Identification of an FMNL2 Interactome by Quantitative Mass Spectrometry.

Formin Homology Proteins (Formins) are a highly conserved family of cytoskeletal regulatory proteins that participate in a diverse range of cellular processes. FMNL2 is a member of the Diaphanous-Related Formin sub-group, and previous reports suggest FMNL2's role in filopodia assembly, force generation at lamellipodia, subcellular trafficking, cell-cell junction assembly, and focal adhesion formation. How FMNL2 is recruited to these sites of action is not well understood. To shed light on how FMNL2 activity is partitioned between subcellular locations, we used biotin proximity labeling and proteomic analysis to identify an FMNL2 interactome. The interactome identified known and new FMNL2 interacting proteins with functions related to previously described FMNL2 activities. In addition, our interactome predicts a novel connection between FMNL2 and extracellular vesicle assembly. We show directly that FMNL2 protein is present in exosomes.

SPECC1L binds the myosin phosphatase complex MYPT1/PP1ß and can regulate its distribution between microtubules and filamentous actin.

The subcellular localization, activity , and substrate specificity of the serine/threonine protein phosphatase 1 catalytic subunit (PP1cat) is mediated through its dynamic association with regulatory subunits in holoenzyme complexes. While some functional overlap is observed for the three human PP1cat isoforms, they also show distinct targeting based on relative preferences for specific regulatory subunits. A well-known example is the preferential association of MYPT1 with PP1ß in the myosin phosphatase complex. In smooth muscle, MYPT1/PP1ß counteracts the muscle contraction induced by phosphorylation of the light chains of myosin by the myosin light chain kinase. This phosphatase complex is also found in nonmuscle cells, where it is targeted to both myosin and nonmyosin substrates and contributes to regulation of the balance of cytoskeletal structure and motility during cell migration and division. Although it remains unclear how MYPT1/PP1ß traffics between microtubule- and actin-associated substrates, our identification of the microtubule- and actin-binding protein SPECC1L in both the PP1ß and MYPT1 interactomes suggests that it is the missing link. Our validation of their association using coimmunoprecipitation and proximity biotinylation assays, together with the strong overlap that we observed for the SPECC1L and MYPT1 interactomes, confirmed that they exist in a stable complex in the cell. We further showed that SPECC1L binds MYPT1 directly and that it can impact the balance of the distribution of the MYPT1/PP1ß complex between the microtubule and filamentous actin networks.

Cooperative assembly of filopodia by the formin FMNL2 and I-BAR domain protein IRTKS.

Filopodia are long finger-like actin-based structures that project out from the plasma membrane as cells navigate and explore their extracellular environment. The initiation of filopodia formation requires release of tension at the plasma membrane followed by the coordinated assembly of long unbranched actin filaments. Filopodia growth is maintained by a tip complex that promotes actin polymerization and protects the growing barbed ends of the actin fibers from capping proteins. Filopodia growth also depends on additional F-actin bundling proteins to stiffen the actin filaments as well as extension of the membrane sheath projecting from the cell periphery. These activities can be provided by a number of actin-binding and membrane-binding proteins including formins such as formin-like 2 (FMNL2) and FMNL3, and Inverse-Bin-Amphiphysin-Rvs (I-BAR) proteins such as IRTKS and IRSp53, but the specific requirement for these proteins in filopodia assembly is not clear. We report here that IRTKS and IRSp53 are FMNL2-binding proteins. Coexpression of FMNL2 with either I-BAR protein promotes cooperative filopodia assembly. We find IRTKS, but not IRSp53, is required for FMNL2-induced filopodia assembly, and FMNL2 and IRTKS are mutually dependent cofactors in this process. Our results suggest that the primary function for FMNL2 during filopodia assembly is binding to the plasma membrane and that regulation of actin dynamics by its formin homology 2 domain is secondary. From these results, we conclude that FMNL2 initiates filopodia assembly via an unexpected novel mechanism, by bending the plasma membrane to recruit IRTKS and thereby nucleate filopodia assembly.

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

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.

Actin-dependent regulation of cilia length by the inverted formin FHDC1.

