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
Phosphorylation of the muscle-specific formin splice variant FHOD3 by CK2 regulates its stability, myofibril targeting, and myofibril integrity.
Members of the formin family are important for actin filament nucleation and elongation. We have identified a novel striated muscle–specific splice variant of the formin FHOD3 that introduces a casein kinase 2 (CK2) phosphorylation site. The specific targeting of muscle FHOD3 to the myofibrils in cardiomyocytes is abolished in phosphomutants or by the inhibition of CK2. Phosphorylation of muscle FHOD3 also prevents its interaction with p62/sequestosome 1 and its recruitment to autophagosomes. Furthermore, we show that muscle FHOD3 efficiently promotes the polymerization of actin filaments in cardiomyocytes and that the down-regulation of its expression severely affects myofibril integrity. In murine and human cardiomyopathy, we observe reduced FHOD3 expression with a concomitant isoform switch and change of subcellular targeting. Collectively, our data suggest that a muscle-specific isoform of FHOD3 is required for the maintenance of the contractile structures in heart muscle and that its function is regulated by posttranslational modification.
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.
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.
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
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.
Members of the formin family of actin filament nucleation factors have been implicated in sarcomere formation, but precisely how these proteins affect sarcomere structure remains poorly understood. Of six formins in the simple nematode Caenorhabditis elegans, only FHOD-1 and CYK-1 contribute to sarcomere assembly in the worm's obliquely striated body-wall muscles. We analyze here the ultrastructure of body-wall muscle sarcomeres in worms with putative null fhod-1 and cyk-1 gene mutations. Contrary to a simple model that formins nucleate actin for thin filament assembly, formin mutant sarcomeres contain thin filaments. Rather, formin mutant sarcomeres are narrower and have deformed thin filament-anchoring Z-line structures. Thus, formins affect multiple aspects of sarcomere structure.
Muscle; Sarcomere; Z-line; Thin filament; Formin; C. elegans
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.
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.
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.
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
We explored a function for tropomyosin (TM) in mammalian myofibril assembly and cardiac development by analyzing a deletion in the mouse TPM1 gene targeting αTM1, the major striated muscle TM isoform.
Mice lacking αTM1 are embryonic lethal at E9.5 with enlarged, misshapen, and non-beating hearts characterized by an abnormally thin myocardium and reduced trabeculae. αTM1-deficient cardiomyocytes do not assemble striated myofibrils, instead displaying aberrant non-striated F-actin fibrils with α-actinin puncta dispersed irregularly along their lengths. αTM1’s binding partner, tropomodulin1 (Tmod1), is also disorganized, and both myomesin-containing thick filaments as well as titin Z1Z2 fail to assemble in a striated pattern. Adherens junctions are reduced in size in αTM1-deficient cardiomyocytes, α-actinin/F-actin adherens belts fail to assemble at apical cell-cell contacts, and cell contours are highly irregular, resulting in abnormal cell shapes and a highly folded cardiac surface. In addition, Tmod1-deficient cardiomyocytes exhibit failure of α-actinin/F-actin adherens belt assembly.
Absence of αTM1 resulting in unstable F-actin may preclude sarcomere formation and/or lead to degeneration of partially assembled sarcomeres due to unregulated actomyosin interactions. Our data also identify a novel αTM1/Tmod1-based pathway stabilizing F-actin at cell-cell junctions, which may be required for maintenance of cell shapes during embryonic cardiac morphogenesis.
α-actinin; cell junction; cardiomyocyte; heart; myofibrillogenesis; myomesin; thin filament; titin; tropomodulin1
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.
Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.
Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis.
Cardiac myocytes; Myofibrillogenesis; Myosin; Obscurin; Sarcomere; Sarcoplasmic reticulum; Z-disks
Experiments are described supporting the proposition that the assembly of stress fibers in non-muscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membrane-associated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cell-substrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, alpha-actin, and sarcomeric alpha-actinin (s-alpha-actinin); (e) AJs are positive for A- CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all alpha-actinin- containing structures invariably are positive for the sarcomeric isoform, alpha-actin and related sarcomeric proteins; they lack non-s- alpha-actinin, gamma-actin, and caldesmon; (g) in fibroblasts all alpha- actinin-containing structures are positive for the non-sarcomeric isoform, gamma-actin, and related non-sarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins.
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
To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A- band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha- actinin and, if actin is added subsequently, the exogenous alpha- actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.
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
An in vitro study reveals how the three actin binding proteins profilin, formin 2, and Spire functionally cooperate by a ping-pong mechanism to regulate actin assembly during reproductive cell division.
In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a “ping-pong” mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase–independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle.
Mammalian reproduction requires successful meiosis, which consists of two strongly asymmetric cell divisions. In meiosis I, movement of the spindle (the subcellular structure that segregates chromosomes during division) toward the oocyte cortex (the outer layer of the egg) is essential for fertility. This process requires that actin filaments assemble in a dynamic mesh, driven by three actin binding proteins, profilin, formin 2, and Spire. To date the molecular mechanisms by which these three proteins cooperate are not known. We now explore this in vitro by a combination of bulk solution and single actin filament assembly assays in the presence of profilin, Spire, and formin 2. Individually, Spire binds to actin filament ends to block their growth, and by itself, formin 2 associates poorly with filament ends, promoting fast processive assembly from the profilin-actin complex. However, when present together, Spire and formin 2 interact with one another (the formin 2 C-terminal binds to the N terminal Spire KIND domain), forming transient complexes at filament ends from which each binds alternately to the filament ends to regulate actin assembly by a ping-pong mechanism. Our in vitro observations are validated by injection studies in mouse oocytes. In oocytes, the additional interaction of Spire and formin 2 with Rab11a-myosin Vb vesicles couples high actin dynamics to vesicle traffic.
Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch-repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, but fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils and contribute insight into mechanisms of myofibrillogenesis and disassembly.
kelch; heart; myofibrillogenesis; α-actinin; actin; myosin
Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells.
Muscle contraction driving voluntary movements and the beating of the heart relies on the contraction of highly regular bundles of actin and myosin filaments, which share a periodic, sarcomeric pattern. We know little about the mechanisms by which these ‘biological crystals’ are assembled and it is a general question how order on a scale of 100 micrometers can emerge from the interactions of micrometer-sized building blocks, such as actin and myosin filaments. In our paper, we consider a computational model for a bundle of actin filaments and discuss physical mechanisms by which periodic order emerges spontaneously. Mutual crosslinking of actin filaments results in the formation and coalescence of growing actin clusters. Active elongation and shrinkage dynamics of actin filaments generates polymerization forces and causes local actin flow that can act like a conveyor belt to sort myosin filaments in place.