Tropomyosin 1 genetically constrains in vitro hematopoiesis.

BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits. RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields. CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.

Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes.

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.

Tropomyosin regulates cell migration during skin wound healing.

Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing.

Tropomyosin modulates erythrocyte membrane stability.

The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a beta-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex.

Modification of the tropomyosin isoform composition of actin filaments in the brain by deletion of an alternatively spliced exon.

Tropomyosin (Tm) in non-muscle cells is involved in stabilisation of the actin cytoskeleton. Some of the 40 isoforms described are found in the brain and exhibit spatial and developmental regulation. Non-muscle isoforms from the gamma Tm gene can be subdivided into three subsets of isoforms differing at the C-terminus, all of which are found throughout the brain and some of which are implicated in different aspects of neuronal function. We have approached the role of different gamma isoforms in neuronal function by knocking out a subset of isoforms. We show here that we can successfully knock out all isoforms containing the brain-specific 9c C-terminus. Brains from these mice did not show any gross abnormalities. Western analysis of adult brains showed that 9c isoforms are reduced in +/- and absent in -/- mice but that a compensation by 9a-containing isoforms resulted in total levels of gamma products remaining the same. No other Tm isoforms were altered. We have therefore specifically altered the Tm composition in these neurons which allows us to study the effects of these changes on the cytoskeleton of neurons during growth, differentiation and maturation and give us insights into the normal roles of these isoforms.

Tropomyosin and troponin are required for ovarian contraction in the Caenorhabditis elegans reproductive system.

Ovulation in the nematode Caenorhabditis elegans is coordinated by interactions between the somatic gonad and germ cells. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells, but the regulatory mechanism of their contraction is unknown. We show that contraction of the ovarian muscle requires tropomyosin and troponin, which are generally major actin-linked regulators of contraction of striated muscle. RNA interference of tropomyosin or troponin C caused sterility by inhibiting ovarian contraction that is required for expelling mature oocytes into the spermatheca where fertilization takes place, thus causing accumulation of endomitotic oocytes in the ovary. Tropomyosin and troponin C were associated with actin filaments in the myoepithelial sheath, and the association of troponin C with actin was dependent on tropomyosin. A mutation in the actin depolymerizing factor/cofilin gene suppressed the ovulation defects by RNA interference of tropomyosin or troponin C. These results strongly suggest that tropomyosin and troponin are the actin-linked regulators for contraction of ovarian muscle in the C. elegans reproductive system.

Gamma tropomyosin gene products are required for embryonic development.

The actin filament system is essential for many cellular functions, including shape, motility, cytokinesis, intracellular trafficking, and tissue organization. Tropomyosins (Tms) are rod-like components of most actin filaments that differentially affect their stability and flexibility. The Tm gene family consists of four genes, alphaTm, betaTm, gammaTm (Tm5 NM, where "NM" indicates "nonmuscle"), and deltaTm (Tm4). Multiple isoforms of the Tm family are generated by alternative splicing of three of these genes, and their expression is highly regulated. Extensive spatial and temporal sorting of Tm isoforms into different cellular compartments has been shown to occur in several cell types. We have addressed the function of the low-molecular-weight Tms encoded by the gammaTm gene by eliminating the corresponding amino-terminal coding sequences from this gene. Heterozygous mice were generated, and subsequent intercrossing of the F1 pups did not result in any viable homozygous knockouts. Genotype analysis of day 2.5 morulae also failed to detect any homozygous knockouts. We have failed in our attempts to delete the second allele and generate in vitro double-knockout cells, although 51 clones displayed homologous recombination back into the originally targeted locus. We therefore conclude that low-molecular-weight products from the gammaTm gene are essential for both embryonic development and cell survival.

Drosophila paramyosin is important for myoblast fusion and essential for myofibril formation.

Paramyosin is a major structural protein of thick filaments in invertebrate muscles. Coiled-coil dimers of paramyosin form a paracrystalline core of these filaments, and the motor protein myosin is arranged on the core surface. To investigate the function of paramyosin in myofibril assembly and muscle contraction, we functionally disrupted the Drosophila melanogaster paramyosin gene by mobilizing a P element located in its promoter region. Homozygous paramyosin mutants die at the late embryo stage. Mutants display defects in both myoblast fusion and in myofibril assembly in embryonic body wall muscles. Mutant embryos have an abnormal body wall muscle fiber pattern arising from defects in myoblast fusion. In addition, sarcomeric units do not assemble properly and muscle contractility is impaired. We confirmed that these defects are paramyosin-specific by rescuing the homozygous paramyosin mutant to adulthood with a paramyosin transgene. Antibody analysis of normal embryos demonstrated that paramyosin accumulates as a cytoplasmic protein in early embryo development before assembling into thick filaments. We conclude that paramyosin plays an unexpected role in myoblast fusion and is important for myofibril assembly and muscle contraction.

