Kalervo Metsikkö

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.

Regulated sarcolemmal localization of the muscle-specific ClC-1 chloride channel

The skeletal muscle-specific ClC-1 is a voltage-gated chloride channel protein. Specific antibodies against ClC-1 revealed in muscle sections a sarcolemmal staining that was absent in the myotonic arrested development of righting response (ADR) mouse muscle. The intensity of the sarcolemmal staining varied from one type of muscle to another and in lateral sections showed a typical mosaic pattern that colocalized with beta-dystroglycan and left the transverse tubule openings clear. Surprisingly, in isolated myofibers, the ClC-1 protein was absent from the sarcolemma. Instead, it localized to intracellular I band areas as soon as the myofibers were isolated. When the isolated myofibers were incubated with the kinase inhibitor staurosporine, the ClC-1 protein shifted back to the sarcolemma. Electric stimulation of the cultivated fibers had a similar effect. Also, myofibers infected with a recombinant Semliki Forest virus (SFV) expressing myc-tagged ClC-1 showed intracellular localization of the protein. The virally expressed mycClC-1 reached the Golgi apparatus but sarcolemmal staining remained nondetectable, and addition of staurosporine into the growth medium recruited the mycClC-1 to the sarcolemma. These data indicate that sarcolemmal targeting of the ClC-1 requires specific signals that are provided by the physiological environment.

Polarity of osteoblasts and osteoblast-like UMR-108 cells

Enveloped viruses, such as vesicular stomatitis virus (VSV) and Influenza virus, have been widely used in studying epithelial cell polarity. Viral particles of VSV‐infected epithelial cells bud from the basolateral membrane, which is in contact with the internal milieu and the blood supply. Influenza‐infected cells bud viral particles from the apical surface facing the external milieu. This feature can be utilized in labeling polarized membrane domains. We studied the polarity of mesenchymal osteoblasts using osteosarcoma cell line UMR‐108 and endosteal osteoblasts in situ in bone tissue cultures. Immunofluorescence confocal microscopy revealed that the VSV glycoprotein (VSV G) was targeted to the culture medium–facing surface. In endosteal osteoblasts, VSV G protein was found in the surface facing bone marrow and circulation. On the contrary, Influenza virus hemagglutinin (HA) was localized to the bone substrate‐facing surface of the UMR‐108 cells. Electron microscopy showed that in the cases where the cells were growing as a single layer, VSV particles were budding from the culture medium–facing surface, whereas Influenza viruses budded from the bone substrate–facing surface. When the cells overlapped, this polarity was lost. Cell surface biotinylation revealed that 55% of VSV G protein was biotinylated, whereas Influenza virus HA was only 22% biotinylated. These findings suggest that osteoblasts are polarized at some point of their life cycle. The bone‐attaching plasma membrane of osteoblasts is apical, and the circulation or bone marrow–facing plasma membrane is basolateral in nature.

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. Muscle Nerve, 2007

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.

Evidence for γ-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.

Purification of human chorionic gonadotropin—receptor complex by immunoaffinity chromatography

Affinity chromatography methods have been applied to purification of several peptide hormone receptors, including insulin [ 11, prolactin [2] and LH(hCG) receptors [3,4]. However, in these methods a good displacement of the receptor from the ligand hormone cannot be achieved because of the almost irreversible nature of the binding between these two entities. Especially, the direct affinity chromatography methods used in LH receptor purification require extreme conditions for dissociation of the receptor and thus LH receptor with all of its original properties is not obtained [3]. We have found that anti-hCG gamma-globulins, covalently linked to CNBr-activated Sepharose 4B, bind solubilized ‘251-labeled hCG-receptor complex without significant dissociation of the complex. The bound hormone-receptor complex can specifically be displaced from the immunoaffinity matrix with an excess of hCG. Here we describe a purification procedure for hCG-receptor complex by the immunoaffmity chromatography developed.

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 DHPRα1 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.

Immunocytochemical localization of receptor human chorionic gonadotropin complexes in rat leydig cells.

Localization of receptor-bound human chorionic gonadotropin (hCG) in rat testis was studied by the peroxidase-antiperoxidase (PAP) complex method. The rats were injected with a single intravenous dose (1000 IU) of hCG. Three, 6, 12, and 24 hr after injection the testes were removed for localization of the hormone. The hormone localized to the periphery of the Leydig cells at all observation points. The intensity of the staining varied between the cells, suggesting that the number of receptors or the accessibility of the receptors to the circulating hormone varies from one cell to another. The staining surrounded the Leydig cells unevenly, but no progressive patching or capping was found. This observation suggests that hCG binds preferentially to the cell surface areas directed toward the capillaries. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. These results are consistent with previous observations that the luteinizing hormone (hCG) receptors accessible to the circulating hormone are located at the surface of the Leydig 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.