Different Localizations and Cellular Behaviors of Leiomodin and Tropomodulin in Mature Cardiomyocyte Sarcomeres

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

Overexpression of Partner of Numb Induces Asymmetric Distribution of the PI4P 5-Kinase Skittles in Mitotic Sensory Organ Precursor Cells in Drosophila

Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::PonLD) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::PonLD is often used as a polarity marker in dividing progenitors

Sarcomeric Pattern Formation by Actin Cluster Coalescence

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

Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation

Relationship Between Anti-DFS70 Autoantibodies and Oxidative Stress

Background: The anti-DFS70 autoantibodies are one of the most commonly and widely described agent of unknown clinical significance, frequently detected in healthy individuals. It is not known whether the DFS70 autoantibodies are protective or pathogenic. One of the factors suspected of inducing the formation of anti-DFS70 antibodies is increased oxidative stress. We evaluated the coexistence of anti-DFS70 antibodies with selected markers of oxidative stress and investigated whether these antibodies could be considered as indirect markers of oxidative stress. Methods: The intensity of oxidative stress was measured in all samples via indices of free-radical damage to lipids and proteins such as total oxidant status (TOS), concentrations of lipid hydroperoxides (LPH), lipofuscin (LPS), and malondialdehyde (MDA). The parameters of the non-enzymatic antioxidant system, such as total antioxidant status (TAS) and uric acid concentration (UA), were also measured, as well as the activity of superoxide dismutase (SOD). Based on TOS and TAS values, the oxidative stress index (OSI) was calculated. All samples were also tested with indirect immunofluorescence assay (IFA) and 357 samples were selected for direct monospecific anti DFS70 enzyme-linked immunosorbent assay (ELISA) testing. Results: The anti-DFS70 antibodies were confirmed by ELISA test in 21.29% of samples. Compared with anti-DFS70 negative samples we observed 23% lower concentration of LPH (P =.038) and 11% lower concentration of UA (P =.005). TOS was 20% lower (P =.014). The activity of SOD was up to 5% higher (P =.037). The Pearson correlation showed weak negative correlation for LPH, UA, and TOS and a weak positive correlation for SOD activity. Conclusion: In samples positive for the anti-DFS70 antibody a decreased level of oxidative stress was observed, especially in the case of samples with a high antibody titer. Anti-DFS70 antibodies can be considered as an indirect marker of reduced oxidative stress or a marker indicating the recent intensification of antioxidant processes

DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin