| 1. |
- Armstrong, Lucas C., et al.
(författare)
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Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism
- 2002
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 13:6, s. 1893-1905
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Tidskriftsartikel (refereegranskat)abstract
- The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell–matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.
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| 2. |
- Bertorello, AM, et al.
(författare)
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Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells
- 2003
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 14:3, s. 1149-1157
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Tidskriftsartikel (refereegranskat)abstract
- Dopamine (DA) increases Na+,K+-ATPase activity in lung alveolar epithelial cells. This effect is associated with an increase in Na+,K+-ATPase molecules within the plasma membrane ( Ridge et al., 2002 ). Analysis of Na+,K+-ATPase motion was performed in real-time in alveolar cells stably expressing Na+,K+-ATPase molecules carrying a fluorescent tag (green fluorescent protein) in the α-subunit. The data demonstrate a distinct (random walk) pattern of basal movement of Na+,K+-ATPase–containing vesicles in nontreated cells. DA increased the directional movement (by 3.5 fold) of the vesicles and an increase in their velocity (by 25%) that consequently promoted the incorporation of vesicles into the plasma membrane. The movement of Na+,K+-ATPase–containing vesicles and incorporation into the plasma membrane were microtubule dependent, and disruption of this network perturbed vesicle motion toward the plasma membrane and prevented the increase in the Na+,K+-ATPase activity induced by DA. Thus, recruitment of new Na+,K+-ATPase molecules into the plasma membrane appears to be a major mechanism by which dopamine increases total cell Na+,K+-ATPase activity.
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| 3. |
- Bjork, P, et al.
(författare)
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A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis
- 2002
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 13:10, s. 3683-3695
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Tidskriftsartikel (refereegranskat)abstract
- Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.
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| 4. |
- Björk, Petra, et al.
(författare)
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A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis
- 2002
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 13:10, s. 3683-3695
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Tidskriftsartikel (refereegranskat)abstract
- Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.
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| 5. |
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| 6. |
- Carmichael, JB, et al.
(författare)
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ago1 and dcr1, two core components of the RNA interference pathway, functionally diverge from rdp1 in regulating cell cycle events in Schizosaccharomyces pombe
- 2004
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 15:3, s. 1425-1435
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Tidskriftsartikel (refereegranskat)abstract
- In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1+, dcr1+, and rdp1+, have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1+and dcr1+but not rdp1+, exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1+and dcr1+are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1+is dispensable for this process, the functions of ago1+and dcr1+in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1+is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1+function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.
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| 7. |
- Carmichael, J B, et al.
(författare)
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Ago1 and Dcr1, two core components of the RNA interference pathway, functionally diverge from Rdp1 in regulating cell cycle events in Schizosaccharomyces pombe
- 2004
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 15:3, s. 1425-1435
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Tidskriftsartikel (refereegranskat)abstract
- In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1(+), dcr1(+), and rdp1(+), have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1(+) and dcr1(+) but not rdp1(+), exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1(+) and dcr1(+) are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1(+) is dispensable for this process, the functions of ago1(+) and dcr1(+) in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1(+) is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1(+) function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.
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| 8. |
- Chou, Wan-Chih, et al.
(författare)
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Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4
- 2003
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 14:3, s. 1279-1294
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Tidskriftsartikel (refereegranskat)abstract
- We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads.
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| 9. |
- Cross, Michael J, et al.
(författare)
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The Shb Adaptor Protein Binds to Tyrosine 766 in the FGFR-1 and Regulatesthe Ras/MEK/MAPK Pathway via FRS2 Phosphorylation in Endothelial Cells
- 2002
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 13:8, s. 2881-2893
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Tidskriftsartikel (refereegranskat)abstract
- Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-γ (PLC-γ). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-α and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-γ activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1–mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-γ activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1–mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1–mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.
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| 10. |
- Edlund, Sofia, et al.
(författare)
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Transforming growth factor-beta-induced mobilization of actin cytoskeleton required signaling by small GTPases Cdc42 and RhoA
- 2002
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Ingår i: Molecular Biology of the Cell. - : The American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 13:3, s. 902-914
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-beta receptors. We studied TGF-beta-induced rearrangements of the actin filament system and found that TGF-beta 1 treatment of PC-3U human prostate carcinoma cells resulted in a rapid formation of lamellipodia. Interestingly, this response was shown to be independent of the Smad signaling pathway; instead, it required the activity of the Rho GTPases Cdc42 and RhoA, because ectopic expression of dominant negative mutant Cdc42 and RhoA abrogated the response. Long-term stimulation with TGF-beta 1 resulted in an assembly of stress fibers; this response required both signaling via Cdc42 and RhoA, and Smad proteins. A known downstream effector of Cdc42 is p38(MAPK); treatment of the cells with the p38(MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38(MAPK), abrogated the TGF-beta-induced actin reorganization. Moreover, treatment of cells with the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and 1-5(-isoquinolinesulfonyl)homopiperazine (HA-1077), as well as ectopic expression of kinase-inactive Rho coiled-coil kinase-1, abrogated the TGF-beta 1-induced formation of stress fibers. Collectively, these data indicate that TGF-beta-induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.
