and posterior seams. (D) Model for potential Rho regulation during MET-like transitions