Caveolin 3, flotillin 1 and influenza virus hemagglutinin reside in distinct domains on the sarcolemma of skeletal myofibers.

We examined the distribution of selected raft proteins on the sarcolemma of skeletal myofibers and the role of cholesterol environment in the distribution. Immunofluorescence staining showed that flotillin-1 and influenza hemagglutinin exhibited rafts that located in the domains deficient of the dystrophin glycoprotein complex, but the distribution patterns of the two proteins were different. Cholesterol depletion from the sarcolemma by means of methyl-ß-cyclodextrin resulted in distorted caveolar morphology and redistribution of the caveolin 3 protein. Concomitantly, the water permeability of the sarcolemma increased significantly. However, cholesterol depletion did not reshuffle flotillin 1 or hemagglutinin. Furthermore, a hemagglutinin variant that lacked a raft-targeting signals exhibited a similar distribution pattern as the native raft protein. These findings indicate that each raft protein exhibits a strictly defined distribution in the sarcolemma. Only the distribution of caveolin 3 that binds cholesterol was exclusively dependent on cholesterol environment.

Mobile ER-to-Golgi but not post-Golgi membrane transport carriers disappear during the terminal myogenic differentiation.

The organelles of the exocytic pathway undergo a profound reorganization during the myogenic differentiation. Here, we have investigated the dynamics of the membrane trafficking at various stages of the differentiation process by using the green fluorescent protein-tagged, temperature-sensitive vesicular stomatitis virus G protein (tsG-GFP) as a marker. At the restrictive temperature of 39°C, the tsG-GFP located to the endoplasmic reticulum (ER) at each stage of differentiation. Mobile membrane containers moving from the ER to the Golgi elements were seen in myoblasts and myotubes upon shifting the temperature to 20°C. In adult myofibers, in contrast, such containers were not seen although the tsG-GFP rapidly shifted from the ER to the Golgi elements. The mobility of tsG-GFP in the myofiber ER was restricted, suggesting localization in an ER sub-compartment. Contrasting with the ER-to-Golgi trafficking, transport from the Golgi elements to the plasma membrane involved mobile transport containers in all differentiation stages. These findings indicate that ER-to-Golgi trafficking in adult skeletal myofibers does not involve long-distance moving membrane carriers as occurs in other mammalian cell types.

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.

Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts.

Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-beta-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.

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.

Restricted distribution of mRNAs encoding a sarcoplasmic reticulum or transverse tubule protein in skeletal myofibers.

Calsequestrin (CSQ) and dihydropyridine receptor (DHPR) are muscle cell proteins that are directed into the endoplasmic reticulum (ER) during translation. The former is subsequently found in the sarcoplasmic reticulum (SR) and the latter in the transverse tubule membrane. To elucidate the potential role of mRNA targeting within muscle cells, we have analyzed the localization of CSQ and DHPR proteins and mRNAs in primary cultured rat myotubes, in skeletal muscle cryosections, and in isolated flexor digitorum brevis muscle fibers. In the myotube stage of differentiation, the mRNAs distributed throughout the cell, mimicking the distribution of the endogenous ER marker proteins. In the adult skeletal myofibers, however, both CSQ and DHPRalpha1 transcripts located perinuclearly and in cross-striations flanking Z lines beneath the sarcolemma, a distribution pattern that sharply contrasted the interfibrillar distribution of typical ER proteins. Interestingly, all nuclei of the myofibers were transcriptionally active. In summary, the mRNAs encoding either a resident SR protein or a transverse tubule protein were located beneath the sarcolemma, implying that translocation of the respective proteins to the lumen of ER takes place at this location.

Distribution of the endoplasmic reticulum and its relationship with the sarcoplasmic reticulum in skeletal myofibers.

