The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1)

Abstract

The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro. In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo. Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.

Distribution of PDLIM1 at actin‐rich structures generated by invasive and adherent bacterial pathogens

“…We and others have shown actin filament crosslinkers (α-actinin 1 (Dold, Sanger, & Sanger, 1994), palladin (Dhanda et al, 2018), nexilin (Law et al, 2012) and transgelin (Chua et al, 2018)) as key determinants of L. monocytogenes and EPEC virulence. Normally, these crosslinking agents function together as part of complexes that organize the cytoskeleton (Chen et al, 2020;Chen, Arora, McCulloch, & Wilde, 2017;Gateva, Tojkander, Koho, Carpen, & Lappalainen, 2014;Piekny & Glotzer, 2008). Key to the proper functioning of these complexes are molecular scaffolds that bring together actin-associated proteins and other molecules (see Garbett & Bretscher, 2014 for a review).…”

Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

“…These data position CLIP170 at the correct location and time to deactivate DIAPH1 via disrupting the DIAPH1-IQGAP1 interaction. We have previously demonstrated that IQGAP1 is specifically involved in the activation of DIAPH1 and not DIAPH3 18 , which utilizes anillin as its enhancer rather than IQGAP1 9 . While we could detect co-precipitation of IQGAP1 and CLIP170, we detected no co-precipitation of anillin and CLIP170 nor DIAPH3 and CLIP170 (Supplementary Fig.…”

“…1b ). We next assessed the integrity of the β- and γ-actin-isoform networks in cells depleted for DIAPH1 or IQGAP1, a DIAPH1 interactor that recruits DIAPH1 to the cell cortex and functions as an enhancer of its actin nucleation activity 18 . siRNA depletion of either DIAPH1 or IQGAP1 reduced the amount of γ-actin at the cortex, but did not disrupt β-actin assembly at the cell equator (Fig.…”

“…1a ). While the initial IQGAP1 binding to DIAPH1 requires RhoA 18 it is not known if IQGAP1 binding to DIAPH1 persists in the absence of RhoA, and if it does, is DIAPH1 maintained in the open active state? To address this question, we performed a series of in vitro biochemical binding assays.…”

“…The DIAPH3 dependent generation of β-actin underlies contractile ring assembly and is required to stabilize the cytokinetic furrow 9 . The cortical γ-actin network is generated through similar molecular principles; the formin DIAPH1 is targeted to the cortex by an enhancer of its activity IQGAP1 18 . The cortical γ-actin network regulates cortical membrane stiffness, furrow positioning 12 , and ingression kinetics.…”