Publications by authors named "Mika Kaakinen"

21 Publications

  • Page 1 of 1

Characterization of nucleic acids from extracellular vesicle-enriched human sweat.

BMC Genomics 2021 Jun 9;22(1):425. Epub 2021 Jun 9.

Faculty of Biochemistry and Molecular Medicine, Disease Networks Research Unit, Laboratory of Developmental Biology, Kvantum Institute, Infotech Oulu, University of Oulu, 90014 University of Oulu, Oulu, Finland.

Background: The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles.

Results: We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7-45%), with 50-60% of those reads mapping to unannotated region of the genome and 30-55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA.

Conclusions: Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.
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http://dx.doi.org/10.1186/s12864-021-07733-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188706PMC
June 2021

The Amino-Terminal Oligomerization Domain of Angiopoietin-2 Affects Vascular Remodeling, Mammary Gland Tumor Growth, and Lung Metastasis in Mice.

Cancer Res 2021 01 9;81(1):129-143. Epub 2020 Oct 9.

Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2), a genetic model for breast cancer and metastasis (MMTV-), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2 promoted destabilization of pulmonary vasculature and lung metastasis. , ANGPT2 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5β1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. SIGNIFICANCE: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer..
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1904DOI Listing
January 2021

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity.

Elife 2020 09 8;9. Epub 2020 Sep 8.

Uppsala University, Department of Immunology, Genetics and Pathology, Uppsala, Sweden.

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.
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http://dx.doi.org/10.7554/eLife.57732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478896PMC
September 2020

Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina.

Elife 2018 11 16;7. Epub 2018 Nov 16.

Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland.

The maintenance of fluid homeostasis is necessary for function of the neural retina; however, little is known about the significance of potential fluid management mechanisms. Here, we investigated angiopoietin-4 (Angpt4, also known as Ang3), a poorly characterized ligand for endothelial receptor tyrosine kinase Tie2, in mouse retina model. By using genetic reporter, fate mapping, and in situ hybridization, we found expression in a specific sub-population of astrocytes at the site where venous morphogenesis occurs and that lower oxygen tension, which distinguishes peripheral and venous locations, enhances Angpt4 expression. Correlating with its spatiotemporal expression, deletion of resulted in defective venous development causing impaired venous drainage and defects in neuronal cells. In vitro characterization of angiopoietin-4 proteins revealed both ligand-specific and redundant functions among the angiopoietins. Our study identifies Angpt4 as the first growth factor for venous-specific development and its importance in venous remodeling, retinal fluid clearance and neuronal function.
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http://dx.doi.org/10.7554/eLife.37776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239434PMC
November 2018

Biallelic mutations in human NHLRC2 enhance myofibroblast differentiation in FINCA disease.

Hum Mol Genet 2018 12;27(24):4288-4302

PEDEGO Research Unit, University of Oulu, Oulu, Finland.

The development of tissue fibrosis is complex and at the present time, not fully understood. Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA disease) have been described in patients with mutations in NHL repeat-containing protein 2 (NHLRC2). However, the molecular functions of NHLRC2 are uncharacterized. Herein, we identified putative interacting partners for NHLRC2 using proximity-labeling mass spectrometry. We also investigated the function of NHLRC2 using immortalized cells cultured from skin biopsies of FINCA patients and normal fibroblasts with NHLRC2 knock-down and NHLRC2 overexpressing gene modifications. Transmission electron microscopy analysis of immortalized cell cultures from three FINCA patients demonstrated multilamellar bodies and distinctly organized vimentin filaments. Additionally, two of three cultures derived from patient skin biopsies contained cells that exhibited features characteristic of myofibroblasts. Altogether, the data presented in this study show for the first time that NHLRC2 is involved in cellular organization through regulation of the cytoskeleton and vesicle transport. We conclude that compound heterozygous p.Asp148Tyr and p.Arg201GlyfsTer6 mutations in NHLRC2 lead to severe tissue fibrosis in humans by enhancing the differentiation of fibroblasts to myofibroblasts.
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http://dx.doi.org/10.1093/hmg/ddy298DOI Listing
December 2018

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease.

Acta Neuropathol 2018 05 8;135(5):727-742. Epub 2018 Feb 8.

PEDEGO Research Unit and Medical Research Center Oulu, University of Oulu and Oulu University Hospital, PO Box 5000, 90014, Oulu, Finland.

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.
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http://dx.doi.org/10.1007/s00401-018-1817-zDOI Listing
May 2018

Cavin-1 deficiency modifies myocardial and coronary function, stretch responses and ischaemic tolerance: roles of NOS over-activity.

