scholarly journals Deficiency of the intermediate filament synemin reduces bone mass in vivo

2016 ◽  
Vol 311 (6) ◽  
pp. C839-C845 ◽  
Author(s):  
Megan C. Moorer ◽  
Atum M. Buo ◽  
Karla P. Garcia-Pelagio ◽  
Joseph P. Stains ◽  
Robert J. Bloch
Keyword(s):  
Intermediate Filament ◽  
Striated Muscle ◽  
Serum Markers ◽  
Intermediate Filament Protein ◽  
Cross Sectional ◽  
Skeletal Tissue ◽  
Genetic Ablation ◽  
Type Iv ◽  
Osteoblast Activity

While the type IV intermediate filament protein, synemin, has been shown to play a role in striated muscle and neuronal tissue, its presence and function have not been described in skeletal tissue. Here, we report that genetic ablation of synemin in 14-wk-old male mice results in osteopenia that includes a more than 2-fold reduction in the trabecular bone fraction in the distal femur and a reduction in the cross-sectional area at the femoral middiaphysis due to an attendant reduction in both the periosteal and endosteal perimeter. Analysis of serum markers of bone formation and static histomorphometry revealed a statistically significant defect in osteoblast activity and osteoblast number in vivo. Interestingly, primary osteoblasts isolated from synemin-null mice demonstrate markedly enhanced osteogenic capacity with a concomitant reduction in cyclin D1 mRNA expression, which may explain the loss of osteoblast number observed in vivo. In total, these data suggest an important, previously unknown role for synemin in bone physiology.

scholarly journals Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

1993 ◽  
Vol 122 (6) ◽  
pp. 1323-1335 ◽  
Author(s):  
GY Ching ◽  
RK Liem
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Neuronal Cell ◽  
Intermediate Filament Protein ◽  
Amino Acid Residues ◽  
Intermediate Filament Proteins ◽  
Transfected Cells ◽  
Type Iv

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.

scholarly journals SPEG binds with desmin and its deficiency causes defects in triad and focal adhesion proteins

2020 ◽  
Author(s):  
Shiyu Luo ◽  
Qifei Li ◽  
Jasmine Lin ◽  
Quinn Murphy ◽  
Isabelle Marty ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Skeletal Muscles ◽  
Striated Muscle ◽  
Calcium Handling ◽  
Intermediate Filament Protein ◽  
Β1 Integrin ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins ◽  
Early Endosomes

Abstract SPEG, a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amounts of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1, and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Isometric tension and myofilamental cross-sectional area in striated muscle

1962 ◽  
Vol 202 (5) ◽  
pp. 824-826 ◽  
Author(s):  
Einar Helander ◽  
Carl-Axel Thulin
Keyword(s):  
Striated Muscle ◽  
Protein Composition ◽  
Isometric Tension ◽  
Cross Sectional Area ◽  
Sectional Area ◽  
Experimental Conditions ◽  
Cross Sectional ◽  
Total N ◽  
Calf Muscles

Isometric tension in tetanically stimulated calf muscles was examined in vivo in 3 rabbits and 18 cats. In two cats the gastrocnemius and soleus muscles were studied separately. After determination of the isometric tension the muscles were dissected and their water content, total N content, and protein composition were analyzed. On this basis it was possible to calculate that part of the cross-sectional area of the muscle fibers which consisted of myofilaments. The recorded maximum isometric tension was related to the myofilamental cross-sectional area. Under the given experimental conditions, the calf muscles developed a tension of 108 ± 5 g/mm2 cross-sectional area. Higher values resulted from tests of individual calf muscles than from combined muscles.

scholarly journals The Intermediate Filament Protein Peripherin Is the Specific Interaction Partner of Mouse BPAG1-n (Dystonin) in Neurons

1999 ◽  
Vol 144 (3) ◽  
pp. 435-446 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Ronald K.H. Liem
Keyword(s):  
Sensory Neurons ◽  
Intermediate Filament ◽  
Expression Patterns ◽  
Specific Interaction ◽  
Binding Domain ◽  
Interaction Partner ◽  
Actin Binding ◽  
Intermediate Filament Protein ◽  
Tail Domain