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| 11. |
- Edlund, Sofia, et al.
(författare)
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Transforming growth factor-beta1-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase kinase 3
- 2003
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Ingår i: Molecular Biology of the Cell. - : The American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 14:2, s. 529-544
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Tidskriftsartikel (refereegranskat)abstract
- The inhibitory Smad7, a direct target gene for transforming growth factor-beta (TGF-beta), mediates TGF-beta1-induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-beta1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-beta-activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-beta1-induced apoptosis. The expression of Smad7 was required for TGF-beta-induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-beta1-induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-beta1-induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.
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| 12. |
- Ferletta, Maria, et al.
(författare)
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Opposing roles of integrin alpha6Abeta1 and dystroglycan in laminin-mediated extracellular signal-regulated kinase activation
- 2003
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 14:5, s. 2088-2103
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Tidskriftsartikel (refereegranskat)abstract
- Laminin-integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed beta1-containing integrins and dystroglycan but lacked integrin alpha6beta4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the alpha3beta1and alpha6beta1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin alpha6beta1 and not by alpha3beta1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin alpha6beta1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin alpha6 splice variants, alpha6A and alpha6B, whereas the nonresponding cell line expressed only alpha6B. Furthermore, ERK activation was seen in cells transfected with the integrin alpha6A subunit, but not in alpha6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin alpha6Abeta1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.
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| 13. |
- Hase, ME, et al.
(författare)
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Amino acid substitutions of coiled-coil protein Tpr abrogate anchorage to the nuclear pore complex but not parallel, in-register homodimerization
- 2001
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 12:8, s. 2433-2452
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Tidskriftsartikel (refereegranskat)abstract
- Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil–forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent α-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ∼7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed.
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| 14. |
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| 15. |
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| 16. |
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| 17. |
- Kniola, Barbara, et al.
(författare)
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The domain structure of centromeres is conserved from fission yeast to humans
- 2001
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 12:9, s. 2767-2775
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Tidskriftsartikel (refereegranskat)abstract
- The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.
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| 18. |
- Koerkamp, M. G., et al.
(författare)
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Dissection of transient oxidative stress response in Saccharomyces cerevisiae by using DNA microarrays
- 2002
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 13:8, s. 2783-2794
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Tidskriftsartikel (refereegranskat)abstract
- Yeast cells were grown in glucose-limited chemostat cultures and forced to switch to a new carbon source, the fatty acid oleate. Alterations in gene expression were monitored using DNA microarrays combined with bioinformatics tools, among which was included the recently developed algorithm REDUCE. Immediately after the switch to oleate, a transient and very specific stress response was observed, followed by the up-regulation of genes encoding peroxisomal enzymes required for fatty acid metabolism. The stress response included up-regulation of genes coding for enzymes to keep thioredoxin and glutathione reduced, as well as enzymes required for the detoxification of reactive oxygen species. Among the genes coding for various isoenzymes involved in these processes, only a specific subset was expressed. Not the general stress transcription factors Msn2 and Msn4, but rather the specific factor Yap1p seemed to be the main regulator of the stress response. We ascribe the initiation of the oxidative stress response to a combination of poor redox flux and fatty acid-induced uncoupling of the respiratory chain during the metabolic reprogramming phase.
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| 19. |
- Kulyte, A, et al.
(författare)
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Characterization of human alpha-dystrobrevin isoforms in HL-60 human promyelocytic leukemia cells undergoing granulocytic differentiation
- 2002
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 13:12, s. 4195-4205
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Tidskriftsartikel (refereegranskat)abstract
- The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.
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| 20. |
- Kulytè, Agnè, et al.
(författare)
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Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation
- 2002
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 13:12, s. 4195-4205
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Tidskriftsartikel (refereegranskat)abstract
- The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.
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| 21. |
- Kurisaki, Akira, et al.
(författare)
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Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner
- 2001
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 12:4, s. 1079-1091
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Tidskriftsartikel (refereegranskat)abstract
- Smad proteins are cytoplasmic signaling effectors of transforming growth factor-beta (TGF-beta) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-beta type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-beta1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-beta1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-beta1. 4) Importin-beta1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-beta receptor leads to enhanced interaction with importin-beta1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.