Basic Res Cardiol 2017 05 25;112(3):24. Epub 2017 Mar 25.

School of Medical Science, Griffith University, Southport, QLD, 4217, Australia.

Caveolae and associated cavin and caveolins may govern myocardial function, together with responses to mechanical and ischaemic stresses. Abnormalities in these proteins are also implicated in different cardiovascular disorders. However, specific roles of the cavin-1 protein in cardiac and coronary responses to mechanical/metabolic perturbation remain unclear. We characterised cardiovascular impacts of cavin-1 deficiency, comparing myocardial and coronary phenotypes and responses to stretch and ischaemia-reperfusion in hearts from cavin-1 and cavin-1 mice. Caveolae and caveolins 1 and 3 were depleted in cavin-1 hearts. Cardiac ejection properties in situ were modestly reduced in cavin-1 mice. While peak contractile performance in ex vivo myocardium from cavin-1 and cavin-1 mice was comparable, intrinsic beating rate, diastolic stiffness and Frank-Starling behaviour (stretch-dependent diastolic and systolic forces) were exaggerated in cavin-1 hearts. Increases in stretch-dependent forces were countered by NOS inhibition (100 µM L-NAME), which exposed negative inotropy in cavin-1 hearts, and were mimicked by 100 µM nitroprusside. In contrast, chronotropic differences appeared largely NOS-independent. Cavin-1 deletion also induced NOS-dependent coronary dilatation, ≥3-fold prolongation of reactive hyperaemic responses, and exaggerated pressure-dependence of coronary flow. Stretch-dependent efflux of lactate dehydrogenase and cardiac troponin I was increased and induction of brain natriuretic peptide and c-Fos inhibited in cavin-1 hearts, while ERK1/2 phospho-activation was preserved. Post-ischaemic dysfunction and damage was also exaggerated in cavin-1 hearts. Diverse effects of cavin-1 deletion reveal important roles in both NOS-dependent and -independent control of cardiac and coronary functions, together with governing sarcolemmal fragility and myocardial responses to stretch and ischaemia.
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http://dx.doi.org/10.1007/s00395-017-0613-6DOI Listing
May 2017

Blue Rubber Bleb Nevus (BRBN) Syndrome Is Caused by Somatic TEK (TIE2) Mutations.

J Invest Dermatol 2017 01 9;137(1):207-216. Epub 2016 Aug 9.

Human Molecular Genetics, de Duve Institute, Université catholique de Louvain, Brussels, Belgium. Electronic address:

Blue rubber bleb nevus syndrome (Bean syndrome) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations; gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with blue rubber bleb nevus syndrome. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal venous malformations. In contrast to common unifocal venous malformation, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, that is, two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in blue rubber bleb nevus, whereas Y897C-R915C is recurrent in sporadically occurring multifocal venous malformation: both cause ligand-independent activation of TIE2, and increase survival, invasion, and colony formation when expressed in human umbilical vein endothelial cells.
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http://dx.doi.org/10.1016/j.jid.2016.07.034DOI Listing
January 2017

Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.

Breast Cancer Res Treat 2016 Jan 18;155(2):261-71. Epub 2016 Jan 18.

Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.
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http://dx.doi.org/10.1007/s10549-016-3683-5DOI Listing
January 2016

Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention.

Oncotarget 2015 Oct;6(30):30035-56

Turku Centre for Biotechnology, University of Turku, Turku, FI-20520, Finland.

Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro.
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http://dx.doi.org/10.18632/oncotarget.5046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745780PMC
October 2015

DNA from dead cancer cells induces TLR9-mediated invasion and inflammation in living cancer cells.

Breast Cancer Res Treat 2013 Dec 10;142(3):477-87. Epub 2013 Nov 10.

Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, SHEL 514, 1825 University Blvd, Birmingham, AL, 35294-3300, USA.

TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple-negative, human MDA-MB-231 breast cancer cells stably expressing control, or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy.
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http://dx.doi.org/10.1007/s10549-013-2762-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238912PMC
December 2013

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell-cell interaction.

Exp Cell Res 2013 Nov 6;319(18):2770-80. Epub 2013 Aug 6.

National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562, Japan.

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.
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http://dx.doi.org/10.1016/j.yexcr.2013.07.023DOI Listing
November 2013

Distribution of mRNA transcripts and translation activity in skeletal myofibers.

Cell Tissue Res 2013 Sep 5;353(3):539-48. Epub 2013 Jun 5.

Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, P.O. Box 5000, Aapistie 7, FI-90014, Oulu, Finland.

