N-terminal Actin-binding Domain Research Articles

FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Abstract P2020: Binding Of Filamin C With Actin Is Essential For Cardiac Development And Function

Filamin C (FLNC), a filamin family member predominantly expressed in striated muscles, is crucial for bridging the extracellular matrix and the cytoskeleton in cardiomyocytes (CMs), and the maintenance of heart integrity and function. FLNC consists of an N-terminal actin-binding domain (ABD) and 24 immunoglobulin (Ig)-like repeat domains at C-terminus. Many pathogenic mutations on N-terminal ABD have been found in patients with cardiomyopathies. However, little is known about how the actin-binding activity intrinsically contributes to the function of FLNC in mammalian hearts. Using structure-based design, we mutated two key actin binding residues F93 and L98 into Ala and Glu respectively in FLNC ABD (F93A/L98E) and show that the mutations completely disrupted the FLNC-actin interaction. We then generated a knock-in mouse model, demonstrating that loss of FLNC-actin interaction in embryonic CMs led to embryonic lethality and cardiac developmental defects displayed by ventricular rupture and reduced CM proliferation. Moreover, disruption of FLNC-actin interaction in adult CMs resulted in severe DCM phenotypes and reduced survival rate, accompanied with aberrant levels of key cytoskeleton components. These phenotypes resemble those of FLNC knockout mouse models, demonstrating a vital role of the FLNC-actin binding in the function of CMs. Our results also provide a foundation for understanding the mutation-induced cardiomyopathies associated with dysregulation of FLNC ABD-actin interaction.

Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function.

FLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC's function in mammalian hearts remains poorly understood. We conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC's actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations. Our findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC's ability to interact with other binding partners ITGB1 (β1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components. Our data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions.

CRISPR-Cas9 correction in the DMD mouse model is accompanied by upregulation of Dp71f protein

Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a deficiency in the dystrophin protein. The most frequent types of disease-causing mutations in the DMD gene are frameshift deletions of one or more exons. Precision genome editing systems such as CRISPR-Cas9 have shown potential to restore open reading frames in numerous animal studies. Here, we applied an AAV-CRISPR double-cut strategy to correct a mutation in the DMD mouse model with exon 8-34 deletion, encompassing the N-terminal actin-binding domain. We report successful excision of the 100-kb genomic sequence, which includes exons 6 and 7, and partial improvement in cardiorespiratory function. While corrected mRNA was abundant in muscle tissues, only a low level of truncated dystrophin was produced, possibly because of protein instability. Furthermore, CRISPR-Cas9-mediated genome editing upregulated the Dp71f dystrophin isoform on the sarcolemma. Given the previously reported Dp71-associated muscle pathology, our results question the applicability of genome editing strategies for some DMD patients with N-terminal mutations. The safety and efficacy of CRISPR-Cas9 constructs require rigorous investigation in patient-specific animal models.

Evolutionary Relationships and Divergence of Filamin Gene Family Involved in Development and Stress in Cotton (Gossypium hirsutum L.)

Filamin protein is characterized by an N-terminal actin-binding domain that is followed by 24 Ig (immunoglobulin)-like repeats, which act as hubs for interactions with a variety of proteins. In humans, this family has been found to be involved in cancer cell invasion and metastasis and can be involved in a variety of growth signal transduction processes, but it is less studied in plants. Therefore, in this study, 54 Filamin gene family members from 23 plant species were investigated and divided into two subfamilies: FLMN and GEX2. Subcellular localization showed that most of the Filamin gene family members were located in the cell membrane. A total of 47 Filamin gene pairs were identified, most of which were whole-genome copies. Through the analyses of cis-acting elements, expression patterns and quantitative fluorescence, it was found that GH_ A02G0519 and GH_ D02G0539 are mainly expressed in the reproductive organs of upland cotton, and their interacting proteins are also related to the fertilization process, whereas GH_A02G0216 and GH_D02G0235 were related to stress. Thus, it is speculated that two genes of the GEX2 subfamily (GH_A02G0519 and GH_D02G0539) may be involved in the reproductive development of cotton and may affect the fertilization process of cotton. This study provides a theoretical basis for the further study of the cotton Filamin gene family.

A commercial ARHGEF17/TEM4 antibody cross-reacts with Nuclear Mitotic Apparatus protein 1 (NuMA).

