A call for action: Improve reporting of research studies to increase the scientific basis for regulatory decision-making.

This is a call for action to scientific journals to introduce reporting requirements for toxicity and ecotoxicity studies. Such reporting requirements will support the use of peer-reviewed research studies in regulatory decision-making. Moreover, this could improve the reliability and reproducibility of published studies in general and make better use of the resources spent in research.

Evidence for gamma-actin as a Z disc component in skeletal myofibers.

We investigated the targeting of the gamma-actin isoform in skeletal myofibers. For this purpose we used expression vectors to produce green fluorescent protein (GFP-) as well as myc-tagged gamma-actin in rat flexor digitorum brevis myofibers. We found that the gamma-actin fusion proteins accumulated into Z discs but not beneath the sarcolemma. Instead, the GFP-tagged skeletal muscle-specific alpha-actin isoform was preferentially incorporated into the pointed ends of thin contractile filaments. The localization pattern of the gamma-actin fusion proteins was completely different from that of the dystrophin glycoprotein complex on the sarcolemma. The results emphasize the role of gamma-actin as a Z disc component but fail to reveal an actin-based sub-sarcolemmal cytoskeleton in skeletal muscle cells.

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers.

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca(2+)-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.

F413C and A531V but not R894X myotonia congenita mutations cause defective endoplasmic reticulum export of the muscle-specific chloride channel CLC-1.

In northern Finland myotonia congenita is caused by three main mutations in the ClC-1 chloride channel. We studied the molecular basis of these mutations (1238T>G/F413C, 1592C>T/A531V, and 2680C>T/R894X). The mutated cDNAs were expressed either in L6 myotubes or in isolated rat myofibers using recombinant Semliki Forest virus. Experiments in L6 cells indicated that A531V and R894X proteins suffered from stability problems in these cells. Analysis in myofibers indicated that the A531V protein was totally retained in the endoplasmic reticulum (ER), whereas the export of the F413C protein was severely reduced. The C-terminal nonsense mutant (R894X), however, was normally transported to the Golgi elements in the myofibers. Defective export or reduced stability of the mutated proteins may thus be reasons for the myotonic symptoms.