Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart, which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2alpha knockout mice showed that phosphorylation by CK2 is required for proper targeting of muscle FHOD3 to the myofibrils also in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in adult heart. Following myofibril disassembly such as in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3.
Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3 and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic for the healthy mature heart and that two distinct phosphorylation events are crucial to regulate its activity in thin filament assembly and maintenance.
myofibril; formin; cardiac cytoarchitecture; heart development
Striated muscle cells display an extremely regular assembly of their actin cytoskeleton that contributes to the contractile elements, the myofibrils. How this assembly is initiated and how these structures are maintained is still unclear. We have recently shown that striated muscle expresses a specific isoform of the formin protein family member FHOD3, which is characterised by the presence of a CK2 phosphorylation site at the C-terminal end of the formin homology domain 2 (FH2). Phosphorylated muscle FHOD3 displays a different subcellular localisation, namely to the myofibrils, and also has increased stability compared to un-phosphorylated or non muscle FHOD3. In addition, we could show that muscle FHOD3 is involved in myofibril maintenance in cultured cardiomyocytes and that its presence dramatically enhances the reconstitution of cardiac actin filaments after depolymerisation. Since FHOD3 expression levels and in particular that of the muscle isoform are also decreased in different types of cardiomyopathy, we postulate a crucial role for this protein in the maintenance of a fully functional cardiac cytoarchitecture.
heart; development; actin filament; formin; sarcomere
The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain.
Our goal was to test whether formin homology protein 1 (FHOD1) plays a significant role in the regulation of SMC differentiation, and if so, whether Rho-kinase (ROCK)-dependent phosphorylation in the diaphanous auto-inhibitory domain is an important signaling mechanism that controls FHOD1 activity in SMC.
Methods and Results
FHOD1 is highly expressed in aortic SMCs and in tissues with a significant SMC component. Exogenous expression of constitutively active FHOD1, but not WT, strongly activated SMC-specific gene expression in 10T1/2 cells. Treatment of SMC with the RhoA activator, sphingosine-1-phosphate (S1P), increased FHOD1 phosphorylation at T1141 and this effect was completely prevented by inhibition of ROCK with Y-27632. Phosphomimetic mutations to ROCK target residues enhanced FHOD1 activity suggesting that phosphorylation interferes with FHOD1 auto-inhibition. Importantly, knock-down of FHOD1 in SMC strongly inhibited S1P-dependent increases in SMC differentiation marker gene expression and actin polymerization suggesting that FHOD1 plays a major role in RhoA-dependent signaling in SMC.
Our results indicate that FHOD1 is a critical regulator of SMC phenotype and is regulated by ROCK-dependent phosphorylation. Thus, further studies on the role of FHOD1 during development and the progression of cardiovascular disease will be important.
Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3−/− mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3−/− embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development.
Actin; Fhod3; Formin; Myofibrillogenesis; Sarcomere
During the second half of embryogenesis, the ellipsoidal Caenorhabditis elegans embryo elongates into a long, thin worm. This elongation requires a highly organized cytoskeleton composed of actin microfilaments, microtubules and intermediate filaments throughout the epidermis of the embryo. This architecture allows the embryonic epidermal cells to undergo a smooth muscle-like actin/myosin-based contraction that is redundantly controlled by LET- 502/Rho kinase and MEL-11/myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21-activated kinase in a parallel pathway(s). Although actin microfilaments surround the embryo, the force for contraction is generated mainly in the lateral (seam) epidermal cells whose actin microfilaments appear qualitatively different from those in their dorsal/ventral neighbors. We have identified FHOD-1, a formin family actin nucleator, which acts in the lateral epidermis. fhod-1 mutants show microfilament defects in the embryonic lateral epidermal cells and FHOD-1 protein is detected only in those cells. fhod-1 genetic interactions with let-502, mel-11, fem-2 and pak-1 indicate that fhod-1 preferentially regulates those microfilaments acting with let-502 and mel-11, and in parallel to fem-2 and pak-1. Thus, FHOD-1 may contribute to the qualitative differences in microfilaments found in the contractile lateral epidermal cells and their non-contractile dorsal and ventral neighbors. Different microfilament populations may be involved in the different contractile pathways.