We examine the distribution of gene products in skeletal myofibers, which are highly differentiated multinucleated cells exhibiting a specific cellular architecture. In situ hybridization studies of adult rat myofibers with a single nucleus infected with influenza virus suggested that the viral mRNA products were distributed beneath the sarcolemma around the nucleus of origin. In situ hybridization studies with a poly-T oligonucleotide probe to detect endogenous mRNAs indicated their concentration around the nuclei and distribution beneath the sarcolemma in a cross-striated fashion at the A-I junctions (costamers). Labeling with bromouridine resulted in a similar distribution pattern. The ribosomal distribution pattern indicated concentration around the myonuclei but an intracellular component was also seen. Localization of the translating ribosomes by puromycylation revealed prominent spots perinuclearly and in the core regions of the myofibers. These spots flanked Golgi elements. Our results thus suggest that the total mRNA pool is heavily concentrated within the perinuclear and subsarcolemmal regions. However, the ribosomes and the translational activity did not follow this distribution pattern, so the mRNA transcripts were not restricted to a region beneath the sarcolemma. Furthermore, experiments utilizing green fluorescent protein showed the rapid movement of proteins within the endomembrane system, which thus facilitated proteins to reach their site of function irrespective of the site of synthesis.
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http://dx.doi.org/10.1007/s00441-013-1659-xDOI Listing
September 2013

New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics.

Neurology 2012 Nov 14;79(22):2194-200. Epub 2012 Nov 14.

Neuromuscular Research Unit, University of Tampere and Tampere University Hospital, Tampere, Finland.

Objective: The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so.

Methods: An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies.

Results: This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing.

Conclusions: We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.
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http://dx.doi.org/10.1212/WNL.0b013e31827595e2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570820PMC
November 2012

Reversible stress-induced lipid body formation in fast twitch rat myofibers.

Exp Cell Res 2012 Oct 6;318(17):2191-9. Epub 2012 Jul 6.

Department of Anatomy and Cell Biology, Institute of Biomedicine, P.O. Box 5000, Aapistie 7, FI 90014 University of Oulu, Finland.

We analyzed the existence of lipid bodies (LBs) in the fast twitch rat flexor digitorum brevis (FDB) myofibers and found that these structures were scarce. However, isolation procedure of the myofibers, heath shock, viral infection or the glycosylation inhibitor tunicamycin induced formation of the LBs, which were stationary structures flanking Z lines. We next infected FDB myofibers with recombinant Semliki Forest virus expressing caveolin 3-yellow fluorescent protein (cav3-YFP) since this chimeric protein was targeted to the LBs facilitating their further analysis. Photobleaching experiments showed that the LBs recovered cav 3-YFP extremely slowly, indicating that they were not continuous with the endoplasmic/sarcoplasmic reticulum. We found, however, that cav3-YFP could move from the LBs to the sarcolemma and this phenomenon was sensitive to Brefeldin A, suggesting that the chimeric protein could be returned from the LBs to the endoplasmic reticulum.
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http://dx.doi.org/10.1016/j.yexcr.2012.06.019DOI Listing
October 2012

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

Biochem Res Int 2012 5;2012:497572. Epub 2012 Mar 5.

Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, P.O. Box 5000, Aapistie 7, 90014 Oulu, Finland.

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-β-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.
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http://dx.doi.org/10.1155/2012/497572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3303869PMC
August 2012

Aquaporin-4 water channel oligomers are associated with the transverse tubules of skeletal myofibers.

Exp Cell Res 2011 Jan 21;317(1):20-8. Epub 2010 Sep 21.

Institute of Biomedicine, Department of Anatomy and Cell Biology, P.O. Box 5000, FIN 90014, University of Oulu, Oulu, Finland.

Transverse (T) tubules comprise a tortuous network inside the skeletal myofibers enclosing a distinct osmotic environment. Here we have examined whether the T tubules contain aquaporin type 4 (AQP4) water channels to mediate rapid transmembrane water flow. Separation of T tubular and sarcolemmal membranes by sucrose density gradient centrifugation revealed that two main isoforms of AQP4, namely M23 and M1, were present in both membrane fractions. Compatible with this, expression of fluorescent Venus-AQP4.M23 in rat muscle showed the protein both in the T tubules and at the sarcolemma. Blue-Native polyacrylamide gel electrophoresis showed that higher order oligomers typical to the AQP4 water channel were present in both membrane compartments. Interestingly, α-syntrophin that mediates binding of AQP4 to the sarcolemmal dystrophin glycoprotein complex was also present in the T tubule fraction. Deletion of the syntrophin-binding sequence of AQP4 increased its mobile fraction at the sarcolemma but not in the T tubules. Taken together, our results strongly suggest that both the sarcolemma and the T tubules harbor higher order oligomers of the AQP4 water channel but the interactions with adjacent macromolecules are different.
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http://dx.doi.org/10.1016/j.yexcr.2010.09.004DOI Listing
January 2011

Influenza virus infection in multinucleated skeletal myofibers.

