Interactome and F-Actin Interaction Analysis of Dictyostelium discoideum Coronin A

Coronin proteins are evolutionary conserved WD repeat containing proteins that have been proposed to carry out different functions. In Dictyostelium, the short coronin isoform, coronin A, has been implicated in cytoskeletal reorganization, chemotaxis, phagocytosis and the initiation of multicellular development. Generally thought of as modulators of F-actin, coronin A and its mammalian homologs have also been shown to mediate cellular processes in an F-actin-independent manner. Therefore, it remains unclear whether or not coronin A carries out its functions through its capacity to interact with F-actin. Moreover, the interacting partners of coronin A are not known. Here, we analyzed the interactome of coronin A as well as its interaction with F-actin within cells and in vitro. Interactome analysis showed the association with a diverse set of interaction partners, including fimbrin, talin and myosin subunits, with only a transient interaction with the minor actin10 isoform, but not the major form of actin, actin8, which was consistent with the absence of a coronin A-actin interaction as analyzed by co-sedimentation from cells and lysates. In vitro, however, purified coronin A co-precipitated with rabbit muscle F-actin in a coiled-coil-dependent manner. Our results suggest that an in vitro interaction of coronin A and rabbit muscle actin may not reflect the cellular interaction state of coronin A with actin, and that coronin A interacts with diverse proteins in a time-dependent manner.

Spata6 is required for normal assembly of the sperm connecting piece and tight head–tail conjunction

“Pinhead sperm,” or “acephalic sperm,” a type of human teratozoospermia, refers to the condition in which ejaculate contains mostly sperm flagella without heads. Family clustering and homogeneity of this syndrome suggests a genetic basis, but the causative genes remain largely unknown. Here we report that Spata6, an evolutionarily conserved testis-specific gene, encodes a protein required for formation of the segmented columns and the capitulum, two major structures of the sperm connecting piece essential for linking the developing flagellum to the head during late spermiogenesis. Inactivation of Spata6 in mice leads to acephalic spermatozoa and male sterility. Our proteomic analyses reveal that SPATA6 is involved in myosin-based microfilament transport through interaction with myosin subunits (e.g., MYL6).

Improved V̇O2 uptake kinetics and shift in muscle fiber type in high-altitude trekkers

The study investigated the effect of prolonged hypoxia on central [i.e., cardiovascular oxygen delivery (Q̇aO2)] and peripheral (i.e., O2 utilization) determinants of oxidative metabolism response during exercise in humans. To this aim, seven male mountaineers were examined before and immediately after the Himalayan Expedition Interamnia 8000–Manaslu 2008, lasting 43 days, among which, 23 days were above 5,000 m. The subjects showed a decrease in body weight ( P < 0.05) and of power output during a Wingate Anaerobic test ( P < 0.05) and an increase of thigh cross-sectional area ( P < 0.05). Absolute maximal O2 uptake (V̇O2max) did not change. The mean response time of V̇O2 kinetics at the onset of step submaximal cycling exercise was reduced significantly from 53.8 s ± 10.9 to 39.8 s ± 10.9 ( P < 0.05), whereas that of Q̇aO2 was not. Analysis of single fibers dissected from vastus lateralis biopsies revealed that the expression of slow isoforms of both heavy and light myosin subunits increased, whereas that of fast isoforms decreased. Unloaded shortening velocity of fibers was decreased significantly. In summary, independent findings converge in indicating that adaptation to chronic hypoxia brings about a fast-to-slow transition of muscle fibers, resulting in a faster activation of the mitochondrial oxidative metabolism. These results indicate that a prolonged and active sojourn in hypoxia may induce muscular ultrastructural and functional changes similar to those observed after aerobic training.

Response of compressed skinned skeletal muscle fibers to conditions that simulate fatigue

Myburgh, Kathryn H., and Roger Cooke. Response of compressed skinned skeletal muscle fibers to conditions that simulate fatigue. J. Appl. Physiol. 82(4): 1297–1304, 1997.—During fatigue, muscles become weaker, slower, and more economical at producing tension. Studies of skinned muscle fibers can explain some but not all of these effects, and, in particular, they are less economical in conditions that simulate fatigue. We investigated three factors that may contribute to the different behavior of skinned fibers. 1) Skinned fibers have increased myofilament lattice spacing, which is reversible by osmotic compression. 2) A myosin subunit becomes phosphorylated during fatigue. 3) Inosine 5′-monophosphate (IMP) accumulates during fatigue. We tested the response of phosphorylated and unphosphorylated single skinned fibers (isometric tension, contraction velocity, and adenosinetriphosphatase activity) to changes in lattice spacing (0–5% dextran) and IMP (0–5 mM) in the presence of altered concentrations of Pi (3–25 mM), H+ (pH 7–6.2), and ADP (0–5 mM). The response of maximally activated skinned fibers to the direct metabolites of ATP hydrolysis is not altered by osmotic compression, phosphorylating myosin subunits, or increasing IMP concentration. These factors, therefore, do not explain the discrepancy between intact and skinned fibers during fatigue.

Identification of sequences necessary for the association of cardiac myosin subunits.

To begin to understand the nature of myosin subunit assembly, we determined the region of a vertebrate sarcomeric myosin heavy chain required for binding of light chain 1. We coexpressed in Escherichia coli segments of the rat alpha cardiac myosin heavy chain which spanned the carboxyl terminus of subfragment 1 and the amino terminus of subfragment 2 with a full-length rat cardiac myosin light chain 1. A 16 amino acid region of the myosin heavy chain (residues 792-808) was shown to be required for myosin light chain 1 binding in an immunoprecipitation assay.