C. elegans; morphogenesis; actin; formin; cytoskeleton; embryo; genetics
Cancer cells can obtain their ability to invade and metastasise by undergoing epithelial-to-mesenchymal transition (EMT). Exploiting this mechanism of cellular plasticity, malignant cells can remodel their actin cytoskeleton and down-regulate proteins needed for cell-cell contacts. The mechanisms of cytoskeletal reorganisation resulting in mesenchymal morphology and increased invasive potential are poorly understood. Actin nucleating formins have been implicated as key players in EMT. Here, we analysed which formins are altered in squamous cell carcinoma related EMT. FHOD1, a poorly studied formin, appeared to be markedly upregulated upon EMT. In human tissues FHOD1 was primarily expressed in mesenchymal cells, with little expression in epithelia. However, specimens from oral squamous cell cancers demonstrated consistent FHOD1 upregulation in mesenchymally transformed cells at the invasive edge. This upregulation was confirmed in an oral squamous carcinoma model, where FHOD1 expression was markedly increased upon EMT in a PI3K signalling dependent manner. In the EMT cells FHOD1 contributed to the spindle-shaped morphology and mesenchymal F-actin organization. Furthermore, functional assays demonstrated that FHOD1 contributes to cell migration and invasion. Finally, FHOD1 depletion reduced the ability of EMT cancer cells to form invadopodia and to degrade extracellular matrix. Our results indicate that FHOD1 participates in cytoskeletal changes in EMT. In addition, we show that FHOD1 upregulation occurs during cancer cell EMT in vivo, which indicates that FHOD1 may contribute to tumour progression.
Two actin-assembling formins, CYK-1 and FHOD-1, are important for muscle cell growth and maintenance of the contractile lattice in striated muscle cells.
Muscle contraction depends on interactions between actin and myosin filaments organized into sarcomeres, but the mechanism by which actin filaments incorporate into sarcomeres remains unclear. We have found that, during larval development in Caenorhabditis elegans, two members of the actin-assembling formin family, CYK-1 and FHOD-1, are present in striated body wall muscles near or on sarcomere Z lines, where barbed ends of actin filaments are anchored. Depletion of either formin during this period stunted growth of the striated contractile lattice, whereas their simultaneous reduction profoundly diminished lattice size and number of striations per muscle cell. CYK-1 persisted at Z lines in adulthood, and its near complete depletion from adults triggered phenotypes ranging from partial loss of Z line–associated filamentous actin to collapse of the contractile lattice. These results are, to our knowledge, the first genetic evidence implicating sarcomere-associated formins in the in vivo organization of the muscle cytoskeleton.
Active positioning of the nucleus is integral to division, migration, and differentiation of mammalian cells1. Fibroblasts polarizing for migration orient their centrosomes by actin-dependent nuclear movement2. This nuclear movement depends on nesprin-2 giant (N2G), a large, actin-binding outer nuclear membrane component of transmembrane actin-associated (TAN) lines that couple nuclei to moving actin cables3. Here, we identify the diaphanous formin FHOD1 as an interaction partner of N2G. Silencing FHOD1 expression or expression of fragments containing binding sites of N2G or FHOD1 disrupted nuclear movement and centrosome orientation in polarizing fibroblasts. Unexpectedly, silencing of FHOD1 expression did not affect the formation or rearward flow of dorsal actin cables required for nuclear positioning. Rather, N2G-FHOD1 interaction provided a second connection to actin cables essential for TAN line formation and thus nuclear movement. These results reveal a unique function for a formin in coupling an organelle to actin filaments for translocation and suggest that TAN lines require multi-point attachments to actin cables to resist the large forces necessary to move the nucleus.
Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE . It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex . Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2 , accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.
► FMNL2 is a novel Cdc42 effector accumulating at lamellipodial and filopodial tips ► FMNL2 is regulated but not localized by N-terminal myristoylation and Cdc42 binding ► FMNL2 processively elongates actin filaments in the presence of profilin ► FMNL2 drives cell migration by increasing the efficiency of lamellipodia protrusion
Vaccinia virus actin–based motility relies on integration of the N-WASP–ARP2/3 and Rac1–FHOD1 pathways.