The Rho family Guanine nucleotide exchange factor (GEF) ARHGEF17 (also known as TEM4) is a large protein with only 3 annotated regions: an N-terminal actin-binding domain, a Rho-specific dbl homology (DH)- pleckstrin homology (PH) type GEF domain and a seven bladed β propeller fold at the C-terminus with unknown function. TEM4 has been implicated in numerous activities that rely on regulation of the cytoskeleton including cell migration, cell-cell junction formation and the spindle assembly checkpoint during mitosis. Here we have assessed the specificity of a TEM4 polyclonal antibody that has been commonly used as a Western blotting and immunocytochemistry probe for TEM4 in mammalian cells. We find that this antibody, in addition to its intended target, cross-reacts with the Nuclear Mitotic Apparatus Protein 1 (NuMA) in Western blotting and immunoprecipitation, and detects NuMA preferentially in immunocytochemistry. This cross-reactivity, with an abundant chromatin- and mitotic spindle-associated factor, is likely to affect the interpretation of experiments that make use of this antibody probe, in particular by immunocytochemistry and immunoprecipitation.

Myosin-18B Regulates Higher-Order Organization of the Cardiac Sarcomere through Thin Filament Cross-Linking and Thick Filament Dynamics

MYO18B loss-of-function mutations and depletion significantly compromise the structural integrity of striated muscle sarcomeres. The molecular function of the encoded protein, myosin-18B (M18B), within the developing muscle is unknown. Here, we demonstrate that recombinant M18B lacks motor ATPase activity and harbors previously uncharacterized N-terminal actin-binding domains, properties that make M18B an efficient actin cross-linker and molecular brake capable of regulating muscle myosin-2 contractile forces. Spatiotemporal analysis of M18B throughout cardiomyogenesis and myofibrillogenesis reveals that this structural myosin undergoes nuclear-cytoplasmic redistribution during myogenic differentiation, where its incorporation within muscle stress fibers coincides with actin striation onset. Furthermore, this analysis shows that M18B is directly integrated within the muscle myosin thick filament during myofibril maturation. Altogether, our data suggest that M18B has evolved specific biochemical properties that allow it to define and maintain sarcomeric organization from within the thick filament via its dual actin cross-linking and motor modulating capabilities.

Homozygous G650del nexilin variant causes cardiomyopathy in mice.

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948–1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn’s function in tubular system organization and normal cardiac function.

Structure and Function of Filamin C in the Muscle Z-Disc.

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction.

The Mechanical Properties of a Utrophin Construct Encoding the Tandem CH Actin Binding Domain through Spectrin Repeat 3 is Altered by the Cell Expression System through Post-Translational Modifications

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by the absence of dystrophin protein. Utrophin is a dystrophin homologue currently under investigation as a replacement therapy for DMD. Dystrophin and utrophin are hypothesized to function as molecular shock absorbers to mechanically stabilize the muscle cell membrane. Recently, we published atomic force microscopy data showing that utrophin is much stiffer than data previously reported for dystrophin. Here we show that the cell expression system employed impacts both the post-translational modification and mechanical behavior of a utrophin construct encoding the N-terminal actin binding domain through spectrin repeat 3 (UtrN-R3).

Tissue-Specificity of Dystrophin-Actin Interactions: Isoform-Specific Thermodynamic Stability and Actin-Binding Function of Tandem Calponin-Homology Domains.

Genetic mutations in Duchenne muscular dystrophy (DMD) gene affecting the expression of dystrophin protein lead to a number of muscle disorders collectively called dystrophinopathies. In addition to muscle dystrophin, mutations in brain-specific dystrophin isoforms, in particular those that are expressed in the brain cortex and Purkinje neurons, result in cognitive impairment associated with DMD. These isoforms carry minor variations in the flanking region of the N-terminal actin-binding domain (ABD1) of dystrophin, which is composed of two calponin-homology (CH) domains in tandem. Determining the effect of these sequence variations is critical for understanding the mechanisms that govern varied symptoms of the disease. We studied the impact of differences in the N-terminal flanking region on the structure and function of dystrophin tandem CH domain isoforms. The amino acid changes did not affect the global structure of the protein but drastically affected the thermodynamic stability, with the muscle isoform more stable than the brain and Purkinje isoforms. Actin binding investigated with actin from different sources (skeletal muscle, smooth muscle, cardiac muscle, and platelets) revealed that the muscle isoform binds to filamentous actin (F-actin) with a lower affinity compared to the brain and Purkinje isoforms, and a similar trend was observed with actin from different sources. In addition, all isoforms showed a higher affinity to smooth muscle actin in comparison to actin from other sources. In conclusion, tandem CH domain isoforms might be using minor sequence variations in the N-terminal flanking regions to modulate their thermodynamic stability and actin-binding function, thus leading to specificity in dystrophin–actin interactions in various tissues.

A Novel Mutation in N-Terminal Actin-Binding Domain of the DMD Gene Presenting Becker Muscular Dystrophy as Recurrent Exertional Rhabdomyolysis: A Case Report.

A Novel Mutation in N-Terminal Actin-Binding Domain of the DMD Gene Presenting Becker Muscular Dystrophy as Recurrent Exertional Rhabdomyolysis: A Case Report.

