FMNL3 is a vertebrate-specific formin protein previously shown to play a role in angiogenesis and cell migration. Here we define the cellular localization of endogenous FMNL3, the dynamics of GFP-tagged FMNL3 during cell migration, and the effects of FMNL3 suppression in mammalian culture cells. The majority of FMNL3 localizes in a punctate pattern, with >95% of these puncta being indistinguishable from the plasma membrane by fluorescence microscopy. A small number of dynamic cytoplasmic FMNL3 patches also exist, which enrich near cell-cell contact sites and fuse with the plasma membrane at these sites. These cytoplasmic puncta appear to be part of larger membranes of endocytic origin. On the plasma membrane, FMNL3 enriches particularly in filopodia and membrane ruffles and at nascent cell-cell adhesions. FMNL3-containing filopodia occur both at the cell-substratum interface and at cell-cell contacts, with the latter being 10-fold more stable. FMNL3 suppression by siRNA has two major effects: decrease in filopodia and compromised cell-cell adhesion in cells migrating as a sheet. Overall our results suggest that FMNL3 functions in assembly of actin-based protrusions that are specialized for cell-cell adhesion.

Focal segmental glomerulosclerosis (FSGS) is a pattern of kidney injury observed either as an idiopathic finding or as a consequence of underlying systemic conditions. Several genes have been identified that, when mutated, lead to inherited FSGS and/or the related nephrotic syndrome. These findings have accelerated the understanding of glomerular podocyte function and disease, motivating our search for additional FSGS genes. Using linkage analysis, we identified a locus for autosomal-dominant FSGS susceptibility on a region of chromosome 14q. By sequencing multiple genes in this region, we detected nine independent nonconservative missense mutations in INF2, which encodes a member of the formin family of actin-regulating proteins. These mutations, all within the diaphanous inhibitory domain of INF2, segregate with FSGS in 11 unrelated families and alter highly conserved amino acid residues. The observation that alterations in this podocyte-expressed formin cause FSGS emphasizes the importance of fine regulation of actin polymerization in podocyte function.

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain unknown. Here, using biochemical fractionation techniques, we observe cholesterol accumulation at the MAM which leads to ACAT1/SOAT1 enrichment in this domain. MAM proteomics data suggests that ACAT1/SOAT1 inhibition strengthens the ER-mitochondria connection. Confocal and electron microscopy confirms that ACAT1/SOAT1 inhibition increases the number of ER-mitochondria contact sites and strengthens this connection by shortening the distance between these two organelles. This work demonstrates how directly manipulating local cholesterol levels at the MAM can alter inter-organellar contact sites and suggests that cholesterol buildup at the MAM is the impetus behind the therapeutic benefits of ACAT1/SOAT1 inhibition.

Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot-Marie-Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain. Interestingly, purified INF2 is not autoinhibited, suggesting the existence of other cellular inhibitors. Here, we purified an INF2 inhibitor from mouse brain tissue, and identified it as a complex of lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP-KAc-actin is dependent on the INF2 diaphanous inhibitory domain (DID). Treatment of CAP-KAc-actin-inhibited INF2 with histone deacetylase 6 releases INF2 inhibition, whereas inhibitors of histone deacetylase 6 block the activation of cellular INF2. Disease-associated INF2 mutants are poorly inhibited by CAP-KAc-actin, suggesting that focal segmental glomerulosclerosis and Charcot-Marie-Tooth disease result from reduced CAP-KAc-actin binding. These findings reveal a role for KAc-actin in the regulation of an actin assembly factor by a mechanism that we call facilitated autoinhibition.

Self-organization of cytoskeletal proteins such as actin and tubulin into filaments and microtubules is frequently assisted by the proteins binding to them. Formins are regulatory proteins that nucleate the formation of new filaments and are essential for a wide range of cellular functions. The vertebrate inverted formin 2 (INF2) has both actin filament nucleating and severing/depolymerizing activities connected to its ability to encircle actin filaments. Using atomic force microscopy, we report that a formin homology 2 (FH2) domain-containing construct of INF2 (INF2-FH1-FH2-C or INF2-FFC) self-assembles into nanoscale ringlike oligomeric structures in the absence of actin filaments, demonstrating an inherent ability to reorganize from a dimeric to an oligomeric state. A construct lacking the C-terminal region (INF2-FH1-FH2 or INF2-FF) also oligomerizes, confirming the dominant role of FH2-mediated interactions. Moreover, INF2-FFC domains were observed to organize into ringlike structures around single actin filaments. This is the first demonstration that formin FH2 domains can self-assemble into oligomers in the absence of filaments and has important implications for observing unaveraged decoration and/or remodeling of filaments by actin binding proteins.

