A Two-Tiered Mechanism Enables Localized Cdc42 Signaling during Enterocyte Polarization

Abstract

Signaling by the small GTPase Cdc42 governs a diverse set of cellular processes that contribute to tissue morphogenesis. Since these processes often require highly localized signaling, Cdc42 activity must be clustered in order to prevent ectopic signaling. During cell polarization, apical Cdc42 signaling directs the positioning of the nascent apical membrane. However, the molecular mechanisms that drive Cdc42 clustering during polarity establishment are largely unknown. Here, we demonstrate that during cell polarization localized Cdc42 signaling is enabled via activity-dependent control of Cdc42 mobility. By performing photoconversion experiments, we show that inactive Cdc42-GDP is 30-fold more mobile than active Cdc42-GTP. This switch in apical mobility originates from a dual mechanism involving RhoGDI-mediated membrane dissociation of Cdc42-GDP and Tuba-mediated immobilization of Cdc42-GTP. Interference with either mechanism affects Cdc42 clustering and as a consequence impairs Cdc42-mediated apical membrane clustering. We therefore identify a molecular network, comprised of Cdc42, the guanine nucleotide exchange factor (GEF) Tuba, and RhoGDI, that enables differential diffusion of inactive and active Cdc42 and is required to establish localized Cdc42 signaling during enterocyte polarization.

Regulation of Cdc42 and its effectors in epithelial morphogenesis

“…This relationship between Cdc42 and apical cargo delivery is well supported by the finding that in 3D MDCK spheroids, Rab11-dependent delivery of apical proteins is required for normal Cdc42 activation (Bryant et al, 2010). Furthermore, reminiscent of the mechanism of Cdc42 polarity in budding yeast, recent work in the Caco-2-derived LS174T-W4 cell line using FRAP experiments has shown that activation of Cdc42 by Tuba leads to a threefold increase in the immobilization of Cdc42 to the apical membrane (Bruurs et al, 2017). This promotes Cdc42 clustering and presumably enables a reaction-diffusion mechanism that is comparable to that operating in budding yeast to determine polarity.…”

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

“…We previously showed that Cdc42 mobility at the apical membrane is highly regulated via a RhoGDI-dependent and Tuba-dependent mechanism and that this regulation is critical for Cdc42 function [ 5 ]. To test whether Par6A contributes to the regulation of Cdc42 mobility we determined the mobility of constitutively active Cdc42(G12V) at the apical membrane of polarized Ls174T:W4 cells and Par6A k.o.…”

“…cells. For this we locally photoconverted Dendra-fused Cdc42(G12V) in the brush border and followed its subsequent loss from the converted region as a consequence of lateral diffusion [ 5 ]. We find that the apical mobility of Cdc42(G12V) is higher in Par6A k.o.…”

“…Apical mobility of Dendra-Cdc42(G12V) was determined as previously described [ 5 ]. In short, transfected cells were polarized and imaged with a Leica SP8x microscope equipped with a temperature- and CO 2 -controlled chamber using a 63x oil objective (HC PL APO 63x/1.40) with Leica LAS AF image acquisition software.…”

“…Using Ls174T:W4 cells, a single cell model for enterocyte polarization in which polarity is induced by forced activation of LKB1 [ 2 ], we previously demonstrated that Cdc42 signaling is required to ensure the formation of a single apical domain [ 4 ]. For this, Cdc42 activity and mobility at the nascent apical plasma membrane is strictly regulated by the Cdc42-specific GEF Tuba [ 5 ]. However, it remains unclear how the GEF Tuba can control Cdc42 mobility and which effector(s) Cdc42 engages to ensure the formation of a single apical domain.…”