A primary cilium is found on most mammalian cells, where it acts as a cellular antenna for the reception of both mechanical and chemical signals. A variety of diseases are associated with defective ciliogenesis, reflecting the ubiquity of the function of cilia and the number of proteins required for their assembly. Proper cilia length is necessary for cilia signaling and is regulated through a poorly understood balance of assembly and disassembly rates. FHDC1 is a unique member of the formin family of cytoskeletal regulatory proteins. Overexpression of FHDC1 induces F-actin accumulation and microtubule stabilization and acetylation. We find that overexpression of FHDC1 also has profound effects on ciliogenesis; in most cells FHDC1 overexpression blocks cilia assembly, but the cilia that are present are immensely elongated. FHDC1-induced cilia growth requires the FHDC1 FH2 and microtubule-binding domain and results from F-actin-dependent inhibition of cilia disassembly. FHDC1 depletion, or treatment with a pan-formin inhibitor, inhibits cilia assembly and induces cilia resorption. Endogenous FHDC1 protein localizes to cytoplasmic microtubules converging on the base of the cilia, and we identify the subdistal appendage protein Cep170 as an FHDC1 interacting protein. Our results suggest that FHDC1 plays a role in coordinating cytoskeletal dynamics during normal cilia assembly.

A specific FMNL2 isoform is up-regulated in invasive cells.

BACKGROUND: Formins are a highly conserved family of cytoskeletal remodeling proteins. A growing body of evidence suggests that formins play key roles in the progression and spread of a variety of cancers. There are 15 human formin proteins and of these the Diaphanous-Related Formins (DRFs) are the best characterized. Included in the DRFs are the Formin-Like proteins, FMNL1, 2 & 3, each of which have been strongly implicated in driving tumorigenesis and metastasis of specific tumors. In particular, increased FMNL2 expression correlates with increased invasiveness of colorectal cancer (CRC) in vivo and for a variety of CRC cell-lines in vitro. FMNL2 expression is also required for invasive cell motility in other cancer cell-lines. There are multiple alternatively spliced isoforms of FMNL2 and it is predicted that the encoded proteins will differ in their regulation, subcellular localization and in their ability to regulate cytoskeletal dynamics. RESULTS: Using RT-PCR we identified four FMNL2 isoforms expressed in CRC and melanoma cell-lines. We find that a previously uncharacterized FMNL2 isoform is predominantly expressed in a variety of melanoma and CRC cell lines; this isoform is also more effective in driving 3D motility. Building on previous reports, we also show that FMNL2 is required for invasion in A375 and WM266.4 melanoma cells. CONCLUSIONS: Taken together, these results suggest that FMNL2 is likely to be generally required in melanoma cells for invasion, that a specific isoform of FMNL2 is up-regulated in invasive CRC and melanoma cells and this isoform is the most effective at facilitating invasion.

Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1.

The Golgi apparatus is the central hub of intracellular trafficking and consists of tethered stacks of cis, medial, and trans cisternae. In mammalian cells, these cisternae are stitched together as a perinuclear Golgi ribbon, which is required for the establishment of cell polarity and normal subcellular organization. We previously identified FHDC1 (also known as INF1) as a unique microtubule-binding member of the formin family of cytoskeletal-remodeling proteins. We show here that endogenous FHDC1 regulates Golgi ribbon formation and has an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression results in defective Golgi assembly and suggests a role for FHDC1 in maintenance of the Golgi-derived microtubule network. Similarly, overexpression of FHDC1 induces dispersion of the Golgi ribbon into functional ministacks. This effect is independent of centrosome-derived microtubules and instead likely requires the interaction between the FHDC1 microtubule-binding domain and the Golgi-derived microtubule network. These effects also depend on the interaction between the FHDC1 FH2 domain and the actin cytoskeleton. Thus our results suggest that the coordination of actin and microtubule dynamics by FHDC1 is required for normal Golgi ribbon formation.

The diaphanous-related formins promote protrusion formation and cell-to-cell spread of Listeria monocytogenes.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen whose virulence depends on its ability to spread from cell to cell within an infected host. Although the actin-related protein 2/3 (Arp2/3) complex is necessary and sufficient for Listeria actin tail assembly, previous studies suggest that other actin polymerization factors, such as formins, may participate in protrusion formation. Here, we show that Arp2/3 localized to only a minor portion of the protrusion. Moreover, treatment of L. monocytogenes-infected HeLa cells with a formin FH2-domain inhibitor significantly reduced protrusion length. In addition, the Diaphanous-related formins 1-3 (mDia1-3) localized to protrusions, and knockdown of mDia1, mDia2, and mDia3 substantially decreased cell-to-cell spread of L. monocytogenes. Rho GTPases are known to be involved in formin activation. Our studies also show that knockdown of several Rho family members significantly influenced bacterial cell-to-cell spread. Collectively, these findings identify a Rho GTPase-formin network that is critically involved in the cell-to-cell spread of L. monocytogenes.

Bacterial subversion of host cytoskeletal machinery: hijacking formins and the Arp2/3 complex.

The host actin nucleation machinery is subverted by many bacterial pathogens to facilitate their entry, motility, replication, and survival. The majority of research conducted in the past primarily focused on exploitation of a host actin nucleator, the Arp2/3 complex, by bacterial pathogens. Recently, new studies have begun to explore the role of formins, another family of host actin nucleators, in bacterial pathogenesis. This review provides an overview of recent advances in the study of the exploitation of the Arp2/3 complex and formins by bacterial pathogens. Secreted bacterial effector proteins seem to manipulate the regulation of these actin nucleators or functionally mimic them to drive bacterial entry, motility and survival within host cells. An enhanced understanding of how formins are exploited will provide us with greater insight into how a fundamental eurkaryotic cellular process is utilized by bacteria and will also advance our knowledge of host-pathogen interactions.

The ability to induce microtubule acetylation is a general feature of formin proteins.

Cytoplasmic microtubules exist as distinct dynamic and stable populations within the cell. Stable microtubules direct and maintain cell polarity and it is thought that their stabilization is dependent on coordinative organization between the microtubule network and the actin cytoskeleton. A growing body of work suggests that some members of the formin family of actin remodeling proteins also regulate microtubule organization and stability. For example, we showed previously that expression of the novel formin INF1 is sufficient to induce microtubule stabilization and tubulin acetylation, but not tubulin detyrosination. An important issue with respect to the relationship between formins and microtubules is the determination of which formin domains mediate microtubule stabilization. INF1 has a distinct microtubule-binding domain at its C-terminus and the endogenous INF1 protein is associated with the microtubule network. Surprisingly, the INF1 microtubule-binding domain is not essential for INF1-induced microtubule acetylation. We show here that expression of the isolated FH1 + FH2 functional unit of INF1 is sufficient to induce microtubule acetylation independent of the INF1 microtubule-binding domain. It is not yet clear whether or not microtubule stabilization is a general property of all mammalian formins; therefore we expressed constitutively active derivatives of thirteen of the fifteen mammalian formin proteins in HeLa and NIH3T3 cells and measured their effects on stress fiber formation, MT organization and MT acetylation. We found that expression of the FH1 + FH2 unit of the majority of mammalian formins is sufficient to induce microtubule acetylation. Our results suggest that the regulation of microtubule acetylation is likely a general formin activity and that the FH2 should be thought of as a dual-function domain capable of regulating both actin and microtubule networks.

The formin FMNL3 is a cytoskeletal regulator of angiogenesis.

The process of angiogenesis requires endothelial cells (ECs) to undergo profound changes in shape and polarity. Although this must involve remodelling of the EC cytoskeleton, little is known about this process or the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of ECs actively undergoing angiogenic morphogenesis. We found that elongation of ECs during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified formin-like 3 (FMNL3; also known as FRL2) as a crucial regulator of EC elongation during angiogenesis. We showed that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for this alignment during angiogenic morphogenesis. FMNL3 was highly expressed in the ECs of zebrafish during development and embryos that were depleted for FMNL3 showed profound defects in developmental angiogenesis that were rescued by expression of the human gene. We conclude that FMNL3 is a new regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent ECs into their elongated angiogenic forms.

Force-induced myofibroblast differentiation through collagen receptors is dependent on mammalian diaphanous (mDia).