Vaccinia virus dissemination relies on the N-WASP–ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP–ARP2/3 and Rac1–FHOD1 pathways.
Seven of 15 different mouse formins localized in diverse patterns to cardiomyocyte sarcomeres. Four were required for proper organization of myofibrils, and two were critical for remodeling and repair of myofibril structure.
Cardiac and skeletal muscle function depends on the proper formation of myofibrils, which are tandem arrays of highly organized actomyosin contractile units called sarcomeres. How the architecture of these colossal molecular assemblages is established during development and maintained over the lifetime of an animal is poorly understood. We investigate the potential roles in myofibril formation and repair of formin proteins, which are encoded by 15 different genes in mammals. Using quantitative real-time PCR analysis, we find that 13 formins are differentially expressed in mouse hearts during postnatal development. Seven formins immunolocalize to sarcomeres in diverse patterns, suggesting that they have a variety of functional roles. Using RNA interference silencing, we find that the formins mDia2, DAAM1, FMNL1, and FMNL2 are required nonredundantly for myofibrillogenesis. Knockdown phenotypes include global loss of myofibril organization and defective sarcomeric ultrastructure. Finally, our analysis reveals an unanticipated requirement specifically for FMNL1 and FMNL2 in the repair of damaged myofibrils. Together our data reveal an unexpectedly large number of formins, with diverse localization patterns and nonredundant roles, functioning in myofibril development and maintenance, and provide the first evidence of actin assembly factors being required to repair myofibrils.
Lmod is a muscle-specific actin nucleator that displays structural similarity to the filament pointed-end–capping protein, Tmod. The mechanisms of localizations of Lmod and Tmod in muscle sarcomeres are strikingly different. Lmod contributes to the organization of mature myofibrils through a mechanism that requires interaction with tropomyosin.
Leiomodin (Lmod) is a muscle-specific F-actin–nucleating protein that is related to the F-actin pointed-end–capping protein tropomodulin (Tmod). However, Lmod contains a unique ∼150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod's localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.
MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor β (TGF-β)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.
The N-terminal region (1–339) of the human FHOD1 protein has been crystallized in two different crystal forms. A crystal of the (C31S,C71S) mutant diffracted to around 2.3 Å resolution.
Formins are key regulators of actin cytoskeletal dynamics that constitute a diverse protein family that is present in all eukaryotes examined. They typically consist of more than 1000 amino acids and are defined by the presence of two conserved regions, namely the formin homology 1 and 2 domains. Additional conserved domains comprise a GTPase-binding domain for activation, a C-terminal autoregulation motif and an N-terminal recognition domain. In this study, the N-terminal region (residues 1–339) of the human formin homology domain-containing protein 1 (FHOD1) was purified and crystallized from 20%(w/v) PEG 4000, 10%(v/v) glycerol, 0.3 M magnesium chloride and 0.1 M Tris–HCl pH 8.0. Native crystals belong to space group P1, with unit-cell parameters a = 35.4, b = 73.9, c = 78.7 Å, α = 78.2, β = 86.2, γ = 89.7°. They contain two monomers of FHOD1 in the asymmetric unit and diffract to a resolution of 2.3 Å using a synchrotron-radiation source.
FHOD1; FH3 domain; diaphanous-related formins
Incomplete penetrance and variable expression of Hypertrophic Cardiomyopathy (HCM) is well appreciated. Common genetic polymorphisms variants that may affect HCM penetrance and expression have been predicted but are not well established.