The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655–665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

scholarly journals Characterization of Osteocrin Expression in Human Bone

2005 ◽  
Vol 53 (10) ◽  
pp. 1181-1187 ◽  
Author(s):  
Sharyn Bord ◽  
Deborah C. Ireland ◽  
Pierre Moffatt ◽  
Gethin P. Thomas ◽  
Juliet E. Compston
Keyword(s):  
High Dose ◽  
Adult Women ◽  
Skeletal Tissue ◽  
Osteoblast Activity ◽  
Bone Biopsies ◽  
Primary Human Osteoblasts ◽  
Osteoblast Lineage

Osteocrin (Ostn), a bone-active molecule, has been shown in animals to be highly expressed in cells of the osteoblast lineage. We have characterized this protein in human cultured primary human osteoblasts, in developing human neonatal bone, and in iliac crest bone biopsies from adult women. In vivo, Ostn expression was localized in developing human neonatal rib bone, with intense immunoreactivity in osteoblasts on bone-forming surfaces, in newly incorporated osteocytes, and in some late hypertrophic chondrocytes. In adult bone, Ostn expression was specifically localized to osteoblasts and young osteocytes at bone-forming sites. In vitro, Ostn expression decreased time dependently ( p<0.02) in osteoblasts cultured for 2, 3, and 6 days. Expression was further decreased in cultures containing 200 nM hydrocortisone by 1.5-, 2.3-, and 3.1-fold ( p<0.05) at the same time points. In contrast, alkaline phosphatase expression increased with osteoblast differentiation ( p<0.05). Low-dose estradiol decreased Ostn expression time dependently ( p<0.05), whereas Ostn expression in cultures treated with high-dose estradiol was not significantly changed. These results demonstrate that Ostn is expressed in human skeletal tissue, particularly in osteoblasts in developing bone and at sites of bone remodeling, suggesting a role in bone formation. Thus, Ostn provides a marker of osteoblast lineage cells and appears to correlate with osteoblast activity.

scholarly journals Artemisinins target the intermediate filament protein vimentin for human cytomegalovirus inhibition

2020 ◽  
Vol 295 (44) ◽  
pp. 15013-15028 ◽  
Author(s):  
Sujayita Roy ◽  
Arun Kapoor ◽  
Fei Zhu ◽  
Rupkatha Mukhopadhyay ◽  
Ayan Kumar Ghosh ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Hcmv Infection ◽  
Intermediate Filament Protein ◽  
Biological Assays ◽  
Human Foreskin Fibroblasts ◽  
Hcmv Replication ◽  
Human Cmv ◽  
Filament Protein

The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentin-deficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virus-induced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti-HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin-binding sites and compound moieties for binding.

scholarly journals A nerve growth factor-regulated messenger RNA encodes a new intermediate filament protein.

1988 ◽  
Vol 106 (1) ◽  
pp. 181-193 ◽  
Author(s):  
D G Leonard ◽  
J D Gorham ◽  
P Cole ◽  
L A Greene ◽  
E B Ziff
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Intermediate Filament ◽  
Predicted Amino Acid Sequence ◽  
Intermediate Filament Protein ◽  
Nerve Growth ◽  
Amino Terminal

Differential screening of a cDNA library from the PC12 rat pheochromocytoma cell line previously revealed a clone, clone 73, whose corresponding mRNA is induced by nerve growth factor (NGF). Induction parallels NGF-stimulated PC12 differentiation from a chromaffinlike phenotype to a sympathetic neuronlike phenotype. We report that DNA sequence analysis reveals that clone 73 mRNA encodes an intermediate filament (IF) protein whose predicted amino acid sequence is distinct from the known sequences of other members of the IF protein family. The sequence has highest homology with desmin and vimentin and includes the highly conserved central alpha-helical rod domain with the characteristic heptad repeat of hydrophobic residues, but has lower homology in the amino-terminal head and carboxyl-terminal tail domains. The head domain contains a large number of serine residues which are potential phosphorylation sites. The expression of clone 73 in vivo in the nervous system of the adult rat was investigated by in situ hybridization of clone 73 probes to tissue sections. The mRNA is expressed at high levels in ganglia of the peripheral nervous system, including the superior cervical ganglion (sympathetic), ciliary ganglion (parasympathetic), and dorsal root ganglion (sensory). In the central nervous system, motor nuclei of cranial nerves III, IV, V, VI, VII, X, and XII as well as ventral horn motor neurons and a restricted set of other central nervous system nuclei express the clone 73 mRNA. Tissues apart from those of the nervous system did not in general express the mRNA, with only very low levels detected in adrenal gland. We discuss the implications of these results for the mechanism of NGF-induced PC12 cell differentiation, the pathways of neuronal development in vivo, and the possible function of the clone 73 IF protein and its relationship to other IF proteins.

