We review some of the problems in determining how myofibrils may be assembled and just as importantly how this contractile structure may be renewed by sarcomeric proteins moving between the sarcomere and the cytoplasm. We also address in this personal review the recent evidence that indicates that the assembly and dynamics of myofibrils are conserved whether the cells are analyzed in situ or in tissue culture conditions. We suggest that myofibrillogenesis is a fundamentally conserved process, comparable to protein synthesis, mitosis, or cytokinesis, whether examined in situ or in vitro.
Luteinizing Hormone Receptor (LHR) expression in the ovary undergoes downregulation in response to LH surge or the administration of a pharmacological dose of hCG. We discovered a trans-factor, designated as LHR mRNA binding protein (LRBP) that selectively binds LHR mRNA, forms an untranslatable complex and targets it for degradation. The sequence of events involved in targeting the ribonucleoprotein complex to the degradative pathway is not understood. Since 5'-decapping of mRNA usually precedes its degradation, the regulated decapping of the LHR mRNA was examined after exposure to hCG. 5-day pseudopregnant female rats were treated with a single dose of hCG (50 IU) or saline (control), ovaries were collected after 0, 4 and 6h and a post-mitochondrial supernatant fraction (S100) was prepared from the homogenates. Our previous studies have shown that LHR mRNA levels begin to decrease at 4h of hCG treatment with no detectable levels seen by 12 hours. Decapping was assayed by immunoprecipitation of the S100 fractions from different hCG treatment intervals with agarose conjugated 7-methyl guanosine antibody which recognizes the 5' cap structure. The capped LHR mRNAs were eluted from the immunoprecipitates and the levels of cap-associated LHR mRNA were quantitated by real time PCR. The results showed that there was a time dependent decrease in LHR mRNA associated with the immunoprecipitates following hCG treatment when compared to control (60 percent and 75 percent decline at 4h and 6h, respectively vs. control). This was confirmed using a second marker of decapping by analyzing the association of LHR mRNA with eukaryotic initiation factor 4E (eIF-4E), since mRNA decapping has been shown to follow decreased association of mRNA with eIF-4E. As expected, a significant decrease in the levels of LHR mRNA associated with eIF-4E was seen after hCG treatment (68 percent and 86 per cent decline at 4h and 6 h, respectively vs. control). These results suggest that during hCG-induced downregulation, LHR mRNA undergoes decapping followed by degradation by 5'-3' exonuclease. Association of LHR mRNA with p bodies, cytoplasmic foci containing 5'-3' exonuclease where mRNAs undergoes degradation following decapping, increased to 4.2-fold and 7.2-fold at 4h and 6h of hCG treatment, respectively. Furthermore, confocal microscopic analysis as well as immunoprecipitation showed a strong co-localization of LRBP and the p body marker protein, DCP1A in the hCG-treated ovaries, but not in the control. These results and our earlier results suggest that LRBP binds to LHR mRNA in the ribosomes, prevents LHR mRNA translation by forming an untranslatable complex followed by its translocation from the ribosomes to the p bodies, where the mRNA undergoes decapping and degradation. These results provide new mechanistic insights on the degradation of LHR mRNA during ligand-induced downregulation. (platform)
It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells.
We have proposed a three‐step model for myofibrillogenesis: premyofibrils to nascent myofibrils to mature myofibrils that involves nonmuscle myosin II presence only in the first two steps. However, it has been reported (Takeda et al., 2000) that antibodies directed against nonmuscle myosin IIs stained the Z‐bands of mature myofibrils and intercalated discs of striated muscle cells. We have used similar nonmuscle myosin II antibodies against intact hearts and cultured cardiac and skeletal muscle cells and did not obtained these results. We have transfected cultures of embryonic avian cardiac and skeletal muscle cells with GFP plasmids encoding the three different isoforms of nonmuscle myosin II (A, B or C). The cells were cotransfected with CeruleanFP plasmids encoding sarcomeric alpha‐actinin, a marker for z‐bodies and Z‐bands. The three different GFP‐nonmuscle myosin II proteins localized to the premyofibrils and nascent myofibrils alternating with z‐bodies (as identified by CeFP‐alpha‐actinin localizations). The nonmuscle myosins never localized to the Z‐bands (identified by CeFP‐alpha‐actinin) or any other region of the mature myofibrils in the muscle cells. Our results support the idea that nonmuscle myosin IIs participate in aligning the z‐bodies and their attached actin filaments in premyofibrils and nascent myofibrils, preceding the formation of the mature myofibril. Research support: NIH.