Methods and Results
We performed a case-control genome wide association (GWA) study to identify common HCM-associated genetic polymorphisms and then asked whether such common variants were more represented in HCM or could explain the heterogeneity of HCM phenotypes. We identified an intronic FHOD3 variant (rs516514) associated with HCM (OR = 2.45 (95% CI 1.76–3.41), p=1.25 × 10−7) and validated this finding in an independent cohort. Next, we tested FHOD3-V1151I (rs2303510), a non-synonymous variant in partial linkage disequilibrium (LD) with rs516514, and we detected an even stronger association with HCM (p=1.76 × 10−9). While HCM patients were more likely to carry these FHOD3 alleles subjects homozygous for FHOD3-1151I had similar HCM phenotypes as carriers of the V1151 allele. FHOD3 expression is increased in the setting of HCM and both alleles of FHOD3-V1151I were detected in HCM myectomy tissue. Previously FHOD3 was found to be required for formation of the sarcomere and here we demonstrate that its fly homolog fhos is required for normal adult heart systolic contraction.
Here we demonstrate the association of a common non-synonymous FHOD3 genetic variant with HCM. This discovery further strengthens the potential role of gene mutations and polymorphisms that alter the amino acid sequence of sarcomere proteins and HCM.
contractility; genome-wide analysis; hypertrophic cardiomyopathy
A homozygous disruption or genetic mutation of the bag3 gene, a member of the Bcl-2-associated athanogene (BAG) family proteins, causes cardiomyopathy and myofibrillar myopathy that is characterized by myofibril and Z-disc disruption. However, the detailed disease mechanism is not yet fully understood.
bag3−/− mice exhibit differences in the extent of muscle degeneration between muscle groups with muscles experiencing the most usage degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests a role for BAG3 in supporting cytoskeletal connections between the Z-disc and myofibrils under mechanical stress. The mechanism by which myofibrillar structure is maintained under mechanical stress remains unclear. The purpose of the study is to clarify the detailed molecular mechanism of BAG3-mediated muscle maintenance under mechanical stress.
Methods and Results
To address the question of whether bag3 gene knockdown induces myofibrillar disorganization caused by mechanical stress, in vitro mechanical stretch experiments using rat neonatal cardiomyocytes and an shRNA-mediated gene knockdown system of the bag3 gene were performed. As expected, mechanical stretch rapidly disrupts myofibril structures in bag3 knockdown cardiomyocytes. BAG3 regulates the structural stability of F-actin through the actin capping protein, CapZβ1, by promoting association between Hsc70 and CapZβ1. BAG3 facilitates the distribution of CapZβ1 to the proper location, and dysfunction of BAG3 induces CapZ ubiquitin-proteasome-mediated degradation. Inhibition of CapZβ1 function by overexpressing CapZβ2 increased myofibril vulnerability and fragmentation under mechanical stress. On the other hand, overexpression of CapZβ1 inhibits myofibrillar disruption in bag3 knockdown cells under mechanical stress. As a result, heart muscle isolated from bag3−/− mice exhibited myofibrillar degeneration, and lost contractile activity after caffeine contraction.
These results suggest novel roles for BAG3 and Hsc70 in stabilizing myofibril structure and inhibiting myofibrillar degeneration in response to mechanical stress. These proteins are possible targets for further research to identify therapies for myofibrillar myopathy or other degenerative diseases.
BAG-family proteins; BAG3; molecular chaperone; myofibrillar myopathy; heart failure
Assembly and maintenance of myofibrils require dynamic regulation of the actin cytoskeleton. In Caenorhabditis elegans, UNC-60B, a muscle-specific actin depolymerizing factor (ADF)/cofilin isoform, is required for proper actin filament assembly in body wall muscle (Ono, S., D.L. Baillie, and G.M. Benian. 1999. J. Cell Biol. 145:491–502). Here, I show that UNC-78 is a homologue of actin-interacting protein 1 (AIP1) and functions as a novel regulator of actin organization in myofibrils. In unc-78 mutants, the striated organization of actin filaments is disrupted, and large actin aggregates are formed in the body wall muscle cells, resulting in defects in their motility. Point mutations in unc-78 alleles change conserved residues within different WD repeats of the UNC-78 protein and cause less severe phenotypes than a deletion allele, suggesting that these mutations partially impair the function of UNC-78. UNC-60B is normally localized in the diffuse cytoplasm and to the myofibrils in wild type but mislocalized to the actin aggregates in unc-78 mutants. Similar Unc-78 phenotypes are observed in both embryonic and adult muscles. Thus, AIP1 is an important regulator of actin filament organization and localization of ADF/cofilin during development of myofibrils.
