Biomedical subjects

Kalervo Metsikkö

Publications and source records attributed to Kalervo Metsikkö.

5 recordsLinked to original sources

PubMed2005

Isolated primary osteocytes express functional gap junctions in vitro.

The osteocyte is the most abundant cell type in bone and is embedded in mineralized bone matrix. Osteocytes are still poorly characterized because of their location and the lack of primary osteocyte isolation methods. Data on the cell biology of osteocytes is especially limited. We have isolated primary osteocytes from rat cortical bone by applying repeated enzymatic digestion and decalcification. The isolated osteocytes expressed typical osteocytic morphology with cell-cell contacts via long protrusions after a 1-day culture. These cells were negative or faintly positive for alkaline phosphatase but expressed high levels of osteocalcin, PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome), and DMP1 (dentin matrix protein 1). These cells also revealed patchy membrane staining for connexin43. For studying the function of gap junctions in isolated osteocytes, we microinjected rhodamine-labeled dextran (MW: 10,000) and Lucifer yellow (MW: 457) and found that Lucifer yellow was rapidly transmitted to several surrounding cells, whereas dextran remained in the injected cells. Heptanol and 18alpha-glycyrrhetinic acid inhibited the transfer of Lucifer yellow. This clearly showed the existence of functional gap junctions in cultured osteocytes. Enveloped viruses, such as vesicular stomatitis virus and influenza A virus, were used for studying cell polarity. We were unable to demonstrate plasma membrane polarization with enveloped viruses in isolated primary osteocytes in culture. Our results suggest that osteocytes do not possess apical and basolateral plasma membrane domains as do osteoblasts, which are their precursors.

Alkaline Phosphatase↗

PubMed2005

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.

Animals↗

PubMed2003

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.

Animals↗

PubMed2003

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.

Animals↗

PubMed2002

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.

Animals↗