Rif-mDia1 Interaction Is Involved in Filopodium Formation Independent of Cdc42 and Rac Effectors

Abstract

Filopodia are cellular protrusions important for axon guidance, embryonic development, and wound healing. The Rho GTPase Cdc42 is the best studied inducer of filopodium formation, and several of its effectors and their interacting partners have been linked to the process. These include IRSp53, N-WASP, Mena, and Eps8. The Rho GTPase, Rif, also drives filopodium formation. The signaling pathway by which Rif induces filopodia is poorly understood, with mDia2 being the only protein implicated to date. It is thus not clear how distinct the Rif-driven pathway for filopodium formation is from the one mediated by Cdc42. In this study, we characterize the dynamics of Rif-induced filopodia by time lapse imaging of live neuronal cells and show that Rif drives filopodium formation via an independent pathway that does not involve the Cdc42 effectors N-WASP and IRSp53, the IRSp53 binding partner Mena, or the Rac effectors WAVE1 and WAVE2. Rif formed filopodia in the absence of N-WASP or Mena and when IRSp53, WAVE1, or WAVE2 was knocked down by RNAi. Rif-mediated filopodial protrusion was instead reduced by silencing mDia1 expression or overexpressing a dominant negative mutant of mDia1. mDia1 on its own was able to form filopodia. Data from acceptor photobleaching FRET studies of protein-protein interaction demonstrate that Rif interacts directly with mDia1 in filopodia but not with mDia2. Taken together, these results suggest a novel pathway for filopodia formation via Rif and mDia1.Filopodia are dynamic, actin-rich cellular protrusions that are important for processes such as cell migration, neuritogenesis, axon guidance, wound healing, angiogenesis, embryonic development, and phagocytosis (1, 2). Elucidating the exact mechanism(s) by which filopodia form will give a greater understanding of these cellular processes and how such structures play a role in pathological conditions such as metastasis (3) and pathogen invasion (4, 5). The Rho GTPase Cdc42 is a key regulator of cell signaling events that lead to filopodium formation in mammalian cells. It binds to and activates IRSp53 (insulin receptor substrate protein 53 kDa) (6 -8). The Cdc42-IRSp53 complex induces filopodia by coupling membrane protrusion with actin dynamics (8). Interacting partners of IRSp53 include N-WASP (neural Wiskott-Aldrich syndrome protein) (8), Mena (mammalian enabled) (7, 9), Eps8 (EGF receptor kinase substrate 8) (10), and the Rac effectors WAVE (WASP family verprolin homology) isoforms WAVE1 (11-14) and WAVE2 (15). Yeast two-hybrid experiments have shown that IRSp53 binds mDia1 (mammalian Diaphanous 1) (16), but little else is known about this interaction. mDia1 and mDia2 (mammalian Diaphanous 2) belong to the formin family of multidomain eukaryotic proteins that are involved in a wide range of cellular processes that require actin polymerization (17). Formins contain an actin-nucleating formin homology 2 domain, and a proline-rich, formin homology 1 domain that binds Src homology 3 and WW domaincontaining proteins and profilin (18). Diaphanous-r...

Three-dimensional aspects of matrix assembly by cells in the developing cornea

“…On a structural basis, filopodialike structures formed by embryonic corneal cells are actin-rich, and of similar diameter, but exceed greatly in length previous descriptions of filopodia. We suspect that they are also more longlived than true filopodia, which, for example in living neuronal cells, had a lifetime of only up to 3.5 min (41). Associations between the keratocyte cytoskeleton and collagen trafficking were previously shown following immunolocalization of actin and type I collagen propeptides in embryonic cornea (42).…”

mDia1 and WAVE2 Proteins Interact Directly with IRSp53 in Filopodia and Are Involved in Filopodium Formation

“…Live Cell Imaging and Analysis of Cell Morphology-Cells were cultured, transfected, and imaged, and images compiled into movies as described in Goh et al (9). For images of fixed cells, as well as time-lapse imaging of live cells, morphological characteristics, including filopodial length and lifetime, were defined and scored as explained in Goh et al (9).…”

“…In addition to these domains, mDia1 and mDia2 also have an N-terminal basic domain that binds phospholipids and allows them to localize to the plasma membrane (17). Both of these formins have been linked to filopodium formation downstream of Rif, with mDia1 binding to this RhoGTPase within the protrusions (9). Under the control of RhoA, mDia1 also plays a role in stress fiber formation (18), neurite outgrowth (19), cell polarity (20), and adherens junction integrity (21).…”

“…These dynamic structures are tubular extensions of the plasma membrane filled with bundles of linear actin filaments, and the cytoskeletal rearrangements leading to their formation are controlled by RhoGTPases such as Cdc42 (5-7) and Rif (8,9).…”

“…Acceptor photobleaching FRET (AP-FRET) experiments revealed that IRSp53 interacts with these two proteins in the filopodia of neuronal cells. In addition, we measured filopodium formation quantitatively using multichannel rapid timelapse imaging of live cells as described in our previous work (7,9) and found that silencing the expression of either mDia1 or WAVE2 reduced the number of IRSp53 filopodia formed. In contrast, knockdown of mDia2 did not affect IRSp53-induced filopodium formation.…”