Oryzocytosis: A Novel Morphological Variant of Hereditary Elliptocytosis Associated with a Novel Mutation in β-Spectrin (SPTB c154 C>T p.Arg52Trp)

Background We here report a family with a variant hereditary elliptocytosis (HE) characterized by previously undescribed mutations in β-spectrin, band 4.2, and the K+ channel ABCB6. The proband was a 2yo boy who presented for evaluation of elliptocytosis. On peripheral blood smear his red cells were elongated, but unusually small and dense in comparison to a classic elliptocytosis. Osmotic gradient ektacytometry (Osmoscan) showed a mild spherocytosis-like pattern and eosin-5-maleimide (EMA) fluorescence test showed a single population of cells with mildly decreased band 3. We termed the morphological pattern "Oryzocytosis" for the rice grain appearance of the cells Methods The proband, 3 siblings (all under 6 yrs of age), and 2 parents were evaluated. Band 3 content was estimated by EMA staining, and membrane deformability was determined by Osmoscan. The following genes were evaluated by next generation sequencing: ANK1, ACTIN,EPB41,EPB42,SLC4A1,SPTA1,SPTB; Ion channel genes- ABCB6, KCNN4, RHAG PIEZO1 (Fulgent Diagnostics, CA). Subsequently, all family members were tested by Sanger sequencing for the identified mutations. RBC ghosts were isolated and profiled on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on non-denaturing gels for spectrin tetramer:dimer ratios. Results Three gene mutations were found in the proband: SPTB c154 C>T p.Arg52Trp affecting the actin binding domain of β-spectrin, EPB42 c70C>T p.Pro24Ser affecting the band 3 binding domain of band 4.2, and ABCB6 c1694T>C p.Phe565Ser affecting a K+ transporter and cellular hydration status. The EPB42 and ABCB6 mutations were inherited from the father. The mother had the SPTB mutation alone, and the most severe phenotype of any family member, with a history of severe neonatal jaundice, multiple transfusions during adolescence, and splenectomy at age 21. She had elliptocytes on peripheral smear, only mild band 3 deficiency, and a normal Osmoscan. Two children inherited the SPTB mutation and were the only children with clinical manifestations by history. The proband inherited all 3 mutations, and had mild neonatal jaundice; a sibling with SPTB and ABCB6 mutations had a history of multiple episodes of mild jaundice. The RBCs of these 2 children were the least deformable on Osmoscan and demonstrated the greatest band 3 deficiencies. The father had both the EPB42 and ABCB6 mutations and passed them on to a third child. Both the father and that child had morphologic spherocytosis but no clinical sequelae. On testing, both had mild band 3 deficiency but no decrease in cell deformability. They had right shifted curves on Osmoscan indicating an altered tolerable range of osmolality, perhaps due to the ABCB6 mutation. The fourth child inherited only the ABCB6 mutation, but the peripheral smear unfortunately was uninterpretable. All 6 individuals had qualitatively high tetramer:dimer ratios on non-denaturing gels, indicating normal tetramerization. Discussion The most clinically significant mutation in this family appears to be in SPTB near the N-terminal actin binding domain. The discordance between the red cell morphology and the combination of decreased band 3 content and spherocytosis-like pattern on Osmoscan (high Omin (S/V), decreased deformability index (DI), and reduced Ohyper) is puzzling (see Figure 1). The unique trapezoidal pattern of classic HE with a double hump in DI max was not seen, nor was the classic hybrid population of RBCs with low and high band 3 content on EMA. Yet all 3 individuals with an SPTB mutation demonstrated some form of elliptocytic morphology. The determinants of elliptocytic shape are not fully defined, but previously described mutations have involved predominantly the spectrin dimer-dimer interaction site or band 4.1. Determining whether our SPTB mutation alters binding of β-spectrin with the junctional complex containing actin and band 4.1 may be key to further defining the mechanism causing Oryzocytosis. Disclosures Ravindranath: Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding.

EP.66A case with dystrophinopathies mimicking metabolic myopathies

Rhabdomyolysis associated with inherited conditions refers to recurrent episodes characterized by minimal exercise, history of malignant hyperthermia, familial history, or hyperCKemia between attacks. We describe a 17-year-old male presented recurrent myalgia following exercise. He had suffered exertional rhabdomyolysis with myoglobinuria and hyperCKemia between attacks, mimicking metabolic myopathies. The neurological examination at his 17 years of age, the muscle weakness was not present. The ischemic forearm exercise test revealed normal response, showing elevated lactate and ammonia. The needle electromyography showed myopathic changes. The immunoreactivity to C-terminal of dystrophin was absent in pathology biopsied from left biceps muscle. Genetic analysis of whole exome sequencing revealed a novel hemizygous c.119T>A (p.Leu40His) nonsense variant in DMD, located in the N-terminal actin-binding domain of dystrophin. In-silico test for the structure of dystrophin was predicted to destabilize interaction in the vicinity at residue 40 by losing hydrophobicity. This report will raise awareness that rhabdomyolysis and exertional myalgia can be seen in patients with a missense mutation of the exons encoding the N-terminal actin-binding domain of dystrophin.