Manipulating the contractile apparatus: genetically defined animal models of cardiovascular disease.

Within the last 10 years via gene targeting and transgenesis, numerous models of cardiovascular disease have been established and used to determine if a protein's presence or absence causes cardiovascular disease. By affecting the heart's protein complement in a defined manner, the function of the different mutated proteins or protein isoforms present in the contractile apparatus can be determined and pathogenic mechanism(s) explored. We can now remodel the cardiac protein profile and effect replacement of even the most abundant contractile proteins. Precise genetic manipulation allows exploration of the structure-function relationships which underlie cardiac function, and the consequences of defined mutations at the molecular, biochemical, cytological and physiologic levels can be determined.

The cardiac mutant Mexican axolotl is a unique animal model for evaluation of cardiac myofibrillogenesis.

Hearts from cardiac mutant Mexican axolotl, Ambystoma mexicanum, do not form organized myofibrils and fail to beat. Though previous biochemical and immunohistochemical experiments showed a possible reduction of cardiac tropomyosin it was not clear that this caused the lack of organized myofibrils in mutant hearts. We used cationic liposomes to introduce both rabbit and chicken tropomyosin protein into whole hearts of embryonic axolotls in whole heart organ cultures. The mutant hearts had a striking increase in the number of well-organized sarcomeric myofibrils when treated with rabbit or chicken tropomyosin. FITC-labeled rabbit tropomyosin was used to examine the kinetics of incorporation of the exogenous protein into mutant hearts and confirmed the uptake of exogenous protein by the cells of live hearts in culture. By 4 h of transfection, both normal and mutant hearts were found to incorporate FITC-labeled tropomyosin into myofibrils. We also delivered an anti-tropomyosin antibody (CH 1) into normal hearts to disrupt the existing cardiac myofibrils which also resulted in reduced heartbeat rates. CH1 antibody was detected within the hearts and disorganization of the myofibrils was apparent when compared to normal controls. Introduction of a C-protein monoclonal antibody (ALD 66) did not result in a disruption of organized myofibrils. The results show clearly that chicken or rabbit tropomyosin could be incorporated by the mutant hearts and that it was sufficient to overcome the factors causing a lack of myofibril formation in the mutant. This finding also suggests that a lack of organized myofibrils is caused primarily by either inadequate levels of tropomyosin or endogenous tropomyosin in mutant hearts is unsuitable for myofibril formation, which we were able to duplicate with the introduction of tropomyosin antibody. Furthermore, incorporation of a specific exogenous protein or antibody into normal and mutant hearts of the Mexican axolotl in whole heart organ culture offers an unique model to evaluate functionalroles of contractile proteins necessary for cardiac development and differentiation.

Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation.

Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton.

Effects of tropomyosin deficiency in flight muscle of Drosophila melanogaster.

We have studied the structure and function of muscle fibers in which tropomyosin stoichiometry has been reduced by genetic mutation. We used a Drosophila melanogaster flightless mutant Ifm(3)3 and a genetic cross of this mutant with wild type flies to achieve a gradation of tropomyosin gene dosage. We measured the flight ability and wingbeat frequency of the live insects and the ultrastructure and mechanochemistry of isolated single flight muscle fibers. Flight ability is impaired when tropomyosin gene dosage is reduced. Wingbeat frequency also depends upon gene dosage as well as the severity of myofilament lattice disruption and the number of myofilaments in the organized core of the myofibrils. A reduction in number of myofilaments appears to result in a reduction in active muscle stiffness without resulting in an appreciable change in kinetics of force production. Ifm(3)3 is trapped in a relaxed state and cannot generate active force. However, tight-binding rigor cross-bridges are able to form; in the absence of ATP, Ifm(3)3 muscle fibers have high stiffness and force.

RNA from normal anterior endoderm/mesoderm-conditioned medium stimulates myofibrillogenesis in developing mutant axolotl hearts.

In the axolotl, Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts do not contain sarcomeric myofibrils nor do they beat. We have previously shown that normal anterior endoderm, medium conditioned by endoderm, or total RNA extracted from endoderm stimulates differentiation of mutant hearts in culture as indicated by the presence of organized myofibrils and rhythmic contractions of the "rescued" mutant heart tube. In this study, to get a more highly purified sample of the "active" molecule, RNA extracted from endoderm-conditioned medium and was assayed for its ability to promote myofibrillogenesis in mutant hearts. Mutant heart mesoderm responded to conditioned-medium RNA in a dose-dependent manner. Proteinase K treatment of the RNA did not affect inductive activity, while digestion with RNase A completely abolished the ability to rescue mutant hearts. Confocal laser scanning microscopy of immunostained, organ-cultured hearts revealed that mutant hearts contain reduced amounts of the sarcomeric protein tropomyosin in an amorphous distribution, whereas normal and corrected mutant hearts contain tropomyosin primarily in organized myofibrils.