Therapies for most malignancies are generally ineffective once metastasis occurs. While tumour cells migrate through tissues using diverse strategies, the signalling networks controlling such behaviours in human tumours are poorly understood. Here we define a role for the Diaphanous-related formin-3 (DIAPH3) as a non-canonical regulator of metastasis that restrains conversion to amoeboid cell behaviour in multiple cancer types. The DIAPH3 locus is close to RB1, within a narrow consensus region of deletion on chromosome 13q in prostate, breast and hepatocellular carcinomas. DIAPH3 silencing in human carcinoma cells destabilized microtubules and induced defective endocytic trafficking, endosomal accumulation of EGFR, and hyperactivation of EGFR/MEK/ERK signalling. Silencing also evoked amoeboid properties, increased invasion and promoted metastasis in mice. In human tumours, DIAPH3 down-regulation was associated with aggressive or metastatic disease. DIAPH3-silenced cells were sensitive to MEK inhibition, but showed reduced sensitivity to EGFR inhibition. These findings have implications for understanding mechanisms of metastasis, and suggest that identifying patients with chromosomal deletions at DIAPH3 may have prognostic value.

Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds actin filaments, and the off-rate of actin-bound Drp1 is significantly increased by unbound Drp1. GDP and GTP accelerate and decelerate Drp1/actin binding dynamics, respectively. Actin has a biphasic effect on Drp1 GTP hydrolysis, increasing at low actin:Drp1 ratio but returning to baseline at high ratio. Drp1 also bundles filaments. Bundles have reduced dynamics but follow the same trends as single filaments. Drp1 preferentially incorporates into bundles at higher ionic strength. We measure Drp1 concentration to be ∼0.5 μM in U2OS cell cytosol, suggesting the actin-binding affinity measured here (Kd = 0.6 μM) is in the physiologically relevant range. The ability of Drp1 to bind actin filaments in a highly dynamic manner provides potential for actin filaments to serve as reservoirs of oligomerization-competent Drp1 that can be accessed for mitochondrial fission.

Mitochondrial damage (MtD) represents a dramatic change in cellular homeostasis, necessitating metabolic changes and stimulating mitophagy. One rapid response to MtD is a rapid peri-mitochondrial actin polymerization termed ADA (acute damage-induced actin). The activation mechanism for ADA is unknown. Here, we use mitochondrial depolarization or the complex I inhibitor metformin to induce ADA. We show that two parallel signaling pathways are required for ADA. In one pathway, increased cytosolic calcium in turn activates PKC-β, Rac, WAVE regulatory complex, and Arp2/3 complex. In the other pathway, a drop in cellular ATP in turn activates AMPK (through LKB1), Cdc42, and FMNL formins. We also identify putative guanine nucleotide exchange factors for Rac and Cdc42, Trio and Fgd1, respectively, whose phosphorylation states increase upon mitochondrial depolarization and whose suppression inhibits ADA. The depolarization-induced calcium increase is dependent on the mitochondrial sodium-calcium exchanger NCLX, suggesting initial mitochondrial calcium efflux. We also show that ADA inhibition results in enhanced mitochondrial shape changes upon mitochondrial depolarization, suggesting that ADA inhibits these shape changes. These depolarization-induced shape changes are not fragmentation but a circularization of the inner mitochondrial membrane, which is dependent on the inner mitochondrial membrane protease Oma1. ADA inhibition increases the proteolytic processing of an Oma1 substrate, the dynamin GTPase Opa1. These results show that ADA requires the combined action of the Arp2/3 complex and formin proteins to polymerize a network of actin filaments around mitochondria and that the ADA network inhibits the rapid mitochondrial shape changes that occur upon mitochondrial depolarization.

During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However, <5 min after bath-applied NMDA, F-actin depolymerizes within spines and polymerizes into stable filaments within the dendrite shaft and soma. A similar actinification occurs after experimental ischemia in culture, and photothrombotic stroke in mouse. Following transient NMDA incubation, actinification spontaneously reverses. Na+, Cl-, water, and Ca2+ influx, and spine F-actin depolymerization are all necessary, but not individually sufficient, for actinification, but combined they induce activation of the F-actin polymerization factor inverted formin-2 (INF2). Silencing of INF2 renders neurons vulnerable to cell death and INF2 overexpression is protective. Ischemia-induced dendritic actin reorganization is therefore an intrinsic pro-survival response that protects neurons from death induced by cell edema.

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.

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.

Mitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through ER-bound inverted formin 2 (INF2) stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER-mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.