scholarly journals Specific interaction of HTLV tax protein and a human type IV neuronal intermediate filament protein

1998 ◽  
Vol 95 (2) ◽  
pp. 702-707 ◽  
Author(s):  
T. R. Reddy ◽  
X. Li ◽  
Y. Jones ◽  
M. H. Ellisman ◽  
G. Y. Ching ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Specific Interaction ◽  
Intermediate Filament Protein ◽  
Human Type ◽  
Tax Protein ◽  
Type Iv ◽  
Htlv Tax ◽  
Filament Protein

Human α-synemin interacts directly with vinculin and metavinculin

2008 ◽  
Vol 409 (3) ◽  
pp. 657-667 ◽  
Author(s):  
Ning Sun ◽  
David R. Critchley ◽  
Denise Paulin ◽  
Zhenlin Li ◽  
Richard M. Robson
Keyword(s):  
Mammalian Cells ◽  
Striated Muscle ◽  
Splice Variants ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Protein Protein Interaction

Synemin is a very large, unique member of the IF (intermediate filament) protein superfamily. Association of synemin with the major IF proteins, desmin and/or vimentin, within muscle cells forms heteropolymeric IFs. We have previously identified interactions of avian synemin with α-actinin and vinculin. Avian synemin, however, is expressed as only one form, whereas human synemin is expressed as two major splice variants, namely α- and β-synemins. The larger α-synemin contains an additional 312-amino-acid insert (termed SNTIII) located near the end of the long C-terminal tail domain. Whether α- and β-synemins have different cellular functions is unclear. In the present study we show, by in vitro protein–protein interaction assays, that SNTIII interacts directly with both vinculin and metavinculin. Furthermore, SNTIII interacts with vinculin in vivo, and this association is promoted by PtdIns(4,5)P2. SNTIII also specifically co-localizes with vinculin within focal adhesions when transiently expressed in mammalian cells. In contrast, other regions of synemin show distinct localization patterns in comparison with those of SNTIII, without labelling focal adhesions. Our results indicate that α-synemin, but not β-synemin, interacts with both vinculin and metavinculin, thereby linking the heteropolymeric IFs to adhesion-type junctions, such as the costameres located within human striated muscle cells.

Cytokeratin expression in rat mesothelial cells in vitro is controlled by the extracellular matrix

1990 ◽  
Vol 95 (1) ◽  
pp. 97-107
Author(s):  
A.M. Mackay ◽  
R.P. Tracy ◽  
J.E. Craighead
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Intermediate Filament ◽  
Culture Conditions ◽  
Mesothelial Cells ◽  
Intermediate Filament Protein ◽  
Cellular Contact ◽  
Filament Protein

Rat mesothelial cells co-express vimentin and the simple epithelial cytokeratins. While cytokeratins predominate in situ, under most culture conditions vimentin is the major intermediate filament protein of the cells. This loss of cytokeratin production upon culture can be partly prevented by growing mesothelial cells on a basement membrane matrix. However, the basement membrane-promoted persistence of cytokeratin synthesis is not accompanied by expression of cytokeratin G (no. 19), the major acidic cytokeratin of mesothelium in vivo. While cells grown on plastic establish a prominent juxtanuclear assemblage of tonofilaments, those cultured on basement membrane exhibit cytokeratin filaments which are distributed throughout the cytoplasm and attach to neighboring cells at the plasma membrane. This latter pattern resembles that seen in the intact mesothelium. Intermediate filaments are markers of cellular differentiation, but their roles are obscure. The response of cultured mesothelial cells to different growth substrata supports the hypothesis that intermediate filament synthesis is influenced by cellular contact with the extracellular matrix.

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