Myosin regulatory light chain is phosphorylated by myosin light chain kinase at conserved serine and threonine residues in a number of species. Phosphorylation of myosin regulatory light chain regulates smooth muscle contraction, but appears to have a modulatory role in striated muscle contraction. We assessed the in vivo role of myosin regulatory light chain phosphorylation in the striated muscles of Drosophila melanogaster by substituting alanine at each or both conserved myosin light chain kinase-dependent phosphorylation sites, serine 66 and serine 67. We report here that myosin light chain kinase-dependent phosphorylation is not required for myofibrillogenesis or for the development of maximal isometric force in indirect flight muscles. However, mutants with substitutions at the major phosphorylation site (serine 66) or with the double substitutions had reduced power output in isolated flight muscle fibres and reduced flight ability, showing that myosin regulatory light chain phosphorylation is a key determinant of the stretch activation response in Drosophila.

We examine how the structure and function of indirect flight muscle (IFM) and the entire flight system of Drosophila melanogaster are affected by phosphorylation of the myosin regulatory light chain (MLC2). This integrated study uses site-directed mutagenesis to examine the relationship between removal of the myosin light chain kinase (MLCK) phosphorylation site, in vivo function of the flight system (flight tests, wing kinematics, metabolism, power output), isolated IFM fiber mechanics, MLC2 isoform pattern, and sarcomeric ultrastructure. The MLC2 mutants exhibit graded impairment of flight ability that correlates with a reduction in both IFM and flight system power output and a reduction in the constitutive level of MLC2 phosphorylation. The MLC2 mutants have wild-type IFM sarcomere and cross-bridge structures, ruling out obvious changes in the ultrastructure as the cause of the reduced performance. We describe a viscoelastic model of cross-bridge dynamics based on sinusoidal length perturbation analysis (Nyquist plots) of skinned IFM fibers. The sinusoidal analysis suggests the high power output of Drosophila IFM required for flight results from a phosphorylation-dependent recruitment of power-generating cross-bridges rather than a change in kinetics of the power generating step. The reduction in cross-bridge number appears to affect the way mutant flies generate flight forces of sufficient magnitude to keep them airborne. In two MLC2 mutant strains that exhibit a reduced IFM power output, flies appear to compensate by lowering wingbeat frequency and by elevating wingstroke amplitude (and presumably muscle strain). This behavioral alteration is not seen in another mutant strain in which the power output and estimated number of recruited cross-bridges is similar to that of wild type.

Background and Aim. Cholangiocarcinoma (CCA) is the most frequent biliary malignancy, which poses high mortality rate due to lack of early detection. Hence, most CCA cases are present at the advanced to late stages with local or distant metastasis at the time of diagnosis. Currently available tumor markers including CA19-9 and CEA are inefficient and of limited usage due to low sensitivity and specificity. Here, we attempt to identify serum tumor markers for CCA that can effectively distinguish CCA from benign biliary tract diseases (BBTDs). Methods. Serum samples from 19 CCA patients and 17 BBTDs were separated by SDS-PAGE followed with LC-MS/MS and were subjected to statistical analysis and cross-validation to identify proteins whose abundance was significantly elevated or suppressed in CCA samples compared to BBTDs. Results. In addition to identifying several proteins previously known to be differentially expressed in CCA and BBTDs, we also discovered a number of molecules that were previously not associated with CCA. These included FAM19A5, MAGED4B, KIAA0321, RBAK, and UPF3B. Conclusions. Novel serum biomarkers to distinguish CCA from BBTDs were identified using a proteomic approach. Further validation of these proteins has the potential to provide a biomarker for differentiating CCA from BBTDs.

A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.