Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity.

Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy-based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

The C-terminal tail extension of myosin 16 acts as a molten globule, including intrinsically disordered regions, and interacts with the N-terminal ankyrin.

The lesser-known unconventional myosin 16 protein is essential in proper neuronal functioning and has been implicated in cell cycle regulation. Its longer Myo16b isoform contains a C-terminal tail extension (Myo16Tail), which has been shown to play a role in the neuronal phosphoinositide 3-kinase signaling pathway. Myo16Tail mediates the actin cytoskeleton remodeling, downregulates the actin dynamics at the postsynaptic site of dendritic spines, and is involved in the organization of the presynaptic axon terminals. However, the functional and structural features of this C-terminal tail extension are not well known. Here, we report the purification and biophysical characterization of the Myo16Tail by bioinformatics, fluorescence spectroscopy, and CD. Our results revealed that the Myo16Tail is functionally active and interacts with the N-terminal ankyrin domain of myosin 16, suggesting an intramolecular binding between the C and N termini of Myo16 as an autoregulatory mechanism involving backfolding of the motor domain. In addition, the Myo16Tail possesses high structural flexibility and a solvent-exposed hydrophobic core, indicating the largely unstructured, intrinsically disordered nature of this protein region. Some secondary structure elements were also observed, indicating that the Myo16Tail likely adopts a molten globule-like structure. These structural features imply that the Myo16Tail may function as a flexible display site particularly relevant in post-translational modifications, regulatory functions such as backfolding, and phosphoinositide 3-kinase signaling.

The discovery of actin: "to see what everyone else has seen, and to think what nobody has thought".

Actin is among the most highly abundant and ubiquitous proteins in eukaryotic cells. The structure, dynamics and functional diversity of actin have continued to mesmerise cell and molecular biologists, biophysicists and physiologists for more than three quarters of a century. The discovery and initial characterization of actin, which took place in the laboratory of Albert Szent-Györgyi by Ilona Banga and Brúnó F. Straub during the second world war in Hungary, is a remarkable and inspiring moment in the history of science. Many of the early thoughts and ideas on the properties and functions of actin and particularly actomyosin, which are referred to in this short historical overview, resonate freshly even today.

Myosin XVI.

Myosin XVI (Myo16), a vertebrate-specific motor protein, is a recently discovered member of the myosin superfamily. The detailed functionality regarding myosin XVI requires elucidating or clarification; however, it appears to portray an important role in neural development and in the proper functioning of the nervous system. It is expressed in the largest amount in neural tissues in the late embryonic-early postnatal period, specifically the time in which neuronal cell migration and dendritic elaboration coincide. The impaired expression of myosin XVI has been found lurking in the background of several neuropsychiatric disorders including autism, schizophrenia and/or bipolar disorders.Two principal isoforms of class XVI myosins have been thus far described: Myo16a, the tailless cytoplasmic isoform and Myo16b, the full-length molecule featuring both cytoplasmic and nuclear localization. Both isoforms contain a class-specific N-terminal ankyrin repeat domain that binds to the protein phosphatase catalytic subunit. Myo16b, the predominant isoform, exhibits a diverse function. In the cytoplasm, it participates in the reorganization of the actin cytoskeleton through activation of the PI3K pathway and the WAVE-complex, while in the nucleus it may possess a role in cell cycle regulation. Based on the sequence, myosin XVI may have a compromised ATPase activity, implying a potential stationary role.

The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones.

Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin-microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in Drosophilamelanogaster primary neurons. In vitro, DAAM binds to F-actin as well as to microtubules and has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of DAAM affects growth cone and microtubule morphology, and several aspects of microtubule dynamics; and biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together, these data suggest that, besides operating as an actin assembly factor, DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth.

Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners.

The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.

The activities of the C-terminal regions of the formin protein disheveled-associated activator of morphogenesis (DAAM) in actin dynamics.

Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of Drosophila DAAM in its interaction with actin with in vitro biochemical and in vivo genetic approaches. We found that the DAD-CT region binds actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context.

Predictive value of serum gelsolin and Gc globulin in sepsis - a pilot study.