We have analyzed the distribution of the endoplasmic reticulum (ER) within isolated rat skeletal muscle flexor digitorum brevis myofibers. Studies with confocal microscopy indicated that the resident ER proteins displayed a perinuclear and cross-striated distribution that extended over the I band areas. Interestingly, two discrete distribution patterns were observed when different receptor or viral marker proteins were blocked in the ER. Accordingly, the vesicular stomatitis virus G protein that lost its efficient export through the Golgi apparatus during myogenesis preferentially marked the A-I junctional areas. The proteins that retained their Golgi processing after myogenesis, on the contrary, concentrated around the myonuclei and over the Z lines. Furthermore, the ER exit site marker sec23 located to Z lines but not to A-I junctions. To analyze the ultrastructural organization of the ER, we infected myofibers with recombinant virus expressing KDEL-tagged peroxidase that is translocated into the ER. With transmission electron microscopy, peroxidase activity was found in perinuclear and Z line-flanking tubular structures, but also within the terminal cisternae of the sarcoplasmic reticulum. The translocon-associated protein exhibited a similar localization. Taken together, the terminal cisternae contained unevenly distributed rough ER structures apparently lacking the export function. The exporting ER comprised perinuclear and Z line-flanking structures.

The architecture of microtubular network and Golgi orientation in osteoclasts--major differences between avian and mammalian species.

In the present study, we analyze multinuclear osteoclasts obtained from several avian and mammalian species and describe the reorganization of their microtubular architecture and Golgi complex orientation during osteoclast differentiation and activation for bone resorption. In nonresorbing quail and chicken multinuclear osteoclasts, microtubules radiate from multiple centrosomal microtubule-organizing centers (MTOCs), whose number is equal to the number of nuclei. However, centrosomal MTOCs disappear at the time of cell activation for bone resorption and the Golgi membranes redistribute to circumscribe nuclei. In contrast to avian osteoclasts, both resorbing and nonresorbing rat, rabbit, and human osteoclasts have no or few centrosomal MTOCs. Instead, after cold-induced depolymerization, regrowing microtubules nucleate from the perinuclear area where immunofluoresce and immunoelectron scanning microscopy reveal pericentriolar matrix protein pericentrin associated with vimentin filaments. Furthermore, the circumnuclear reorganization of MTOCs and the Golgi is a result of mammalian osteoclast maturation and occur before any resorptive activity of the mononuclear osteoclasts and their fusion into multinucleated cells. Our results show that unlike previously suggested, the nuclear surfaces of mammalian osteoclasts act as the microtubule anchoring sites similarly to nuclear surfaces in multinucleated myotubes and suggest the role of perinuclear intermediate filament network in orchestrating the microtubular cytoskeleton.

Retargeting of viral glycoproteins into a non-exporting compartment during the myogenic differentiation of rat L6 cells.

We have analysed protein trafficking during the differentiation of rat L6 myoblasts into myotubes. Different proteins were found to lose different amounts of their processing by the Golgi apparatus during the myogenic differentiation, indicating that they were transported to this organelle with differing efficiencies. In order to investigate the destination of the nonprocessed glycoproteins we analysed the behaviour of vesicular stomatitis virus (VSV) and Semliki Forest virus glycoproteins in the presence of Brefeldin A, which returns the enzymes of the Golgi apparatus to the ER. Such experiments indicated that during myogenesis a fraction of both glycoproteins was shunted into a compartment that did not participate recycling with the Golgi apparatus. Immunofluorescence studies with the mutant VSV tsO45 G protein suggested that this compartment was diffusively distributed. We investigated whether the cytoplasmic tail had a role in the myogenic transport modulation by analysing the behaviour of recombinant VSV G proteins. Exchanging the cytoplasmic tail or the tail plus the membrane anchor had no effect, suggesting that the luminal portion was responsible for the diverted transport. Taken together, the results suggest that during the myogenesis of L6 myoblasts, varying fractions of different viral glycoproteins were sorted from the ER into a specific compartment that did not recycle with the Golgi apparatus.