Mitochondrial damage represents a dramatic change in cellular homeostasis. One rapid response is perimitochondrial actin polymerization, termed acute damage-induced actin (ADA). The consequences of ADA are not understood. In this study, we show evidence suggesting that ADA is linked to rapid glycolytic activation upon mitochondrial damage in multiple cells, including mouse embryonic fibroblasts and effector CD8+ T lymphocytes. ADA-inducing treatments include CCCP, antimycin, rotenone, oligomycin, and hypoxia. The Arp2/3 complex inhibitor CK666 or the mitochondrial sodium-calcium exchanger (NCLX) inhibitor CGP37157 inhibits both ADA and the glycolytic increase within 5 min, supporting ADA's role in glycolytic stimulation. Two situations causing chronic reductions in mitochondrial ATP production, mitochondrial DNA depletion and mutation to the NDUFS4 subunit of complex 1 of the electron transport chain, cause persistent perimitochondrial actin filaments similar to ADA. CK666 treatment causes rapid mitochondrial actin loss and a drop in ATP in NDUFS4 knock-out cells. We propose that ADA is necessary for rapid glycolytic activation upon mitochondrial impairment, to re-establish ATP production.

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex-nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.

Focal adhesions (FAs) and stress fibers (SFs) act in concert during cell motility and in response to the extracellular environment. Although the structures of mature FAs and SFs are well studied, less is known about how they assemble and mature de novo during initial cell spreading. In this study using live-cell Airyscan microscopy, we find that FAs undergo "splitting" during their assembly, in which the FA divides along its longitudinal axis. Before splitting, FAs initially appear as assemblies of multiple linear units (FAUs) of 0.3-μm width. Splitting occurs between FAUs, resulting in mature FAs of either a single FAU or of a small number of FAUs that remain attached at their distal tips. Variations in splitting occur based on cell type and extracellular matrix. Depletion of adenomatous polyposis coli (APC) or vasodilator-stimulated phosphoprotein (VASP) results in reduced splitting. FA-associated tension increases progressively during splitting. Early in cell spreading, ventral SFs are detected first, with other SF sub-types (transverse arcs and dorsal SFs) being detected later. Our findings suggest that the fundamental unit of FAs is the fixed-width FAU, and that dynamic interactions between FAUs control adhesion morphology.

Mitochondria are dynamic organelles, undergoing both fission and fusion regularly in interphase cells. Mitochondrial fission is thought to be part of a quality-control mechanism whereby damaged mitochondrial components are segregated from healthy components in an individual mitochondrion, followed by mitochondrial fission and degradation of the damaged daughter mitochondrion. Fission also plays a role in apoptosis. Defects in mitochondrial dynamics can lead to neurodegenerative diseases such as Alzheimer's disease. Mitochondrial fission requires the dynamin GTPase Drp1, which assembles in a ring around the mitochondrion and appears to constrict both outer and inner mitochondrial membranes. However, mechanisms controlling Drp1 assembly on mammalian mitochondria are unclear. Recent results show that actin polymerization, driven by the endoplasmic reticulum-bound formin protein INF2, stimulates Drp1 assembly at fission sites. Here, we show that myosin II also plays a role in fission. Chemical inhibition by blebbistatin or small interfering RNA (siRNA)-mediated suppression of myosin IIA or myosin IIB causes an increase in mitochondrial length in both control cells and cells expressing constitutively active INF2. Active myosin II accumulates in puncta on mitochondria in an actin- and INF2-dependent manner. In addition, myosin II inhibition decreases Drp1 association with mitochondria. Based on these results, we propose a mechanistic model in which INF2-mediated actin polymerization leads to myosin II recruitment and constriction at the fission site, enhancing subsequent Drp1 accumulation and fission.

Drp1 is a dynamin guanosine triphosphatase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51, and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is associated with ER in mammalian cells and is distinct from mitochondrial or peroxisomal Drp1 populations. Subpopulations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, and ER bound) and mitochondrial division, whereas Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division.

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.

Actin depolymerizing factor-homology (ADF-H) family proteins regulate actin filament dynamics at multiple cellular locations. Herein, we have investigated the function of the ADF-H family member coactosin-like 1 (COTL1) in the regulation of actin dynamics at the T cell immune synapse (IS). We initially identified COTL1 in a genetic screen to identify novel regulators of T cell activation, and subsequently found that it associates with F-actin and localizes at the IS in response to TCR+CD28 stimulation. Live cell microscopy showed that depletion of COTL1 protein impaired T cell spreading in response to TCR ligation and abrogated lamellipodial protrusion at the T cell - B cell contact site, producing only a band of F-actin. Significantly, re-expression of wild type COTL1, but not a mutant deficient in F-actin binding could rescue these defects. In addition, COTL1 depletion reduced T cell migration. In vitro studies showed that COTL1 and cofilin compete with each other for binding to F-actin, and COTL1 protects F-actin from cofilin-mediated depolymerization. While depletion of cofilin enhanced F-actin assembly and lamellipodial protrusion at the IS, concurrent depletion of both COTL1 and cofilin restored lamellipodia formation. Taken together, our results suggest that COTL1 regulates lamellipodia dynamics in part by protecting F-actin from cofilin-mediated disassembly.