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| 22. |
- Liu, Hebin, et al.
(författare)
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Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1
- 2002
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 13:12, s. 4497-4507
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Tidskriftsartikel (refereegranskat)abstract
- The multipotent cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in particular in the physiological response to infection and in inflammatory responses. GM-CSF is produced by many cell types, including T lymphocytes responding to T-cell receptor activation and mantle zone B lymphocytes. B-cell receptor and T-cell receptor activation generates two major signals: an increase in intracellular Ca(2+) concentration and a protein kinase cascade. Previous studies have shown that the Ca(2+)/calmodulin-dependent phosphatase calcineurin mediates stimulation of GM-CSF transcription in response to Ca(2+). In this study, we show that Ca(2+) signaling also regulates GM-CSF transcription negatively through Ca(2+)/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Wild-type Ets1 negatively affects GM-CSF transcription on Ca(2+) stimulation in the presence of cyclosporin A, which inhibits calcineurin. Conversely, Ets1 with mutated CaMK II target serines showed an increase in transactivation of the GM-CSF promoter/enhancer. Moreover, constitutively active CaMK II inhibited transactivation of GM-CSF by wild-type Ets1 but not by Ets1 with mutated CaMK II sites. Mutation of CaMK II target serines in Ets1 also relieves inhibition of cooperative transactivation of GM-CSF with the Runx1/AML1 transcription factor. In addition, the Ca(2+)-dependent phosphorylation of Ets1 reduces the binding of Ets1 to the GM-CSF promoter in vivo.
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| 23. |
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| 24. |
- Los, Marek Jan, et al.
(författare)
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Activation and caspase-mediated inhibition of PARP : A molecular switch between fibroblast necrosis and apoptosis in death receptor signaling
- 2002
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 13:3, s. 978-988
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Tidskriftsartikel (refereegranskat)abstract
- Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-I mutant were more sensitive to TNF than wild-tvpe cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death.
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| 25. |
- Ridge, KM, et al.
(författare)
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Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta
- 2002
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 13:4, s. 1381-1389
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Tidskriftsartikel (refereegranskat)abstract
- The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 μM DA or a dopaminergic D1agonist, fenoldopam, but not with the dopaminergic D2agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase α1 and β1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-δ, or isozyme-specific inhibitor peptides for PKC-δ or PKC-ε inhibited the DA-mediated increase in Na,K-ATPase. PKC-δ and PKC-ε, but not PKC-α or -β, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-δ– and PKC-ε–specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-δ and PKC-ε.
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| 26. |
- Sabri, N, et al.
(författare)
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Evidence for a Posttranscription Role of a TFIIIC-like Protein in Chironomus tentans.
- 2002
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 13, s. 1765-1777
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Tidskriftsartikel (refereegranskat)abstract
- We have cloned and sequenced a cDNA that encodes for a nuclear protein of 238 kDa in the dipteran Chironomus tentans. This protein, that we call p2D10, is structurally similar to the α subunit of the general transcription factor TFIIIC. Using immunoelectron microscopy we have shown that a fraction of p2D10 is located at sites of transcription, which is consistent with a possible role of this protein in transcription initiation. We have also found that a large fraction of p2D10 is located in the nucleoplasm and in the nuclear pore complexes. Using gel filtration chromatography and coimmunoprecipitation methods, we have identified and characterized two p2D10-containing complexes that differ in molecular mass and composition. The heavy p2D10-containing complex contains at least one other component of the TFIIIC complex, TFIIIC-ε. Based on its molecular mass and composition, the heavy p2D10-containing complex may be the Pol III holoenzyme. The light p2D10-containing complex contains RNA together with at least two proteins that are thought to be involved in mRNA trafficking, RAE1 and hrp65. The observations reported here suggest that this new TFIIIC-α-like protein is involved in posttranscriptional steps of premRNA metabolism in Chironomus tentans.
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| 27. |
- Sabri, Nafiseh, et al.
(författare)
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Evidence for a posttranscriptional role of a TFIIICalpha-like protein in Chironomus tentans
- 2002
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 13:5, s. 1765-1777
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Tidskriftsartikel (refereegranskat)abstract
- We have cloned and sequenced a cDNA that encodes for a nuclear protein of 238 kDa in the dipteran Chironomus tentans. This protein, that we call p2D10, is structurally similar to the alpha subunit of the general transcription factor TFIIIC. Using immunoelectron microscopy we have shown that a fraction of p2D10 is located at sites of transcription, which is consistent with a possible role of this protein in transcription initiation. We have also found that a large fraction of p2D10 is located in the nucleoplasm and in the nuclear pore complexes. Using gel filtration chromatography and coimmunoprecipitation methods, we have identified and characterized two p2D10-containing complexes that differ in molecular mass and composition. The heavy p2D10-containing complex contains at least one other component of the TFIIIC complex, TFIIIC-epsilon. Based on its molecular mass and composition, the heavy p2D10-containing complex may be the Pol III holoenzyme. The light p2D10-containing complex contains RNA together with at least two proteins that are thought to be involved in mRNA trafficking, RAE1 and hrp65. The observations reported here suggest that this new TFIIIC-alpha-like protein is involved in posttranscriptional steps of premRNA metabolism in Chironomus tentans.
