Formin INF2 Research Articles

The Disease-Associated Formin INF2/EXC-6 Organizes Lumen and Cell Outgrowth during Tubulogenesis by Regulating F-Actin and Microtubule Cytoskeletons

We investigate how outgrowth at the basolateral cell membrane is coordinated with apical lumen formation in the development of a biological tube by characterizing exc-6, a gene required for C. elegans excretory cell (EC) tubulogenesis. We show that EXC-6 is orthologous to the human formin INF2, which polymerizes filamentous actin (F-actin) and binds microtubules (MTs) in vitro. Dominant INF2 mutations cause focal segmental glomerulosclerosis (FSGS), a kidney disease, and FSGS+Charcot-Marie-Tooth neuropathy. We show that activated INF2 can substitute for EXC-6 in C. elegans and that disease-associated mutations cause constitutive activity. Using genetic analysis and live imaging, we show that exc-6 regulates MT and F-actin accumulation at EC tips and dynamics of basolateral-localized MTs, indicating that EXC-6 organizes F-actin and MT cytoskeletons during tubulogenesis. The pathology associated with INF2 mutations is believed to reflect misregulation of F-actin, but our results suggest alternative or additional mechanisms via effects on MT dynamics.

Novel roles for actin in mitochondrial fission.

Mitochondrial dynamics, including fusion, fission and translocation, are crucial to cellular homeostasis, with roles in cellular polarity, stress response and apoptosis. Mitochondrial fission has received particular attention, owing to links with several neurodegenerative diseases. A central player in fission is the cytoplasmic dynamin-related GTPase Drp1, which oligomerizes at the fission site and hydrolyzes GTP to drive membrane ingression. Drp1 recruitment to the outer mitochondrial membrane (OMM) is a key regulatory event, which appears to require a pre-constriction step in which the endoplasmic reticulum (ER) and mitochondrion interact extensively, a process termed ERMD (ER-associated mitochondrial division). It is unclear how ER-mitochondrial contact generates the force required for pre-constriction or why pre-constriction leads to Drp1 recruitment. Recent results, however, show that ERMD might be an actin-based process in mammals that requires the ER-associated formin INF2 upstream of Drp1, and that myosin II and other actin-binding proteins might be involved. In this Commentary, we present a mechanistic model for mitochondrial fission in which actin and myosin contribute in two ways; firstly, by supplying the force for pre-constriction and secondly, by serving as a coincidence detector for Drp1 binding. In addition, we discuss the possibility that multiple fission mechanisms exist in mammals.

Actin Monomers Activate Inverted Formin 2 by Competing with Its Autoinhibitory Interaction

INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.

An Actin-Dependent Step in Mitochondrial Fission Mediated by the ER-Associated Formin INF2

Mitochondrial fission is fundamentally important to cellular physiology. The dynamin-related protein Drp1 mediates fission, and interaction between mitochondrion and endoplasmic reticulum (ER) enhances fission. However, the mechanism for Drp1 recruitment to mitochondria is unclear, although previous results implicate actin involvement. Here, we found that actin polymerization through ER-localized inverted formin 2 (INF2) was required for efficient mitochondrial fission in mammalian cells. INF2 functioned upstream of Drp1. Actin filaments appeared to accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. Thus, INF2-induced actin filaments may drive initial mitochondrial constriction, which allows Drp1-driven secondary constriction. Because INF2 mutations can lead to Charcot-Marie-Tooth disease, our results provide a potential cellular mechanism for this disease state.

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

T cell antigen receptor-proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor-induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells.

Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized in an actin-independent manner, INF2-non-CAAX localizes in an actin-dependent meshwork pattern distinct from ER. INF2-non-CAAX is loosely attached to this meshwork, being extracted by brief digitonin treatment. Suppression of INF2-non-CAAX causes fragmentation of the Golgi apparatus. This effect is counteracted by treatment with the actin monomer-sequestering drug latrunculin B. We also find discrete patches of actin filaments in the peri-Golgi region, and these patches are reduced upon INF2 suppression. Our results suggest that the non-CAAX isoform of INF2 serves a distinct cellular function from that of the CAAX isoform.

The Formin INF2 Regulates Basolateral-to-Apical Transcytosis and Lumen Formation in Association with Cdc42 and MAL2

Transcytosis is a widespread pathway for apical targeting in epithelial cells. MAL2, an essential protein of the machinery for apical transcytosis, functions by shuttling in vesicular carriers between the apical zone and the cell periphery. We have identified INF2, an atypical formin with actin polymerization and depolymerization activities, which is a binding partner of MAL2. MAL2-positive vesicular carriers associate with short actin filaments during transcytosis in a process requiring INF2. INF2 binds Cdc42 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are necessary for apical transcytosis and the formation of lateral lumens in hepatoma HepG2 cells. INF2 and MAL2 are also essential for the formation of the central lumen in organotypic cultures of epithelial MDCK cells. Our results reveal a functional mechanism whereby Cdc42, INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation.

The Inverted Formin INF2 Sorts It Out

A new study published in this issue of Developmental Cell by Madrid et al. surprisingly reveals that both the actin polymerization and depolymerization activities of human inverted formin INF2 and its interactions with MAL2 and active Cdc42 are critical for basolateral-to-apical transcytosis and lumen formation in polarized epithelial cells.