Identification of Filamin A Mechanobinding Partner I: Smoothelin Specifically Interacts with the Filamin A Mechanosensitive Domain 21.

Filamin A (FLNA) is a ubiquitously expressed actin cross-linking protein and a scaffold of numerous binding partners to regulate cell proliferation, migration, and survival. FLNA is a homodimer, and each subunit has an N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats (R). FLNA mediates mechanotransduction by force-induced conformational changes of its cryptic integrin-binding site on R21. Here, we identified two novel FLNA-binding partners, smoothelins (SMTN A and B) and leucine zipper protein 1 (LUZP1), using stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics followed by an in silico screening for proteins having a consensus FLNA-binding domain. We found that, although SMTN does not interact with full-length FLNA, it binds to FLNA variant 1 (FLNAvar-1) that exposes the cryptic CD cleft of R21. Point mutations on the C strand that disrupt the integrin binding also block the SMTN interaction. We identified FLNA-binding domains on SMTN using mutagenesis and used the mutant SMTN to investigate the role of the FLNA-SMTN interaction on the dynamics and localization of SMTN in living cells. Fluorescence recovery after photobleaching (FRAP) of GFP-labeled SMTN in living cells demonstrated that the non-FLNA-binding mutant SMTN diffuses faster than wild-type SMTN. Moreover, inhibition of Rho-kinase using Y27632 also increases the diffusion. These data demonstrated that SMTN specifically interacts with FLNAvar-1 and mechanically activated FLNA in cells. The companion report (Wang and Nakamura, 2019) describes the interactions of FLNA with the transcript of the LUZP1 gene.

CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene.

ABSTRACTExon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.

Dynamics of Dystrophin’s Actin-Binding Domain

We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin’s N-terminal actin-binding domain (ABD1) and compare it to utrophin’s ABD1. Like other members of the spectrin superfamily, dystrophin’s ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin’s ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin’s and utrophin’s ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6–7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.

Sarcomeric and nonmuscle α-actinin isoforms exhibit differential dynamics at skeletal muscle Z-lines.

The α-actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α-actinin-1 and α-actinin-4 crosslink actin filaments in the cytoskeleton, while sarcomeric α-actinin-2 and α-actinin-3 serve a crucial role in anchoring actin filaments to the muscle Z-line. To assess the difference in turnover dynamics and structure/function properties between the α-actinin isoforms at the sarcomeric Z-line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α-actinin-2/α-actinin-3 had the slowest turn over, α-actinin-1 recovered to an intermediate degree, and α-actinin-4 had the fastest recovery. Interestingly, the isoforms' patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α-actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z-line. Protein domain-specific investigations using α-actinin-2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N-terminal actin-binding domain, the spectrin-like linker region and the C-terminal calmodulin-like EF hand domain. Together, these findings demonstrate that α-actinin isoforms are unique in binding dynamics at the Z-line and suggest differentially evolved interactive and Z-line association capabilities of each functional domain.

The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

In animals and fungi, cytoplasmic dynein is a processive minus-end-directed motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1, but not CC2, forms the coiled-coil to enable OsKCH2 dimerization. Instead, our results reveal that removing CC2 renders OsKCH2 a nonprocessive motor. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

FLNa negatively regulated proliferation and metastasis in lung adenocarcinoma A549 cells via suppression of EGFR.

Filamin A (FLNa) is a ubiquitously expressed cytoplasmic protein, which composes of an N-terminal actin binding domain (ABD) followed by 24 Ig-like repeats. FLNa functions as a cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and serves as a signaling intermediate. Recent studies have identified FLNa as a scaffold protein that interacts with over 90 proteins and plays vital roles in cellular signaling transduction. Mutations or defects in human FLNa gene have been shown to cause numerous developmental defects. Moreover, aberrant expression of FLNa has been observed in many cancers, such as parathyroid tumor, cervical cancer, and breast cancer. However, its role in lung adenocarcinoma has seldom been discussed. In the present study, our in vitro and in vivo studies demonstrated that silencing FLNa expression in lung cancer cell line A549 cells promoted proliferation, migration, and invasiveness of A549 cells by enhancing the activation of epidermal growth factor receptor and ERK signaling pathway. These results shed light on novel functions of FLNa in lung cancer and uncovered novel mechanisms, these results provided possible targets for the prediction and treatment for lung adenocarcinoma.