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| 28. |
- Schafer, J C, et al.
(författare)
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XBX-1 encodes a dynein light intermediate chain required for retrograde intraflagellar transport and cilia assembly in Caenorhabditis elegans
- 2003
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 14:5, s. 2057-2070
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Tidskriftsartikel (refereegranskat)abstract
- Intraflagellar transport (IFT) is a process required for flagella and cilia assembly that describes the dynein and kinesin mediated movement of particles along axonemes that consists of an A and a B complex, defects in which disrupt retrograde and anterograde transport, respectively. Herein, we describe a novel Caenorhabditis elegans gene, xbx-1, that is required for retrograde IFT and shares homology with a mammalian dynein light intermediate chain (D2LIC). xbx-1 expression in ciliated sensory neurons is regulated by the transcription factor DAF-19, as demonstrated previously for genes encoding IFT complex B proteins. XBX-1 localizes to the base of the cilia and undergoes anterograde and retrograde movement along the axoneme. Disruption of xbx-1 results in cilia defects and causes behavioral abnormalities observed in other cilia mutants. Analysis of cilia in xbx-1 mutants reveals that they are shortened and have a bulb like structure in which IFT proteins accumulate. The role of XBX-1 in IFT was further confirmed by analyzing the effect that other IFT mutations have on XBX-1 localization and movement. In contrast to other IFT proteins, retrograde XBX-1 movement was detected in complex A mutants. Our results suggest that the DLIC protein XBX-1 functions together with the CHE-3 dynein in retrograde IFT, downstream of the complex A proteins.
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| 29. |
- Schafer, JC, et al.
(författare)
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XBX-1 encodes a dynein light intermediate chain required for retrograde intraflagellar transport and cilia assembly in Caenorhabditis elegans
- 2003
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 14:5, s. 2057-2070
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Tidskriftsartikel (refereegranskat)abstract
- Intraflagellar transport (IFT) is a process required for flagella and cilia assembly that describes the dynein and kinesin mediated movement of particles along axonemes that consists of an A and a B complex, defects in which disrupt retrograde and anterograde transport, respectively. Herein, we describe a novel Caenorhabditis elegans gene, xbx-1, that is required for retrograde IFT and shares homology with a mammalian dynein light intermediate chain (D2LIC). xbx-1 expression in ciliated sensory neurons is regulated by the transcription factor DAF-19, as demonstrated previously for genes encoding IFT complex B proteins. XBX-1 localizes to the base of the cilia and undergoes anterograde and retrograde movement along the axoneme. Disruption of xbx-1 results in cilia defects and causes behavioral abnormalities observed in other cilia mutants. Analysis of cilia in xbx-1 mutants reveals that they are shortened and have a bulb like structure in which IFT proteins accumulate. The role of XBX-1 in IFT was further confirmed by analyzing the effect that other IFT mutations have on XBX-1 localization and movement. In contrast to other IFT proteins, retrograde XBX-1 movement was detected in complex A mutants. Our results suggest that the DLIC protein XBX-1 functions together with the CHE-3 dynein in retrograde IFT, downstream of the complex A proteins.
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| 30. |
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| 31. |
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| 32. |
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| 33. |
- Thorn, Hans, et al.
(författare)
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Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes
- 2003
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 14:10, s. 3967-3976
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Tidskriftsartikel (refereegranskat)abstract
- Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.
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| 34. |
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| 35. |
- Zeidman, Ruth, et al.
(författare)
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Protein Kinase Cepsilon Actin-binding Site Is Important for Neurite Outgrowth during Neuronal Differentiation.
- 2002
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1939-4586 .- 1059-1524. ; 13:1, s. 12-24
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Tidskriftsartikel (refereegranskat)abstract
- We have previously shown that protein kinase Cepsilon (PKCepsilon) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCepsilon-mediated neurite induction. We show an increased association of PKCepsilon to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCepsilon is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCepsilon actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCepsilon overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCepsilon independent of both serum and the actin-binding site, and PKCepsilon colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCepsilon in a pathway leading to neurite outgrowth. Localization of PKCepsilon to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.
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| 36. |
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