The development of fibrosis promotes the differentiation of myofibroblasts, pro-fibrotic cells, which contribute to tissue dysfunction. Myofibroblast differentiation is dependent on actin assembly, which in response to force, is mediated by various actin-binding proteins including the mammalian Diaphanous-related formins (mDia). We examined the role of mDia in the mechano-sensing pathway that leads to force-induced expression of alpha-smooth muscle actin (SMA), a marker and critical determinant of myofibroblast differentiation. In cells treated with siRNA to knockdown mDia and then subjected to tensile force using collagen-coated magnetite beads attached to beta1 integrins, actin assembly was inhibited at bead contact sites. Force-induced nuclear translocation of MRTF-A, a transcriptional co-activator of SMA, was reduced 50% by mDia knockdown. The expression of the transcriptional co-activator of SMA, serum response factor, was reduced by 50% after siRNA knockdown of mDia or by 100% in cells transfected with catalytically inactive mDia. Force-induced activation of the SMA promoter and SMA expression were blocked by knockdown of siRNA of mDia. In anchored collagen gel assays to measure myofibroblast-mediated contraction, knockdown of mDia reduced contraction by 50%. We conclude that mDia plays an important role in the development of force-induced transcriptional activation of SMA and myofibroblast differentiation.

Formins in cell signaling.

The founding formin homology protein family members were implicated early on as being involved in regulating cytoskeletal remodeling pathways, as formin protein mutations in Drosophila and yeast lead to obvious actin cytoskeleton defects. The discovery that these proteins associated directly with small Rho family GTPases confirmed these results and greatly enhanced our understanding of their function. The mammalian diaphanous-related formins (DRFs) were subsequently recognized as being involved in activation of serum response factor (SRF), tying formins to transcriptional regulation. In the past few years, much progress has been made in demonstrating how DRFs act as both downstream effectors and upstream modulators of Rho GTPase signaling. These functions are important for regulation of both actin and microtubule cytoskeletal structures, and affect cellular processes such as the establishment of polarity, vesicle movement, and focal adhesion remodeling. The connection of DRFs to the SH3 domain-containing protein, Src, has also been described as being important to several basic cellular functions. While still unresolved, extensive work has been carried out on how DRFs mediate SRF activation, and the importance of this to the regulation of cytoskeletal structure. This review will focus on the role of formins in cytoplasmic signal transduction pathways and the downstream effects on the regulation of gene expression.

Interaction of the N- and C-terminal autoregulatory domains of FRL2 does not inhibit FRL2 activity.

Formin homology proteins are a highly conserved family of cytoskeletal remodeling proteins best known for their ability to induce the formation of long unbranched actin filaments. They accomplish this by nucleating the de novo polymerization of F-actin and also by acting as F-actin barbed end "leaky cappers" that allow filament elongation while antagonizing the function of capping proteins. More recently, it has been reported that the FH2 domains of FRL1 and mDia2 and the plant formin AFH1 are able to bind and bundle actin filaments via distinct mechanisms. We find that like FRL1, FRL2 and FRL3 are also able to bind and bundle actin filaments. In the case of FRL3, this activity is dependent upon a proximal DAD/WH2-like domain that is found C-terminal to the FH2 domain. In addition, we show that, like other Diaphanous-related formins, FRL3 activity is subject to autoregulation mediated by the interaction between its N-terminal DID and C-terminal DAD. In contrast, the DID and DAD of FRL2 also interact in vivo and in vitro but without inhibiting FRL2 activity. These data suggest that current models describing DID/DAD autoregulation via steric hindrance of FH2 activity must be revised. Finally, unlike other formins, we find that the FH2 and N-terminal dimerization domains of FRL2 and FRL3 are able to form hetero-oligomers.

INF1 is a novel microtubule-associated formin.

Formin proteins, characterized by the presence of conserved formin homology (FH) domains, play important roles in cytoskeletal regulation via their abilities to nucleate actin filament formation and to interact with multiple other proteins involved in cytoskeletal regulation. The C-terminal FH2 domain of formins is key for actin filament interactions and has been implicated in playing a role in interactions with microtubules. Inverted formin 1 (INF1) is unusual among the formin family in having the conserved FH1 and FH2 domains in its N-terminal half, with its C-terminal half being composed of a unique polypeptide sequence. In this study, we have examined a potential role for INF1 in regulating microtubule structure. INF1 associates discretely with microtubules, and this association is dependent on a novel C-terminal microtubule-binding domain. INF1 expressed in fibroblast cells induced actin stress fiber formation, coalignment of microtubules with actin filaments, and the formation of bundled, acetylated microtubules. Endogenous INF1 showed an association with acetylated microtubules, and knockdown of INF1 resulted in decreased levels of acetylated microtubules. Our data suggests a role for INF1 in microtubule modification and potentially in coordinating microtubule and F-actin structure.

