Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3(-/-) erythroblasts, and cell-cycle analysis reveals that there are more Ter119(hi) cells in S-phase in Tmod3(-/-) embryos. Notably, enucleating Tmod3(-/-) erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3(-/-) late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3(-/-) fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.

Force production in skeletal muscle is proportional to the amount of overlap between the thin and thick filaments, which, in turn, depends on their lengths. Both thin- and thick-filament lengths are precisely regulated and uniform within a myofibril. While thick-filament lengths are essentially constant across muscles and species (∼1.65 μm), thin-filament lengths are highly variable both across species and across muscles of a single species. Here, we used a high-resolution immunofluorescence and image analysis technique (distributed deconvolution) to directly test the hypothesis that thin-filament lengths vary across human muscles. Using deltoid and pectoralis major muscle biopsies, we identified thin-filament lengths that ranged from 1.19 ± 0.08 to 1.37 ± 0.04 μm, based on tropomodulin localization with respect to the Z-line. Tropomodulin localized from 0.28 to 0.47 μm further from the Z-line than the NH 2 -terminus of nebulin in the various biopsies, indicating that human thin filaments have nebulin-free, pointed-end extensions that comprise up to 34% of total thin-filament length. Furthermore, thin-filament length was negatively correlated with the percentage of type 2X myosin heavy chain within the biopsy and shorter in type 2X myosin heavy chain-positive fibers, establishing the existence of a relationship between thin-filament lengths and fiber types in human muscle. Together, these data challenge the widely held assumption that human thin-filament lengths are constant. Our results also have broad relevance to musculoskeletal modeling, surgical reattachment of muscles, and orthopedic rehabilitation.

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.