myofibrils; AIP1; ADF/cofilin; WD repeats; actin filament dynamics
Matrix adhesions provide critical signals for cell growth or differentiation. They form through a number of distinct steps that follow integrin binding to matrix ligands. In an early step, integrins form clusters that support actin polymerization by an unknown mechanism. This raises the question of how actin polymerization occurs at the integrin clusters. We report here that a major formin in mouse fibroblasts, FHOD1 is recruited to integrin clusters, resulting in actin assembly. Using cell-spreading assays on lipid bilayers, solid substrates and high-resolution force sensing pillar arrays, we find that knockdown of FHOD1 impairs spreading, coordinated application of adhesive force and adhesion maturation. Finally we show that targeting of FHOD1 to the integrin sites depends on the direct interaction with Src family kinases, and is upstream of the activation by Rho Kinase. Thus our findings provide insights into the mechanisms of cell migration with implications for development and disease.
The structure of the NH2-terminal region of troponin T (TnT) is hypervariable among the muscle type-specific isoforms and is also regulated by alternative RNA splicing. This region does not contain binding sites for other thin filament proteins but alteration of its structure affects the Ca2+-regulation of muscle contraction. Here we report a truncated cardiac TnT produced during myocardial ischemia-reperfusion. Amino acid sequencing and protein fragment reconstruction determined that it is generated by a posttranslational modification selectively removing the NH2-terminal variable region and preserving the conserved core structure of TnT. Triton X-100 extraction of cardiac muscle fibers promoted production of the NH2-terminal truncated cardiac TnT (cTnT-ND), indicating a myofibril-associated proteolytic activity. μ-Calpain is a myofibril-associated protease and is known to degrade TnT. Supporting a role of μ-calpain in producing cTnT-ND in myocardial ischemia-reperfusion, calpain inhibitors decreased the level of cTnT-ND in Triton-extracted myofibrils. μ-Calpain treatment of cardiac myofibril and troponin complex specifically reproduced cTnT-ND. In contrast, μ-calpain treatment of isolated cardiac TnT resulted in non-specific degradation, suggesting that this structural modification is relevant to physiological structures of the myofilament. Triton X-100 treatment of transgenic mouse cardiac myofibrils over-expressing fast skeletal muscle TnT produced similar NH2-terminal truncations of the endogenous and exogenous TnT, despite different amino acid sequences at the cleavage site. With the functional consequences of removing the NH2-terminal variable region of TnT, the μ-calpain-mediated proteolytic modification of TnT may act as an acute mechanism to adjust muscle contractility under stress conditions.
Tropomodulin (Tmod) is an actin pointed-end capping protein that regulates actin dynamics at thin filament pointed ends in striated muscle. Although pointed-end capping by Tmod controls thin filament lengths in assembled myofibrils, its role in length specification during de novo myofibril assembly is not established. We used the Drosophila Tmod homologue, sanpodo (spdo), to investigate Tmod's function during muscle development in the indirect flight muscle. SPDO was associated with the pointed ends of elongating thin filaments throughout myofibril assembly. Transient overexpression of SPDO during myofibril assembly irreversibly arrested elongation of preexisting thin filaments. However, the lengths of thin filaments assembled after SPDO levels had declined were normal. Flies with a preponderance of abnormally short thin filaments were unable to fly. We conclude that: (a) thin filaments elongate from their pointed ends during myofibril assembly; (b) pointed ends are dynamically capped at endogenous levels of SPDO so as to allow elongation; (c) a transient increase in SPDO levels during myofibril assembly converts SPDO from a dynamic to a permanent cap; and (d) developmental regulation of pointed-end capping during myofibril assembly is crucial for specification of final thin filament lengths, myofibril structure, and muscle function.
