Abstract

Skeletal muscles are composed of long, multinucleated cells called myofibers, which are highly differentiated cells and therefore unique in structure. In the present study the organization of the endocytic and exocytic pathways in isolated rat skeletal myofibers was defined with confocal and electron microscopic methods.

In isolated myofibers the I band areas were shown to be active in endocytosis. The sorting endosomes were distributed in a cross-striated fashion while the recycling and late endosomal compartments were located to perinuclear areas and interfibrillar spaces, where they followed the course of microtubules.

Protein trafficking in the different stages of muscle cell differentation was also analyzed. The studies with L6 myoblasts and myotubes showed that during myogenesis varying fractions of different viral glycoproteins were sorted from the endoplasmic reticulum (ER) into a specific compartment that did not recycle with the Golgi apparatus. This compartment is suggested to be the sarcoplasmic reticulum (SR).

The studies with living muscle cells showed further changes in vesicle trafficking taking place during myogenesis. With GFP-tagged tsO45G protein, transport containers were detected in 20% of the infected myofibers, while all infected L6 myoblasts or myotubes showed intense movement of corresponding structures. We also detected significant differences between the pre-and post-Golgi traffickings in myofibers.

When the distribution of the ER in adult myofibers was studied, the confocal microscopic data showed that the labeling patterns of the rough endoplasmic reticulum (RER) and the SR markers were different. Blocking of different cargo proteins in the RER revealed two discrete distribution patterns, neither of them identical with the SR. The collected electron microscopic data supported the idea that in mature myofibers there are two separate RER compartments. We suggest that the RER compartment capable of export function located around the myonuclei and on the Z lines, while the non-exporting RER compartment localized to terminal cisternae and probably took care of the synthesis of the SR proteins.