The diaphanous inhibitory domain/diaphanous autoregulatory domain interaction is able to mediate heterodimerization between mDia1 and mDia2.

Formins are multidomain proteins that regulate numerous cytoskeleton-dependent cellular processes. These effects are mediated by the presence of two regions of homology, formin homology 1 and FH2. The diaphanous-related formins (DRFs) are distinguished by the presence of interacting N- and C-terminal regulatory domains. The GTPase binding domain and diaphanous inhibitory domain (DID) are found in the N terminus and bind to the diaphanous autoregulatory domain (DAD) found in the C terminus. Adjacent to the DID is an N-terminal dimerization motif (DD) and coiled-coil region (CC). The N terminus of Dia1 is also proposed to contain a Rho-independent membrane-targeting motif. We undertook an extensive structure/function analysis of the mDia1 N terminus to further our understanding of its role in vivo. We show here that both DID and DD are required for efficient autoinhibition in the context of full-length mDia1 and that the DD of mDia1 and mDia2, like formin homology 2, mediates homo- but not heterodimerization with other DRF family members. In contrast, our results suggest that the DID/DAD interaction mediates heterodimerization of full-length mDia1 and mDia2 and that the auto-inhibited conformation of DRFs is oligomeric. In addition, we also show that the DD/CC region is required for the Rho-independent membrane targeting of the isolated N terminus.

An unusual Zn-finger/FH2 domain protein controls a left/right asymmetric neuronal fate decision in C. elegans.

Gene regulatory networks that control the terminally differentiated state of a cell are, by and large, only superficially understood. In a mutant screen aimed at identifying regulators of gene batteries that define the differentiated state of two left/right asymmetric C. elegans gustatory neurons, ASEL and ASER, we have isolated a mutant, fozi-1, with a novel mixed-fate phenotype, characterized by de-repression of ASEL fate in ASER. fozi-1 codes for a protein that functions in the nucleus of ASER to inhibit the expression of the LIM homeobox gene lim-6, neuropeptide-encoding genes and putative chemoreceptors of the GCY gene family. The FOZI-1 protein displays a highly unusual domain architecture, that combines two functionally essential C2H2 zinc-finger domains, which are probably involved in transcriptional regulation, with a formin homology 2 (FH2) domain, normally found only in cytosolic regulators of the actin cytoskeleton. We demonstrate that the FH2 domain of FOZI-1 has lost its actin polymerization function but maintains its phylogenetically ancient ability to homodimerize. fozi-1 genetically interacts with several transcription factors and micro RNAs in the context of specific regulatory network motifs. These network motifs endow the system with properties that provide insights into how cells adopt their stable terminally differentiated states.

Homo-oligomerization is essential for F-actin assembly by the formin family FH2 domain.

Formin proteins regulate the actin and microtubule cytoskeletons and also control the activity of the SRF transcription factor through depletion of the G-actin pool. Although the conserved formin homology 2 (FH2) domains of the mDia1 and Bni1 formins can nucleate actin polymerization in vitro, the activity of other FH2 domains and the relationship between actin polymerization and microtubule reorganization have been controversial. We show that, similar to the mDia1 FH2 domain, the FH2 domains of mDia2 and ld are sufficient for SRF activation in vivo. We demonstrate that an mDia1 mutant defective for microtubule rearrangement in vivo is also defective in SRF activation in vivo as well as actin polymerization in vitro and that the mDia2 FH2 domain promotes actin polymerization in vitro. Using co-immunoprecipitation, we show that mDia1 is oligomeric in its inactive autoinhibited state in vivo, that the active mDia1 and mDia2 FH2 domains form homo- but not hetero-oligomers in vivo, and that oligomerization is abolished by inactivating FH2 deletion and point mutations. Nevertheless, inactive mDia1 FH2 domain mutants retain the ability to interfere with cellular mDia activity. Our results show that self-oligomerization is essential for SRF activation in vivo and F-actin assembly in vitro and provide strong support for recent structural models of the FH2 domain.

A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity.

Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.

The diaphanous-related formin mDia1 controls serum response factor activity through its effects on actin polymerization.

SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF mDia1. We show that the ability of mDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the mDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive mDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of mDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin.