| 1. |
- Tingström, Anders, et al.
(författare)
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Distribution and dynamics of cell surface-associated cellCAM 105 in cultured rat hepatocytes
- 1989
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 185:1, s. 132-142
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Tidskriftsartikel (refereegranskat)abstract
- The cellular location of cellCAM 105 was studied by indirect immunofluorescence microscopy of primary rat hepatocytes grown in monolayer culture. Staining corresponding to cellCAM 105 was seen both in cell-cell contact areas and on the upper surfaces of the cells. In the cell-cell contact areas the antigen was not accessible to the antibodies unless the cells were either permeabilized with detergent or incubated in a calcium-free medium. Removal of calcium from the medium caused the cells to separate from each other. Within a few minutes wide intercellular clefts were formed, and upon further incubation the cells became stellate-shaped and finally remained in contact with each other only via thin cellular processes. These processes were cellCAM 105-positive and at sites where they attached to the bodies of the contracted cells a granular fluorescence pattern appeared. After 24-48 h of culture, intercellular channels resembling bile canaliculi were sometimes formed in the hepatocyte monolayers. The membranes of these intercellular channels were stained for cellCAM 105. After culture for several days the hepatocytes lost their polygonal shape and gradually acquired a more fibroblast-like morphology. This morphological change was accompanied by a decrease in cellCAM 105-specific fluorescence, both in the cell-cell contact areas and on the free cell surfaces.
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| 2. |
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| 3. |
- Imreh, Gabriela, et al.
(författare)
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ER retention may play a role in sorting of the nuclear pore membrane protein POM121
- 2003
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Ingår i: Experimental Cell Research. - 0014-4827 .- 1090-2422. ; 284:2, s. 173-184
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Tidskriftsartikel (refereegranskat)abstract
- Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 mum(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15degreesC. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.
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| 4. |
- Lennartsson, Johan, et al.
(författare)
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Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk
- 2003
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Ingår i: Experimental Cell Research. - 0014-4827 .- 1090-2422. ; 288:1, s. 110-118
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Tidskriftsartikel (refereegranskat)abstract
- We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.
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| 5. |
- Melhus, Håkan, et al.
(författare)
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Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol
- 1991
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Ingår i: Experimental Cell Research. - 0014-4827 .- 1090-2422. ; 197:1, s. 119-124
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Tidskriftsartikel (refereegranskat)abstract
- To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.
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| 6. |
- Rennel, Emma, et al.
(författare)
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Regulation of endothelial cell differentiation and transformation by H-Ras
- 2003
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Ingår i: Experimental Cell Research. - 0014-4827 .- 1090-2422. ; 291:1, s. 189-200
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Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis is regulated by growth factors which activate tyrosine kinase receptors leading to the activation of a number of intracellular signaling pathways. The specific function of H-Ras during FGF-2 stimulated endothelial cell differentiation, defined as invasive growth and formation of branching networks in fibrin gels, was investigated by using conditionally immortalized endothelial cell lines induced to express H-Ras mutants. Expression of inhibitory N17Ras did not impair differentiation in response to FGF-2 and TNF-alpha. The farnesyltransferase inhibitor FTI-277 inhibited farnesylation of Ras but did not inhibit differentiation of human microvascular endothelial cells or mouse brain endothelial cells. In contrast, activated V12Ras inhibited endothelial cell differentiation and cells displayed a transformed phenotype with an increased rate of proliferation and loss of contact inhibited growth. Furthermore, V12Ras expressing endothelial cells grew as solid tumors when injected subcutaneously into mice. Our data suggest that, in endothelial cells, H-Ras activity is not required for differentiation. However, this activity must be tightly regulated as aberrant activity can disturb the ability of endothelial cells to undergo differentiation.
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| 7. |
- Siwicki, Jan Konrad, et al.
(författare)
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Telomere maintenance and cell cycle regulation in spontaneously immortalized T-cell lines from Nijmegen breakage syndrome patients
- 2003
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Ingår i: Experimental Cell Research. - 0014-4827 .- 1090-2422. ; 287:1, s. 178-189
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Tidskriftsartikel (refereegranskat)abstract
- Nijmegen breakage syndrome (NBS) is a rare genetic instability syndrome associated with a high incidence of lymphoid malignancies. The NBS1 protein has been implicated in telomere biology suggesting that cells from NBS patients might have deficient telomere maintenance capacity. In this study we characterized spontaneously immortalized T-cell lines derived from three NBS patients regarding growth characteristics, telomere biology, expression of cell-cycle regulators, and response to DNA damage to understand the role of NBS1 in the immortalization process. In all the NBS T-cell lines the acquisition of an immortal phenotype was associated with telomere length stabilization, high telomerase activity, and increased mRNA expression of the catalytic subunit of telomerase (hTERT), together with c-myc up-regulation. Our findings provide evidence that telomere length maintenance was intact in the T lymphocytes in the absence of a full-length NBS protein, presumably due to the presence of an alternatively transcribed NBS protein of 70 kDa. Normal protein expression patterns for pRb and p53 in all the immortal lines coincided with altered expression of some cell-cycle proteins as well as with an impaired G1/S arrest after gamma irradiation, despite a seemingly normal p53/p21 pathway. The here described, spontaneously immortalized NBS derived T-cell lines can be useful in future analysis of the biologic effects in the NBS.
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| 8. |
- Tågerud, Sven, et al.
(författare)
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Receptor-mediated uptake of horseradish peroxidase in innervated and denervated skeletal muscle
- 1985
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 160:1, s. 95-105
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Tidskriftsartikel (refereegranskat)abstract
- The in vitro uptake of [3H]inulin and horseradish peroxidase (HRP) has been studied in innervated and 6 days denervated extensor digitorum longus muscle of the mouse. Both markers were taken up at a higher rate in denervated muscle. The increase in uptake after denervation was, however, larger for HRP than for [3H]inulin. After 2 h incubation at 37 °C, pH 7.3, in the presence of equimolar concentrations of HRP and [3H]inulin (approx. 2.1 μM), the uptake of HRP was approx. 8 times as great as the uptake of [3H]inulin in the same innervated muscles. In denervated muscle the HRP uptake was approx. 19 times as great as the [3H]inulin uptake in the same muscles. Various possible explanations of these differences in uptake have been considered and tested experimentally. [3H]Inulin uptake in skeletal muscle has previously been shown to obey bulk kinetics. The present investigation shows the HRP uptake to obey saturation kinetics. The HRP uptake shows dependency on divalent cations and is reduced if incubation is carried out at pH 6.4. The uptake of HRP, when used at a low, non-saturating concentration (10 μg/ml approx. 0.25 μM), is inhibited 60% by yeast mannan (0.1 mg/ml), ribonuclease B (0.1 mg/ml, approx. 7.4 μM), mannose (30 mM), monodansylcadaverine (1 mM), chloroquine (100 μM), trifluoperazine (25 μM) or maleic acid (2 mM). It is concluded that HRP is taken up in innervated and denervated skeletal muscle by a process of receptor-mediated endocytosis and that this uptake is under neurotrophic control.
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| 9. |
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| 10. |
- Ageberg, Malin, et al.
(författare)
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Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines.
- 2011
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 317, s. 1179-1191
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Tidskriftsartikel (refereegranskat)abstract
- Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line- based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitises DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor, FTI-277, or the geranylgeranyl transferase I inhibitor, GGTI-298, indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.
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| 11. |
- Ahrén, Bo
(författare)
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GLP-1 for type 2 diabetes
- 2011
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 317:9, s. 1239-1245
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Forskningsöversikt (refereegranskat)abstract
- Glucagon-like peptide-1 (GLP-1)-based therapy of type 2 diabetes is executed either by GLP-1 receptor agonists, which stimulate the GLP-1 receptors, or by dipeptidyl peptidase-4 (DPP-4) inhibitors, which prevent the inactivation of endogenous GLP-1 thereby increasing the concentration of endogenous active GLP-1. GLP-1 activates pancreatic receptors resulting in improved glycemia through glucose-dependent stimulation of insulin secretion and inhibition of glucagon secretion. There is also a potential beta cell preservation effect, as judged from rodent studies. GLP-1 receptors are additionally expressed in extrapancreatic tissue, having potential for the treatment to reduce body weight and to potentially have beneficial cardio- and endothelioprotective effects. Clinical trials in subjects with type 2 diabetes have shown that in periods of 12 weeks or more, these treatments reduce HbA1c by approximate to 0.8-1.1% from baseline levels of 7.7-8.5%, and they are efficient both as monotherapy and in combination therapy with metformin, sulfonylureas, thiazolidinediones or insulin. Furthermore, GLP-1 receptor agonists reduce body weight, whereas DPP-4 inhibitors are body weight neutral. The treatment is safe with very low risk for adverse events, including hypoglycaemia. GLP-1 based therapy is thus a novel and now well established therapy of type 2 diabetes, with a particular value in combination with metformin in patients who are inadequately controlled by metformin alone. (C) 2011 Elsevier Inc. All rights reserved.
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| 12. |
- Aifa, Sami, 1967-, et al.
(författare)
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A basic peptide within the juxtamembrane region is required for EGF receptor dimerization
- 2005
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 302:1, s. 108-114
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Tidskriftsartikel (refereegranskat)abstract
- The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (ΔTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function. © 2004 Elsevier Inc. All rights reserved.
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| 13. |
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| 14. |
- Almkvist, Jenny, 1971, et al.
(författare)
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Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe
- 2004
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Ingår i: Experimental cell research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 298:1, s. 74-82
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Tidskriftsartikel (refereegranskat)abstract
- Human neutrophils are activated by the beta-galactoside-binding lectin galectin-3, provided that the cells are primed by in vivo extravasation or by in vitro preactivation with, for example, LPS. Removal of terminal sialic acid can change neutrophil functionality and responsiveness due to exposure of underlying glycoconjugate receptors or change in surface charge. Here, we investigated whether such alteration of the cell surface carbohydrate composition can alter the responsiveness of the cells to galectin-3. Neutrophils were treated with neuraminidases (NA) of different origins: Clostridium perfringens (CP), Salmonella typhimurium, Vibrio cholerae, and Newcastle disease virus (NDV). In the presence of NDV-NA, but no other NA, the otherwise non-responding neutrophils responded readily to galectin-3 by activation of the NADPH-oxidase. The galectin-3 priming effect was inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid. Earlier studies have shown that priming of the neutrophil response to galectin-3 with, for example, LPS is paralleled by degranulation of intracellular vesicles and granules and upregulation of potential galectin-3 receptors. Also, NDV-NA (but not CP-NA) treatment induced degranulation, shown as an upregulation of complement receptor 3. Since not only the galectin response but also the response to the chemoattractant fMLF was primed, NDV-NA appears to induce a general priming phenomenon, possibly due to receptor upregulation by degranulation.
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| 15. |
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| 16. |
- Andersson, Elin, et al.
(författare)
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Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells.
- 2007
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 313:6, s. 1172-1180
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's Disease (PD) is a debilitating motor function disorder due primarily to a loss of midbrain dopaminergic neurons and a subsequent reduction in dopaminergic innervation of the striatum. Several attempts have been made to generate dopaminergic neurons from progenitor cell populations in vitro for potential use in cell replacement therapy for PD. However, expanding cells from fetal brain with retained potential for dopaminergic differentiation has proven to be difficult. In this study, we sought to generate mesencephalic dopaminergic (mesDA) neurons from an expanded population of fetal mouse ventral midbrain (VM) progenitors through the use of retroviral gene delivery. We over-expressed Ngn2 and Nurr1, two genes present in the ventral midbrain and important for normal development of mesDA neurons, in multipassaged neurosphere-expanded midbrain progenitors. We show that over-expression of Ngn2 in these progenitors results in increased neuronal differentiation but does not promote mesDA formation. We also show that over-expression of Nurr1 alone is sufficient to generate tyrosine hydroxylase (TH) expressing cells with an immature morphology, however the cells do not express any additional markers of mesDA neurons. Overexpression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers. (c) 2006 Elsevier Inc. All rights reserved.
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| 17. |
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| 18. |
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| 19. |
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| 20. |
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| 21. |
- Aspenström, Pontus, et al.
(författare)
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Taking Rho GTPases to the next level : the cellular functions of atypical Rho GTPases
- 2007
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 313:17, s. 3673-3679
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Forskningsöversikt (refereegranskat)abstract
- The Rho GTPases are influential regulators of signalling pathways that control vital cellular processes such as cytoskeletal dynamics, gene transcription, cell cycle progression and cell transformation. A vast majority of the studies involving Rho GTPases have been focused to the famous triad, Cdc42, Rac1 and RhoA, but this protein family actually harbours 20 members. Recently, the less known Rho GTPases have received increased attention. Many of the less studied Rho GTPases have structural, as well as, functional features which makes it pertinent to classify them as atypical Rho GTPases. This review article will focus on the critical aspects of the atypical Rho GTPases, RhoH, Wrch-1, Chp and RhoBTB. These proteins are involved in a broad spectre of biological processes, such as cytoskeletal dynamics, T-cell signalling and protein ubiquitinylation. We will also discuss the roles of atypical Rho GTPases as oncogenes or tumour suppressors, as well as their potential involvement in human diseases.
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| 22. |
- Aspenström, Pontus, et al.
(författare)
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The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics
- 2006
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 312:12, s. 2180-2194
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Tidskriftsartikel (refereegranskat)abstract
- Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.
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| 23. |
- Aspenström, Pontus
(författare)
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The mammalian verprolin homologue WIRE participates in receptor-mediated endocytosis and regulation of the actin filament system by distinct mechanisms
- 2004
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 298:2, s. 485-498
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Tidskriftsartikel (refereegranskat)abstract
- The mammalian verprolin family consists of three family members: WIP, WIRE and CR16. WIRE was recently found to bind to WASP and N-WASP and to have roles in regulating actin dynamics downstream of the platelet-derived growth factor beta-receptor. In the current study, the WASP-binding domain of WIRE was identified, with the core of the binding motif encompassing amino acid residues 408-412. A stretch of aromatic amino acid residues close to the core motif also participates in WASP binding. Amino acid substitutions in each of these motifs abrogated WASP binding, suggesting that both motifs are involved in the binding of WIRE to WASP. Interestingly, WIRE mutants unable to bind WASP were still able to induce a reorganisation of the actin filament system, indicating that WASP did not participate in the signalling pathway that link WIRE to actin dynamics. In cells ectopically expressing WIRE, the endocytosis of the platelet-derived growth factor beta-receptor was drastically reduced. However, in contrast to the effect on the actin filament system, the WIRE-induced ablation of the receptor endocytosis required an intact WASP-binding domain. Moreover, WIRE was more efficient than WIP in inhibiting the receptor endocytosis, implicating that these two mammalian verprolins have distinct roles in mammalian cells.
