A Tuba/Cdc42/Par6A complex is required to ensure singularity in apical domain formation during enterocyte polarization

Abstract

Apico-basal polarity establishment is a seminal process in tissue morphogenesis. To function properly it is often imperative that epithelial cells limit apical membrane formation to a single domain. We previously demonstrated that signaling by the small GTPase Cdc42, together with its guanine nucleotide exchange factor (GEF) Tuba, is required to prevent the formation of multiple apical domains in polarized Ls174T:W4 cells, a single cell model for enterocyte polarization. To further chart the molecular signaling mechanisms that safeguard singularity during enterocyte polarization we generated knockout cells for the Cdc42 effector protein Par6A. Par6A loss results in the formation of multiple apical domains, similar to loss of Cdc42. In Par6A knockout cells, we find that active Cdc42 is more mobile at the apical membrane compared to control cells and that wild type Cdc42 is more diffusely localized throughout the cell, indicating that Par6A is required to restrict Cdc42 signaling. Par6A, Cdc42 and its GEF Tuba bind in a co-immunoprecipitation experiment and they partially colocalize at the apical membrane in polarized Ls174T:W4 cells, suggesting the formation of a trimeric complex. Indeed, in a rescue experiment using Par6A mutants, we show that the ability to establish this trimeric complex correlates with the ability to restore singularity in Par6A knockout cells. Together, these experiments therefore indicate that a Tuba/Cdc42/Par6A complex is required to ensure the formation of a single apical domain during enterocyte polarization.

Multifaceted Rho GTPase Signaling at the Endomembranes

“…Earlier report suggested that the GEF Tuba might play a role in modulating Cdc42 activity at the Golgi (Kodani et al, 2009). However, four independent groups have failed to localize Tuba to the Golgi, but rather found it to be confined to cytoplasmic vesicles or the cell surface (Salazar et al, 2003; Kovacs et al, 2006; Baschieri et al, 2014; Bruurs et al, 2018). Very recently, the Golgi matrix protein GCC88 was found to interact with the long form of intersectin-1, a Cdc42 GEF (Makhoul et al, 2019).…”

Tuba Activates Cdc42 during Neuronal Polarization Downstream of the Small GTPase Rab8a

“…Tuba is located below the apical cortex and is essential for Cdc42 enrichment at this site (Qin et al, 2010). On the same line, Par6a, Cdc42, and Tuba form a trimeric complex and colocalize in the apical domain of polarized enterocytes (Bruurs et al, 2018), where Tuba is responsible for immobilizing active Cdc42 to ensure its enrichment at apical plasma membrane (Bruurs et al, 2017). In epithelial polarity, Tuba is integrated in a complex molecular network, downstream of Rab11a-Rabin8-Rab8a cascade (Bryant et al, 2010) and upstream of Cdc42 and its effectors that includes the Par polarity complex and the actin regulator N-WASP (Otani et al, 2006;Kovacs et al, 2011).…”

Spatiotemporal Control of Intracellular Membrane Trafficking by Rho GTPases

“…Despite these reports, the localization of RhoGEFs at Golgi membranes is still a matter of debate. In many reports, endogenous and overexpressed Tuba failed to be detected at the Golgi but rather was present on vesicle-like structures [33,[74][75][76] questioning the specificity of the antibodies used in the former studies. Additionally, a recent study confirmed a perinuclear/vesicular localization for overexpressed intersectin-1, while FGD1 was mainly cytosolic in primary endothelial cells, although it was not investigated whether the RhoGEFs activate Cdc42 at these sites [76].…”