The Binding in Vitro of Modified LDL to the Intermediate Filament Protein Vimentin

2000 ◽  
Vol 267 (1) ◽  
pp. 49-53 ◽  
Author(s):  
Anne K. Heidenthal ◽  
Peter C. Weber ◽  
Friedrich Lottspeich ◽  
Nina Hrboticky
Keyword(s):  
Intermediate Filament ◽  
Intermediate Filament Protein ◽  
Modified Ldl ◽  
Filament Protein

scholarly journals Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons.

1991 ◽  
Vol 112 (6) ◽  
pp. 1205-1213 ◽  
Author(s):  
D E Weinstein ◽  
M L Shelanski ◽  
R K Liem
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Cell Lines ◽  
Intermediate Filament ◽  
Neuronal Cell ◽  
Acidic Protein ◽  
Intermediate Filament Protein ◽  
Astrocytoma Cell ◽  
Filament Protein ◽  
U251 Cells

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.

scholarly journals Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

2005 ◽  
Vol 171 (3) ◽  
pp. 431-436 ◽  
Author(s):  
Tomoya Yamaguchi ◽  
Hidemasa Goto ◽  
Tomoya Yokoyama ◽  
Herman Silljé ◽  
Anja Hanisch ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Rho Kinase ◽  
Direct Interaction ◽  
Enzyme Reaction ◽  
Aurora B ◽  
Intermediate Filament Protein ◽  
Cleavage Furrow ◽  
Filament Protein ◽  
Vimentin Filaments

Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at ∼1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

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 Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55

1999 ◽  
Vol 10 (6) ◽  
pp. 1997-2015 ◽  
Author(s):  
Patric Turowski ◽  
Timothy Myles ◽  
Brian A. Hemmings ◽  
Anne Fernandez ◽  
Ned J. C. Lamb
Keyword(s):  
Intermediate Filament ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Selective Inhibition ◽  
Major Protein ◽  
Intermediate Filament Protein ◽  
Regulatory Subunits ◽  
Phosphatase 2A ◽  
Filament Protein

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.

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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