Abstract Abstract 84 Tropomodulin1 (Tmod1) binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild sphero-elliptocytic anemia due to mis-regulation of actin filament lengths and a disrupted membrane skeleton. The mild phenotype may be explained by the compensation of Tmod3, which is not found in wild-type RBCs but exists in Tmod1-null RBCs (one-fifth level of Tmod1). Our experiments with human erythroblasts show that the expression of Tmod1 increases during terminal differentiation while the level of Tmod3 is relatively constant, only decreasing at a very late stage. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) with a gene-trap vector insertion into intron 1 of Tmod3. Both RT-PCR and western-blot results show that the expression of Tmod3 is abolished in Tmod3−/− mice. Tmod3+/− mice are viable and fertile, while Tmod3−/− animals are embryonic lethal, with most nulls dying between E16.5-E17.5. Tmod3−/− embryos are pale and anemic with a smaller fetal liver, suggesting that the lethality might be due to defective definitive erythropoiesis. This is supported by analysis of peripheral blood, which shows fewer definitive enucleated erythroblasts in Tmod3-null embryos. Flow-cytometry of fetal liver erythroblasts labeled with Ter119 and CD71 indicates that the late stage R3 population is reduced by about one-third in absence of Tmod3, while R1-R2 populations are somewhat increased. In addition, Annexin V staining shows a two-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. These data indicate that definitive erythropoiesis is defective due to impaired erythroblast terminal differentiation in absence of Tmod3. To determine the underlying mechanisms, we used histology and confocal fluorescence microscopy to investigate the morphology and actin cytoskeleton of erythroblasts in process of enucleation. These experiments show abnormal nuclear morphology in orthochromatic Tmod3-null fetal liver erythroblasts, as well as defective F-actin contractile ring assembly in Tmod3−/− erythroblasts in process of nuclear expulsion, suggesting a role for Tmod3 in enucleation. Since macrophages are required for production of definitive erythroblasts and enucleation in vivo, we explored the role of macrophages in the Tmod3−/− phenotype. Immunofluorescence staining of fetal liver cryosections with F4/80, Ter119 and Hoechst reveals that macrophages display strikingly less dendritic morphologies in the Tmod3−/− mice, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Isolation of native erythroblast-macrophage islands from fetal liver demonstrates that islands isolated from Tmod3−/− fetal livers contain fewer erythroblasts compared with those from wild-type fetal liver. Further, reconstitution experiments indicate that erythroblasts from Tmod3−/− fetal liver are unable to form normal islands, indicating that Tmod3 function is required in erythroblasts. In conclusion, our study shows that knockout of Tmod3 leads to defective definitive erythropoiesis and embryonic lethality in mice, due to defects in island formation and abnormal enucleation. These data suggest that Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion as well as contractile ring assembly during erythroblast enucleation. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1036 Tropomodulin1 binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild compensated, sphero-elliptocytic anemia with fragile RBCs, due to mis-regulation of actin filament lengths and a disrupted membrane skeleton lattice (Moyer et al. 2010. Blood 116:2590–2599). The mild phenotype may be explained by the appearance of Tmod3, which is not found in wild-type RBCs but is in Tmod1-null RBCs, albeit at only 1/5th of normal Tmod1 levels. Tmod3 is ubiquitously expressed, including in erythroblasts, and thus the presence of Tmod3 in Tmod1-null RBCs is likely due to aberrant persistence of Tmod3 during terminal differentiation and maturation. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) in which intron 1 of the Tmod3 locus was disrupted by retroviral-mediated insertion of a gene-trap vector. The insertion creates a novel fusion transcript joining sequences from exons 5' to the insertion site to the beta-galactosidase marker. Tmod3+/− mice are viable and fertile, but Tmod3−/− animals are embryonic lethal, with 94% of nulls dying between E13.5 – E15.5 (175 total embryos, E12.5-E18.5). Tmod3−/− embryos are pale and anemic starting at E13.5, with much smaller livers. Tmod3 mRNA and protein are reduced in Tmod3+/− and absent in Tmod3−/−, demonstrating that this is a true null. Tmod1 mRNA and protein are also reduced, consistent with anemia, fewer erythroblasts and RBCs, indicating that Tmod1 expression does not compensate for absence of Tmod3. The peripheral blood of wild-type E13.5-E15.5 embryos contains abundant large primitive and smaller definitive RBCs, but few or no definitive enucleated RBCs are observed in Tmod3-null embryos. Fetal livers from Tmod3−/− embryos were about ½ the size of wild-type livers, and histology showed altered cellularity. However, the proliferative potential of erythroid progenitors appears not to be impaired based on similar numbers of BFU-E and CFU-E colonies from Tmod3+/+ and Tmod3−/− fetal livers. Cytospins of dissociated fetal liver cells from E13.5-E14.5 embryos reveal the presence of erythroblast/macrophage islands in both genotypes, but with some macrophages containing abundant ingested cellular debris in the Tmod3−/− cytospins. High magnification images show increased erythroblast blebbing in absence of Tmod3, suggesting a weaker cortex, and/or apoptosis of erythroblasts. Flow cytometry of E14.5 fetal liver erythroblasts labeled with Ter119 and CD71 indicate that the late stage R3 population is reduced by about 40–50% in absence of Tmod3, while R1-R2 populations are somewhat increased (possibly as a compensatory response to fetal anemia). In addition, Annexin V staining shows a 2-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. To explore the role of macrophages in the Tmod3−/− phenotype, we performed immunofluorescence staining and confocal microscopy of fetal liver cryosections stained with F4/80 (macrophages), Ter119 (erythroblasts) and Hoechst (nuclei). Macrophages displayed strikingly less dendritic morphologies in the Tmod3−/−, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Our data show that definitive erythropoiesis is impaired during terminal differentiation in absence of Tmod3, and we speculate that this is due to defective erythroblast-macrophage interactions during terminal differentiation. Experiments are in progress to determine whether Tmod3 function is required in erythroblasts or macrophages, and to identify the molecular pathway in which Tmod3 functions in erythropoiesis. Supported by NIH/NHLBI grant R01HL083464 to V.M.F. Disclosures: No relevant conflicts of interest to declare.

During myofibril assembly, thin filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify thin filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and thin filament lengths are normal in the absence of Tmod1. Tmod4 localizes to thin filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology.

The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.