BACKGROUND: Simultaneous determination of the two main actin scavenger proteins in sepsis has not been investigated until now. In our pilot study, we elucidated the predictive values of Gc globulin and gelsolin (GSN) in sepsis by comparing them to classic laboratory and clinical parameters. METHODS: A 5-day follow-up was performed, including 46 septic patients, 28 non-septic patients and 35 outpatients as controls. Serum Gc globulin and GSN levels were determined by automated immune turbidimetric assay on a Cobas 8000/c502 analyzer. Patients were retrospectively categorized according to the sepsis-3 definitions, and 14-day mortality was also investigated. RESULTS: First-day GSN also differentiated sepsis from non-sepsis (AUC: 0.88) similarly to C-reactive protein (AUC: 0.80) but was slightly inferior to procalcitonin (PCT) (AUC: 0.98) with a cutoff value of GSN at 22.29 mg/L (sensitivity: 83.3%; specificity: 86.2%). Only first-day SOFA scores (0.88) and GSN (0.71) distinguished septic survivors from non-survivors, whereas lactate (0.99), Gc globulin (0.76) and mean arterial pressure (MAP) (0.74) discriminated septic shock from sepsis. Logistic regression analyses revealed SOFA scores and GSN being significant factors regarding 14-day mortality. First-day GSN levels were higher (p<0.05) in septic survivors than in non-survivors. Gc globulin levels remained higher (p<0.01) in sepsis when compared with septic shock during the follow-up period. CONCLUSIONS: Both serum GSN and Gc globulin may have predictive values in sepsis. Considering the small sample size of our study, further measurements are needed to evaluate our results. Measurement of Gc globulin and GSN maybe useful in assessment of sepsis severity and in therapeutic decision-making.

Validation of an automated immune turbidimetric assay for serum gelsolin and its possible clinical utility in sepsis.

BACKGROUND: Studies showing the potential predictive value of the actin-binding protein gelsolin, in critically ill patients are scarce. Moreover, even up to now a rapid automated measurement of gelsolin has still remained a challenge. Therefore, we developed and validated an automated serum gelsolin immune turbidimetric assay for possible clinical use. METHODS: Validation of serum gelsolin assay was performed on a Cobas 8000/c502 analyzer (Roche) according to the second edition of Eurachem guidelines. Furthermore, we also studied the diagnostic value of serum gelsolin in sepsis when investigating sera of septic (n = 25), systemic inflammatory response syndrome (SIRS; n = 8) and control patients (n = 14). We compared our previously published Western blot data with those of the new turbidimetric assay. RESULTS: The sample volume was 7 µL and the assay time was 10 minutes. The detection limit was 0.72 mg/L, intra- and inter-assay imprecision remained in most cases less than 5% expressed as CV. Recovery was found to be 84.56%-93.52% and linearity study gave an appropriate correlation coefficient by linear regression analysis (r2  = .998). Septic patients exhibited lower (P = .015) first-day serum gelsolin levels than SIRS patients, which confirmed our previous Western blot results. The determined cut-off point for serum gelsolin was 14.05 mg/L (sensitivity: 75%; specificity: 60%) when investigating its diagnostic value in sepsis. CONCLUSION: Based on the results, our immune turbidimetric measurement offers a rapid and accurate quantitation of gelsolin in human serum samples. Serum gelsolin seems a promising additional diagnostic marker of sepsis which has to be further investigated.

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein.

Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich syndrome protein homology 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcomeric actin filaments. Here, we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer-filament equilibrium toward the monomeric actin. In addition, SALS-WH2 can bind to but fails to depolymerize phalloidin- or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with the following two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning.

DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

How tropomyosin regulates lamellipodial actin-based motility: a combined biochemical and reconstituted motility approach.

At the leading edge of migrating cells, protrusive forces are developed by the assembly of actin filaments organised in a lamellipodial dendritic array at the front and a more distal lamellar linear array. Whether these two arrays are distinct or functionally linked and how they contribute to cell migration is an open issue. Tropomyosin severely inhibits lamellipodium formation and facilitates the lamellar array while enhancing migration, by a mechanism that is not understood. Here we show that the complex in vivo effects of tropomyosin are recapitulated in the reconstituted propulsion of neural Wiskott-Aldrich syndrome protein (N-WASP)-functionalised beads, which is based on the sole formation of a dendritic array of actin-related protein (Arp)2/3-branched filaments. Actin-depolymerising factor (ADF) and tropomyosin control the length of the actin tail. By competing with Arp2/3 during filament branching, tropomyosin displays opposite effects on propulsion depending on the surface density of N-WASP. Tropomyosin binding to the dendritic array is facilitated following filament debranching, causing its enrichment at the rear of the actin tail, like in vivo. These results unveil the mechanism by which tropomyosin generates two morphologically and dynamically segregated actin networks from a single one.

Myosin and tropomyosin stabilize the conformation of formin-nucleated actin filaments.

