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

Abstract

Plasma membrane (PM) blebs are dynamic actin-rich cell protrusions that occur, e.g., during cytokinesis, amoeboid cell motility and cell attachment. Using a targeted siRNA screen against 21 actin nucleation factors, we identify a novel and essential role of the human diaphanous formin DIAPH3 in PM blebbing during cell adhesion. Suppression of DIAPH3 inhibited blebbing to promote rapid cell spreading involving β1-integrin. Multiple isoforms of DIAPH3 were detected on the mRNA and protein level of which isoforms 3 and 7 were the largest and most abundant isoforms that however did not induce formation of actin-rich protrusions. Rather, PM blebbing specifically involved the low abundance isoform 1 of DIAPH3 and activation of isoform 7 by deletion of the diaphanous-autoregulatory domain caused the formation of filopodia. Dimerization and actin assembly activity were essential for induction of specific cell protrusions by DIAPH3 isoforms 1 and 7. Our data suggest that the N-terminal region comprising the GTPasebinding domain determined the subcellular localization of the formin as well as its protrusion activity between blebs and filopodia. We propose that isoform-selective actin assembly by DIAPH3 exerts specific and differentially regulated functions during cell adhesion and motility.

Membrane bleb: A seesaw game of two small GTPases

“…In addition to the activation of ezrin, active RhoA also activates another RhoA effector protein, formins, which are essential for the regrowth of actin filaments at the retracting membrane. 18,19 In the expanding phase of membrane blebs, we found that Rnd3 is recruited to the expanding plasma membrane. The membrane localization of Rnd3 was gradually lost when reassembly of actin filaments started (Fig.…”

“…Subsequently, Stastna et al and Bovellan et al demonstrated that formin family proteins, key regulators of actin nucleation, are recruited to the membrane blebs and essential for the reassembly of actin filaments. 18,19 In addition, Logue et al showed that Eps8, a plus end capping protein, is recruited to the plasma membrane on a similar timescale as ezrin and essential for the formation of membrane blebs. 20 Thus, important regulators of actin cortex during membrane blebs were identified; however how these proteins are coordinated during reassembly of actin filaments in membrane blebs has remained unclear.…”

Formin-like 2 Promotes β1-Integrin Trafficking and Invasive Motility Downstream of PKCα

“…Most formins function as effectors for Rho-GTPases and are regulated by the intramolecular binding between the Diaphanous inhibitory (DID) and autoregulatory (DAD) domains (Faix and Grosse, 2006). Formins are involved in a variety of actin-dependent membrane processes ranging from filopodia formation (Schirenbeck et al, 2005), cell spreading (Iskratsch et al, 2013), and non-apoptotic blebbing (Kitzing et al, 2007;Stastna et al, 2012) to engulfment and phagocytosis Seth et al, 2006). FMNL2 is unique among formins as it is upregulated in several metastatic cancers and is involved in cancer cell behavior and progression (Li et al, 2010;Liang et al, 2013;Zhu et al, 2011Zhu et al, , 2008, but the underlying molecular mechanisms are not well understood.…”

Formins at a glance

“…Formins also have essential roles in physiological processes ranging from cell motility in the immune system (Yayoshi-Yamamoto et al, 2000;Eisenmann et al, 2007;Shi et al, 2009), to gastrulation and neural tube closure (Habas et al, 2001;Sato et al, 2006;Lai et al, 2008), heart morphogenesis (Iskratsch et al, 2010;Li et al, 2011), kidney morphogenesis (Brown et al, 2010;Boyer et al, 2011a;Boyer et al, 2011b), and dendritic spine formation in neurons. Some of these functions depend on the actin nucleation and elongation activities of formins (Bartolini et al, 2008;Andrés-Delgado et al, 2010;Madrid et al, 2010;Andrés-Delgado et al, 2012;Ramabhadran et al, 2012;Stastna et al, 2012), which have been demonstrated by investigating point mutations that impair in these activities Lu et al, 2007;Ramabhadran et al, 2012). However, for many of the other in vivo functions of formins, it has not yet been determined which of their activities are required.…”