Exp Cell Res 2010 Jul 1;316(11):1784-94. Epub 2010 Apr 1.

Institute of Biomedicine, Department of Anatomy and Cell Biology, PO Box 5000, FIN-90014 University of Oulu, Finland.

We examined the progression of the WSN influenza virus infection in isolated, multinucleated rat skeletal myofibers. Contrary to mononucleated cells, the adsorbed virions showed markedly delayed entry kinetics. Viral budding occurred on the sarcolemma, but the hemagglutinin envelope glycoprotein matured inefficiently and was poorly cleaved. Compatible with this, plaque assays indicated that infective viral particles were not formed. In situ hybridization studies showed that at low-dose infection, viral RNA production was restricted to one or a few nuclei within a myofiber. Dual in situ hybridization indicated that two different viral RNAs usually co-localized in the same nucleus or nuclei, suggesting that different viral genome segments replicated in the same nucleus. Newly synthesized viral ribonucleoprotein particles (vRNPs) did not re-enter virgin nuclei. Therefore, a single infected nucleus was able to support viral protein production, and notably, these proteins could reach hundreds of micrometers from the nucleus of origin. These results suggest that after viral disassembly in the endosome, the genome segments remained glued together and entered a myonucleus as a package. Spreading of the infection into virgin nuclei either by vRNPs or newly made virions did not occur, and thus the infection was abortive.
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http://dx.doi.org/10.1016/j.yexcr.2010.03.020DOI Listing
July 2010

Evidence for gamma-actin as a Z disc component in skeletal myofibers.

Exp Cell Res 2009 Jan 31;315(2):218-25. Epub 2008 Oct 31.

Institute of Biomedicine, Department of Anatomy and Cell Biology, P.O. Box 5000 (Aapistie 7), FIN-90014 University of Oulu, Finland.

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.
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http://dx.doi.org/10.1016/j.yexcr.2008.10.021DOI Listing
January 2009

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers.

Exp Cell Res 2008 Jan 22;314(2):237-45. Epub 2007 Oct 22.

Department of Anatomy and Cell Biology, P.O. Box 5000 (Aapistie 7), FIN-90014 University of Oulu, Finland.

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca(2+)-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.
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http://dx.doi.org/10.1016/j.yexcr.2007.10.009DOI Listing
January 2008

Distribution of aquaporin 4 on sarcolemma of fast-twitch skeletal myofibres.

Cell Tissue Res 2007 Sep 26;329(3):529-39. Epub 2007 Jun 26.

Department of Anatomy and Cell Biology, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland.

The aquaporin 4 (AQP4) water channel is present on the sarcolemma of fast-twitch-type skeletal myofibres. We have examined the distribution of AQP4 in relation to sarcolemmal domain structure and found that AQP4 protein is not evenly distributed on the sarcolemma. Immunofluorescence staining of isolated single myofibres indicated a punctate staining pattern overlapping with the dystrophin glycoprotein complex, but with the transverse tubule openings being left clear. Myotendinous and neuromuscular junctions also lacked AQP4, despite their high content of the dystrophin glycoprotein complex. The destruction of caveoli with methyl-beta-cyclodextrin did not change the distribution of AQP4 at the sarcolemma. Moreover, AQP4 did not float with the caveolar marker caveolin-3 in sucrose gradients after Triton X-100 extraction at 4 degrees C. These data indicated that AQP4 was not associated with caveoli. Surprisingly, m. flexor digitorum brevis fibres, although of the fast-twitch type, often lacked AQP4. Furthermore, those fibres harbouring AQP4 at the sarcolemma showed a regionalized distribution, suggesting that large areas were devoid of the protein. Blockage of the synthesized proteins in the endoplasmic reticulum with brefeldin A showed that, in spite of its regionalized sarcolemmal distribution, AQP4 was synthesized along the entire length of the fibres. These results suggest functional differences in the water permeability of the sarcolemma not only between the fast-twitch muscles, but also within single muscle fibres.
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http://dx.doi.org/10.1007/s00441-007-0442-2DOI Listing
September 2007
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