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.

Molecular Dissection of DAAM Function during Axon Growth in Drosophila Embryonic Neurons.

Axonal growth is mediated by coordinated changes of the actin and microtubule (MT) cytoskeleton. Ample evidence suggests that members of the formin protein family are involved in the coordination of these cytoskeletal rearrangements, but the molecular mechanisms of the formin-dependent actin-microtubule crosstalk remains largely elusive. Of the six Drosophila formins, DAAM was shown to play a pivotal role during axonal growth in all stages of nervous system development, while FRL was implicated in axonal development in the adult brain. Here, we aimed to investigate the potentially redundant function of these two formins, and we attempted to clarify which molecular activities are important for axonal growth. We used a combination of genetic analyses, cellular assays and biochemical approaches to demonstrate that the actin-processing activity of DAAM is indispensable for axonal growth in every developmental condition. In addition, we identified a novel MT-binding motif within the FH2 domain of DAAM, which is required for proper growth and guidance of the mushroom body axons, while being dispensable during embryonic axon development. Together, these data suggest that DAAM is the predominant formin during axonal growth in Drosophila, and highlight the contribution of multiple formin-mediated mechanisms in cytoskeleton coordination during axonal growth.

Characterization of the biochemical properties and biological function of the formin homology domains of Drosophila DAAM.

We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.

The effect of toxins on inorganic phosphate release during actin polymerization.

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.

Urinary actin, as a potential marker of sepsis-related acute kidney injury: A pilot study.

INTRODUCTION: A major complication of sepsis is the development of acute kidney injury (AKI). Recently, it was shown that intracellular actin released from damaged tissues appears in the urine of patients with multiple organ dysfunction syndrome. Our aims were to measure urinary actin (u-actin) concentrations of septic and control patients and to test if u-actin levels could predict AKI and mortality. METHODS: Blood and urine samples were collected from septic and sepsis-related AKI patients at three time points (T1-3): T1: within 24 hours after admission; T2: second day morning; T3: third day morning of follow-up. Patients with malignancies needing palliative care, end-stage renal disease or kidney transplantation were excluded. Serum and u-actin levels were determined by quantitative Western blot. Patients were categorized by the Sepsis-3 and KDIGO AKI classifications. RESULTS: In our study, 17 septic, 43 sepsis-induced AKI and 24 control patients were enrolled. U-actin levels were higher in septic patients compared with controls during follow-up (p<0.001). At T1, the septic and sepsis-related AKI groups also showed differences (p<0.001), yet this increase was not statistically significant at T2 and T3. We also detected significantly elevated u-actin concentrations in AKI-2 and AKI-3 septic patients compared with AKI-1 septic patients (p<0.05) at T1 and T3, along with a significant increase in AKI-2 septic patients compared with AKI-1 septic patients at T2 (p<0.01). This tendency remained the same when referring u-actin to urine creatinine. Parameters of first-day septic patient samples could discriminate AKI from non-AKI state (AUC ROC, p<0.001): u-actin: 0.876; se-creatinine: 0.875. Derived cut-off value for u-actin was 2.63 µg/L (sensitivity: 86.0%, specificity: 82.4%). CONCLUSION: U-actin may be a complementary diagnostic biomarker to se-creatinine in sepsis-related AKI while higher u-actin levels also seem to reflect the severity of AKI. Further investigations may elucidate the importance of u-actin release in sepsis-related AKI.

Myosin XVI in the Nervous System.

The myosin family is a large inventory of actin-associated motor proteins that participate in a diverse array of cellular functions. Several myosin classes are expressed in neural cells and play important roles in neural functioning. A recently discovered member of the myosin superfamily, the vertebrate-specific myosin XVI (Myo16) class is expressed predominantly in neural tissues and appears to be involved in the development and proper functioning of the nervous system. Accordingly, the alterations of MYO16 has been linked to neurological disorders. Although the role of Myo16 as a generic actin-associated motor is still enigmatic, the N-, and C-terminal extensions that flank the motor domain seem to confer unique structural features and versatile interactions to the protein. Recent biochemical and physiological examinations portray Myo16 as a signal transduction element that integrates cell signaling pathways to actin cytoskeleton reorganization. This review discusses the current knowledge of the structure-function relation of Myo16. In light of its prevalent localization, the emphasis is laid on the neural aspects.

The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics.

Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. Our work identifies the first three gelsolin homology domains (1-3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4-6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (∼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼µM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4-6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1-3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4-6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.