actin-capping protein; thin filaments; myofibril; sanpodo; tropomodulin
Monoclonal antibodies specific for the muscle protein titin have been used in conjunction with muscle-specific antibodies against myofibrillar myosin heavy chains (MHCs) and desmin to study myogenesis in cultured cells. Desmin synthesis is initiated in replicating presumptive myoblasts, whereas the synthesis of titin and MHC is initiated simultaneously in their progeny, the postmitotic, mononucleated myoblasts. Both titin and MHC are briefly localized to nonstriated and thereafter to definitively striated myofibrils. At no stage during myofibrillogenesis is either protein observed as part of a sequence of mini-sarcomeres. Titin antibodies bind to the A-I junction, MHC antibodies to the A bands in nascent, maturing, and mature myofibrils. In contrast, desmin remains distributed as longitudinal filaments until well after the definitive myofibrils have aligned laterally. This tight temporal and topographical linkage between titin and myosin is also observed in postmitotic, mononucleated myoblasts and multinucleated myotubes when myofibrillogenesis is perturbed with Colcemid or taxol. Colcemid induces elongating postmitotic mononucleated myoblasts and multinucleated myotubes to round up and form Colcemid myosacs. The myofibrils that emerge in these rounded cells are deployed in convoluted circles. The time required for their nonstriated myofibrils to transform into striated myofibrils is greatly protracted. Furthermore, as Colcemid induces immense desmin intermediate filament cables, the normal spatial relationships between emerging individual myofibrils is distorted. Despite these disturbances at all stages, the characteristic temporal and spatial relationship observed in normal myofibrils between titin and MHC is observed in myofibrils assembling in Colcemid-treated cells. Newly born postmitotic mononucleated myoblasts, or maturing myotubes, reared in taxol acquire a star-shaped configuration and are induced to assemble "pseudo- striated myofibrils." Pseudo-striated myofibrils consist of laterally aggregated 1.6-micron long, thick filaments that interdigitate, not with thin filaments, but with long microtubules. These atypical myofibrils lack Z bands. Despite the absence of thin filaments and Z bands, titin localizes with its characteristics sarcomeric periodicity in pseudo-striated myofibrils. We conclude that the initiation and subsequent regulation of titin and myosin synthesis, and their spatial deployment within developing sarcomeres are tightly coupled events. These findings are discussed in terms of a model that proposes interaction between two relatively autonomous "organizing centers" in the assembly of each sarcomere.
Capping protein nucleates the assembly of actin filaments and stabilizes actin filaments by binding to their barbed ends. We describe here a novel isoform of the beta subunit of chicken capping protein, the beta 2 isoform, which arises by alternative splicing. The chicken beta 1 isoform and the beta 2 isoform are identical in their amino acid sequence except for a short region at the COOH terminus; this region of the beta subunit has been implicated in binding actin. Human and mouse cDNAs of the beta 1 and beta 2 isoforms also were isolated and among these vertebrates, the COOH-terminal region of each isoform is highly conserved. In contrast, comparison of the sequences of the vertebrate beta subunit COOH-termini to those of lower eukaryotes shows no similarities. The beta 2 isoform is the predominant isoform of nonmuscle tissues and the beta 1 isoform, which was first characterized in studies of capping protein from chicken muscle, is the predominant isoform of muscle tissues, as shown by immunoblots probed with isoform- specific antibodies and by RNAse protection analysis of mRNAs. The beta 2 isoform also is a component of dynactin complex from brain, which contains the actin-related protein Arp1. Both beta-subunit isoforms are expressed in cardiac muscle but they have non-overlapping subcellular distributions. The beta 1 isoform is at Z-discs of myofibrils, and the beta 2 isoform is enriched at intercalated discs; in cardiac myocytes grown in culture, the beta 2 isoform also is a component of cell-cell junctions and at sites where myofibrils contact the sarcolemma. The biochemical basis for the differential distribution of capping protein isoforms is likely due to interaction with specific proteins at Z-discs and cell-cell junctions, or to preferential association with different actin isoforms. Thus, vertebrates have developed isoforms of capping protein that associate with distinct actin-filament arrays.
In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate. © 2010 Wiley-Liss, Inc.
myofibrils; sarcomeres; actin turnover; congenital myopathy; stabilization; depolymerization; capping
In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate.
Myofibrils; sarcomeres; actin turnover; congenital myopathy; stabilization; depolymerization; capping