N-ethylmaleimide-sensitive factor is associated with the nuclear envelope.

N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg(2+)-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg(2+)-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle.

Skeletal muscle regeneration induced by chorio-allantoic grafting.

To examine whether the expression pattern of fast-muscle type troponin-T (TnT) isoforms was fixed in cell lineage, breast muscle pieces (pectoralis major) from chick embryos and young and adult chickens were grafted on to chorio-allantoic membrane of 9-day-old chick embryos and cultured until the host embryos hatched out. Muscle fibre formation of the grafts was investigated by histological and immunohistochemical methods with anti-fast-muscle type and anti-slow-muscle type TnT sera, and the expression of fast-muscle type TnT in the grafts from chick embryos and young chickens was studied by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and immunoblotting. In the chorio-allantoic grafting, the breast muscle initially degenerated forming pyknotic nuclei and hyaline cytoplasm. The surviving cells, which were supposed to be satellite cells, regenerated new muscle fibres of the same type as those of the grafted muscle in respect of TnT isoform expression. Therefore, we considered that the ability to express specific isoforms of TnT was fixed in the satellite cells, and that chorio-allantoic grafting was a useful technique for studying muscle differentiation.

Stability of chicken troponin T expression in cultured muscle cells.

Cells prepared from chicken skeletal muscles of early developmental stages were cultured to study their troponin T isoform expression, using antisera specific to fast- and slow-muscle-type isoforms, and compared with the cells from later stages described in the previous study (Mashima at al., 1996). We found that cultured myogenic cells from chickens and chick embryos could be classified, as in the previous study, into two types, fast type and fast/slow type in which fast- and slow-muscle-type isoforms were coexpressed. Ratios of these two types of muscle cells varied depending on their origins and developmental stages, and fast/slow type cells were in the majority at early stages. Since two distinct populations of cells committed to myogenic cell lineages were supposed to give rise to the two types of myotubes, we investigated the intrinsic stability of troponin T expression of the cultured myogenic cells using the serial subcloning method. The results of clonal analysis suggested that the expression pattern of troponin T isoform in cultured muscle cells is stable and that myogenic cell lineages play an important role in giving rise to different muscle types.

Expression of chicken troponin T isoforms in cultured muscle cells.

Cells prepared from chicken skeletal muscles of different developmental stages were cultured to study their troponin T isoform expression, using antisera specific to the fast- and slow-muscle-type isoforms. We found that the cultured myogenic cells from chickens and chick embryos were classified into two types, fast type and fast/slow type in which fast- and slow-muscle-type isoforms were coexpressed. Cells expressing only slow-muscle-type troponin T isoforms could not be found. Most cells prepared from pectoralis major (fast muscle) and gastrocnemius (mixed muscle) of 11-day old embryos belonged to the latter, with only a small fraction belonging to the former. The percentage of fast type cells in those cells prepared from pectoralis major increased along development to over 90% by the 17th day of incubation, while, in the cells prepared from gastrocnemius, it reached a plateau of 30-40% by the 13th day of incubation. All the cells from anterior latissimus dorsi (slow muscle) belonged to the fast/slow type. Ratios of these two types of muscle cells varied depending on their origins and stages. The in vitro expression of troponin T isoforms was different from the in vivo expression, and each muscle seems to be determined differently in the composition of cell types during the developmental course.