Background: The actin-binding protein Flightless-I (Flii) has not been quantified in the human serum yet. We aimed to determine serum Flii levels in healthy individuals and to investigate Flii as a potential marker in patients with sepsis focusing on diagnosis, organ failures, and short-term mortality. Methods: A total of 30 controls and 64 septic and 22 non-septic patients were enrolled in this follow-up study. Serum Flii levels were quantified by using the capillary electrophoresis-based Simple Western™ system with chemiluminescent detection. The assay was calibrated by applying dilution series of a purified recombinant human Flii standard and a parallel internal standard. Results: Flii levels of healthy controls were found between 3.5 and 8.8 mg/L, while septic and non-septic patients showed significantly lower values (p < 0.001). First-day Flii could differentiate sepsis from the non-septic inflammatory state (AUC: 0.667; p < 0.05) and indicated acute respiratory distress syndrome (ARDS) among septic patients (AUC: 0.686; p < 0.05). Furthermore, a combination of Flii and other sepsis markers seemed to offer an improved diagnostic performance (sepsis vs. non-sepsis, AUC of Flii + gelsolin (GSN) + Gc-globulin + procalcitonin: 0.974; p < 0.001 and ARDS vs. non-ARDS, AUC of Flii + GSN + presepsin: 0.776; p < 0.001) compared with single markers even in the prediction of 14-day mortality (AUC of Flii + GSN + Gc-globulin: 0.76; p < 0.001). Conclusions: We adapted a properly precise and reproducible automated Western blot method to determine concentrations of Flii in human serum. Our results revealed the relationship between Flii and sepsis; however, Flii alone did not appear to be a prominent sepsis marker. When combined with other biomarkers, measurement of serum Flightless-I may provide additional value supporting patient care.

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.

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.

The monomeric (G-actin) and polymer (F-actin) forms of actin play important role in muscle development and contraction, cellular motility, division, and transport processes. Leiomodins 1–3 (Lmod1–3) are crucial for the development of muscle sarcomeres. Unlike tropomodulins that localize only at the pointed ends, the striated muscle specific Lmod2 shows diffuse distribution along the entire length of the thin filaments. The G-actin-binding profilin (Pro) facilitates the nucleotide exchange on monomeric actin and inhibits the polymerization at the barbed end, therefore contributes to the maintenance of the intracellular pool of polymerization competent ATP-G-actin. Cyclophosphamide (CP) is a cytostatic drug that can have potential side effects on muscle thin filaments at the level of actin in myofilaments. Here, we aimed at investigating the influence of CP on actin and its complexes with actin-binding proteins by using differential scanning calorimetry (DSC). We found that upon CP treatment, the denaturation of the Pro-G-actin and Lmod2-F-actin complexes was characterized by an increased enthalpy change. However, after the CP treatment, the melting temperature of F-actin was the same as in the presence of Lmod2, seems like Lmod2 does not have any effect on the structure of the CP alkylated F-actin. In case of Pro bound G-actin the melting temperature did not respond to the CP addition. The intracellular function of Lmod2 in muscle cells can be modified within CP drug treatment.

The lesser-known unconventional myosin 16 protein is crucial in 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, as well as downregulates the actin dynamics at the postsynaptic site of dendritic spines and involved in the organization of the presynaptic axon terminals.

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.

IntroductionA 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.MethodsBlood 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.ResultsIn 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%).ConclusionU-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.

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 background of 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. 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. Flightless-I was found to interact with actin and affect actin dynamics in a calcium-independent fashion in vitro, suggesting the lack of calcium-mediated activation and conformational change of protein. For the comparative structural analysis of the six gelsolin homology domains (GH16) of gelsolin and Flightless-I, we used a combination of biophysical and biochemical approaches. The use of both internal (tryptophans) and external (8-anilinonaphthalene-1-sulfonic acid; ANS) fluorophores revealed that calcium-binding induces structural changes in gelsolin but the conformational behavior of Flightless-I GH16 was not significantly affected by the divalent cation. Different kinetics of limited proteolysis observed for gelsolin and Flightless-I GH16 further strengthened this conclusion. Our experimental findings are supported by bioinformatics analysis predicting that the sequence elements responsible for Ca2+-activation of GSN are not conserved in Flightless-I GH16.

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 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 ofMYO16has 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.

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.

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.

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.

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 nonsepsis (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.

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.

Gelsolin is a severing and capping protein that targets filamentous actin and regulates filament lengths near plasma membranes, contributing to cell movement and plasma membrane morphology. Gelsolin binds to the plasma membrane via phosphatidylinositol 4,5-bisphosphate (PIP2) in a state that cannot cap F-actin, and gelsolin-capped actin filaments are uncapped by PIP2 leading to filament elongation. The process by which gelsolin is removed from PIP2 at the plasma membrane is currently unknown. Gelsolin also binds ATP with unknown function. Here we characterize the role of ATP on PIP2-gelsolin complex dynamics. Fluorophore-labeled PIP2 and ATP were used to study their interactions with gelsolin using steady-state fluorescence anisotropy, and Alexa488-labeled gelsolin was utilized to reconstitute the regulation of gelsolin binding to PIP2-containing phospholipid vesicles by ATP. Under physiological salt conditions ATP competes with PIP2 for binding to gelsolin, while calcium causes the release of ATP from gelsolin. These data suggest a cycle for gelsolin activity. Firstly, calcium activates ATP-bound gelsolin allowing it to sever and cap F-actin. Secondly, PIP2-binding removes the gelsolin cap from F-actin at low calcium levels, leading to filament elongation. Finally, ATP competes with PIP2 to release the calcium-free ATP-bound gelsolin, allowing it to undergo a further round of severing.

Leiomodin proteins are vertebrate homologues of tropomodulin, having a role in the assembly and maintenance of muscle thin filaments. Leiomodin2 contains an N-terminal tropomodulin homolog fragment including tropomyosin-, and actin-binding sites, and a C-terminal Wiskott-Aldrich syndrome homology 2 actin-binding domain. The cardiac leiomodin2 isoform associates to the pointed end of actin filaments, where it supports the lengthening of thin filaments and competes with tropomodulin. It was recently found that cardiac leiomodin2 can localise also along the length of sarcomeric actin filaments. While the activities of leiomodin2 related to pointed end binding are relatively well described, the potential side binding activity and its functional consequences are less well understood. To better understand the biological functions of leiomodin2, in the present work we analysed the structural features and the activities of Rattus norvegicus cardiac leiomodin2 in actin dynamics by spectroscopic and high-speed sedimentation approaches. By monitoring the fluorescence parameters of leiomodin2 tryptophan residues we found that it possesses flexible, intrinsically disordered regions. Leiomodin2 accelerates the polymerisation of actin in an ionic strength dependent manner, which relies on its N-terminal regions. Importantly, we demonstrate that leiomodin2 binds to the sides of actin filaments and induces structural alterations in actin filaments. Upon its interaction with the filaments leiomodin2 decreases the actin-activated Mg2+-ATPase activity of skeletal muscle myosin. These observations suggest that through its binding to side of actin filaments and its effect on myosin activity leiomodin2 has more functions in muscle cells than it was indicated in previous studies.