The Diaphanous-related Formin FHOD1 Associates with ROCK1 and Promotes Src-dependent Plasma Membrane Blebbing

Abstract

Diaphanous-related formins (DRFs) mediate GTPase-triggered actin rearrangements to regulate central cellular processes, such as cell motility and cytokinesis. The DRF FHOD1 interacts with the Rho-GTPase Rac1 and mediates formation of actin stress fibers in its deregulated form; the physiologically relevant activities and molecular mechanisms of endogenous FHOD1, however, are still unknown. Here we report that FHOD1 physically associates via the N-terminal part of its FH2 domain with the central domain of ROCK1. Although FHOD1 does not affect the kinase activity of ROCK1, the DRF is an efficient substrate for phosphorylation by ROCK1. Co-expression of FHOD1 and ROCK1 results in the generation of nonapoptotic plasma membrane (PM) blebs, to which the DRF is efficiently recruited. Blebbing induced by FHOD1 and ROCK1 depends on F-actin integrity, the Rho-ROCK cascade, and Src activity and is reminiscent of the recently described PM blebs triggered by expression of Src homology 4 (SH4) domain PM targeting signals. Consistently, endogenous FHOD1 is required in SH4 domain expressing cells for efficient PM blebbing and rounded cell morphology in two-dimensional cultures or three-dimensional matrices, respectively. Efficient association of FHOD1 with ROCK1, as well as recruitment of the DRF to blebs, depends on Src activity, suggesting that the functional interaction between both proteins is regulated by Src. These results define a role for endogenous FHOD1 in SH4 domain-induced blebbing and suggest that its activity is regulated by ROCK1 in a Src-dependent manner.

Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation

“…Consistent with the requirement for ROCK, PM blebbing of adhering HeLa cells strictly required the activity of the GTPase and ROCK regulator RhoA, while activity of Rac1 and Cdc42 GTPases was dispensable (Supplementary information, Figure S1A and S1B). Finally and in line with previous results on blebs observed upon SH4-domain expression or filamin A deficiency, PM blebs were enriched in p-MLC, p-ERM and p-Src that displayed distinct localizations at blebs (Supplementary information, Figure S1C) [12,20]. Together, these results establish PM blebbing of adhering HeLa cells as a model system that mirrors the general characteristics of PM blebbing and in which blebbing is governed by Rho-ROCK signaling and F-actin remodeling.…”

“…Only few studies previously addressed the involvement of specific actin nucleators in PM blebbing. Using experimental systems in which PM blebbing was induced by genetic manipulation or protein overexpression, nucleators such as mDia1, mDia2, FHOD1, Arp2/3 that are also involved in the regulation of diverse additional cellular actin remodeling events were implicated in blebbing [12,[41][42][43]. In this present study, we conducted a comprehensive analysis of the involvement of endogenously expressed human actin nucleators in PM blebbing of adhering HeLa cells.…”

“…In addition to these domains, that mediate actin polymerization, the socalled diaphanous formins are subject to autoinhibition [11] mediated by interaction of their C-terminal diaphanous autoregulatory domain (DAD) with an N-terminal region also referred to as diaphanous inhibitory domain (DID) that is part of the FH3 module. Activation through release of autoinhibition can occur via phosphorylation within the DAD, as demonstrated for FHOD1 [12,13], or by interaction of the N-terminal GTPase-binding domain (GBD) with GTPases of the Rho family as exemplified for mDia1 [11].…”

“…Blebbing can also be induced experimentally, e.g. by disruption of the genes encoding the actin-membrane crosslinker filamin A or the tumor suppressor p53, or by overexpression of SH4 domain membrane targeting signals [12,[20][21][22][23][24][25]. Despite their distinct physiological context and triggers, PM blebs typically display high dynamics of bleb expansion and retraction.…”

Fhos encodes a Drosophila Formin‐Like Protein participating in autophagic programmed cell death

“…Run-on transcription assays revealed that the amount of nascent RNA is drastically diminished in the cells expressing the C-terminal portion, which in turn, is translocated to the nucleus (Ménard et al, 2006). Hannemann et al (2008) described that FHOD1, associated to ROCK1 is required for the membrane blebbing, which is dependent on F-actin integrity. Also, mammalian and Drosophila formins perform different roles during development and differentiated cells (Homem and Pfeifer, 2009;Massarwa et al, 2009;Matusek et al, 2006Matusek et al, , 2008.…”