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| 24. |
- Aspenström, Pontus
(författare)
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The WASP-binding protein WIRE has a role in the regulation of the actin filament system downstream of the platelet-derived growth factor receptor
- 2002
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 279:1, s. 21-33
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Tidskriftsartikel (refereegranskat)abstract
- Activation of growth factor receptors, such as platelet-derived growth factor (PDGF) receptors, has a major impact on the motile behavior of vertebrate cells. The WASP family of proteins has been recognized as important regulators of actin polymerization via the activation of the Arp2/3 complex. The activity of the WASP proteins has, in turn, been shown to be governed by a number of associated proteins, including the WASP interacting protein (WIP). This report presents a novel WIP-like protein, WIRE (for WIP-related). WIRE was shown to bind to the WH1 domain of WASP and N-WASP. WIRE was localized to actin filaments in transiently transfected PAE/PDGFRbeta cells, and in cells simultaneously expressing WIRE and WASP, WIRE relocalized WASP to actin filaments, a relocalization that required direct interaction between the two proteins. In addition, WIRE was able to bind the PDGF receptor substrate Nckbeta. PDGF treatment of cells ectopically expressing WIRE resulted in formation of peripheral protrusions composed of filopodia and lamellipodia-like structures. In cells expressing both WIRE and WASP, PDGF treatment induced a translocation of WASP to the cell margin, an effect that required the presence of WIRE. Taken together, the data presented indicate that WIRE has a role in the WASP-mediated organization of the actin cytoskeleton and that WIRE is a potential link between the activated PDGF receptor and the actin polymerization machinery.
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| 25. |
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| 26. |
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| 27. |
- Babakov, V.N., et al.
(författare)
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RelA/NF-?B transcription factor associates with a-actinin-4
- 2008
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 314:5, s. 1030-1038
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Tidskriftsartikel (refereegranskat)abstract
- The NF-?B/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-?B in the cytoplasm and the transport mechanism to the nucleus. We found that NF-?B is associated with the actin-binding protein a-actinin-4. NF-?B and a-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-a, a-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-a led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-?B co-immunoprecipitated a-actinin-4 from A431 cell lysates and nuclear extracts, but a-actinin-1 and ß-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-?B can bind to matrix-bound chicken gizzard a-actinin. We suggest that the a-actinin-4 is important for the NF-?B nuclear translocation and its functions inside the nucleus. © 2007 Elsevier Inc. All rights reserved.
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| 28. |
- Baniol, Marion, et al.
(författare)
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Identification and characterization of distinct cell cycle stages in cardiomyocytes using the FUCCI transgenic system
- 2021
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 408:2
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the regulatory mechanism by which cardiomyocyte proliferation transitions to endoreplication and cell cycle arrest during the neonatal period is crucial for identifying proproliferative factors and developing regenerative therapies. We used a transgenic mouse model based on the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to isolate and characterize cycling cardiomyocytes at different cell cycle stages at a single-cell resolution. Single-cell transcriptome analysis of cycling and noncycling cardiomyocytes was performed at postnatal days 0 (P0) and 7 (P7). The FUCCI system proved to be efficient for the identification of cycling cardiomyocytes with the highest mitotic activity at birth, followed by a gradual decline in the number of cycling and mitotic cardiomyocytes during the neonatal period. Cardiomyocytes showed premature cell cycle exit at G1/S shortly after birth and delayed G1/S progression during endoreplication at P7. Single-cell RNA-seq confirmed previously described signaling pathways involved in cardiomyocyte proliferation (Erbb2 and Hippo/YAP), and maturation-related transcriptional changes during postnatal development, including the metabolic switch from glycolysis to fatty acid oxidation in cardiomyocytes. Importantly, we generated transcriptional profiles specific to cell division and endoreplication in cardiomyocytes at different developmental stages that may facilitate the identification of genes important for adult cardiomyocyte proliferation and heart regeneration. In conclusion, the FUCCI mouse provides a valuable system to study cardiomyocyte cell cycle activity at single cell resolution that can help to decipher the switch from cardiomyocyte proliferation to endoreplication, and to revert this process to facilitate endogenous repair.
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| 29. |
- Bauerschmidt, Christina, et al.
(författare)
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Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells
- 2011
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 317:3, s. 330-337
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Tidskriftsartikel (refereegranskat)abstract
- Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.
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| 30. |
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| 31. |
- Benedito, Rui, et al.
(författare)
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Notch as a hub for signaling in angiogenesis
- 2013
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 319:9, s. 1281-1288
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Forskningsöversikt (refereegranskat)
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| 32. |
- Berglund, Erik, et al.
(författare)
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Evidence for Ca2+-regulated ATP release in gastrointestinal stromal tumors
- 2013
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 319:8, s. 1229-1238
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Tidskriftsartikel (refereegranskat)abstract
- Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca2+ concentration ([Ca(2+)1](i)), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca2+-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca2+ influx since exclusion of extracellular Ca2+ diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.
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| 33. |
- Berglund, Erik, et al.
(författare)
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Functional role of the Ca2+-activated Cl- channel DOG1/TMEM16A in gastrointestinal stromal tumor cells
- 2014
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 326:2, s. 315-325
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Tidskriftsartikel (refereegranskat)abstract
- DOG1, a Ca2+-activated Cl- channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl- currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.
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| 34. |
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| 35. |
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| 36. |
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| 37. |
- Bergström, J.Daniel, et al.
(författare)
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Epidermal growth factor receptor signaling activates Met in human anaplastic thyroid carcinoma cells
- 2000
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 259:1, s. 293-299
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.
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| 38. |
- Berthold, Jessica, et al.
(författare)
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Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes--evidence for an autoregulatory mechanism
- 2008
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 314:19, s. 3453-3465
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Tidskriftsartikel (refereegranskat)abstract
- RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.
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| 39. |
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| 40. |
- Björnsdottir, Halla, et al.
(författare)
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Inhibition of phospholipase A(2) abrogates intracellular processing of NADPH-oxidase derived reactive oxygen species in human neutrophils.
- 2013
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Ingår i: Experimental cell research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 319:5, s. 761-774
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Tidskriftsartikel (refereegranskat)abstract
- Upon activation of human neutrophils, superoxide can be produced at two cellular sites; either in the plasma membrane, giving extracellular release of oxidants, or in intracellular organelles, resulting in oxidants being retained in the cell. The involvement of phospholipase A(2) (PLA(2)) in phorbol myristate acetate (PMA)-induced activation of the two pools of NADPH-oxidase was investigated using a variety of PLA(2) inhibitors and the oxidase activity was measured by luminol/isoluminol-amplified chemiluminescence (CL). Two of the seven inhibitors were without effect, two inhibitors inhibited both intra- and extracellular ROS production equally, and three inhibitors inhibited intracellular but not extracellular CL. Using another technique to measure ROS, PHPA oxidation, we found that intracellular ROS production was unaltered with the three last inhibitors, indicating that PLA(2) is not involved in the NADPH-oxidase activity per se, but in the intracellular processing of the radicals necessary for the CL reaction to take place. The PLA(2) inhibitors did not abolish the activity of myeloperoxidase (MPO), an enzyme necessary for intracellular CL to occur. Instead, we suggest that these PLA(2) inhibitors block heterotypic granule fusion and prohibit the colocalization of ROS and MPO needed for intracellular CL activity.
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| 41. |
- Blom, M., et al.
(författare)
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RhoD is a Golgi component with a role in anterograde protein transport from the ER to the plasma membrane
- 2015
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 333:2, s. 208-219
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Tidskriftsartikel (refereegranskat)abstract
- RhoD is a member of the Rho GTPase family and it coordinates actin dynamics and membrane trafficking. Activation of RhoD results in formation of filopodia, dissolution of stress fibers, and the subsequent formation of short actin bundles. In addition, RhoD localizes to early endosomes and recycling endosomes, and has a regulatory role in endosome trafficking. In this study, we report on a function of RhoD in the regulation of Golgi homeostasis. We show that manipulation of protein and activation levels of RhoD, as well as of its binding partner WHAMM, result in derailed localization of Golgi stacks. Moreover, vesicle trafficking from the endoplasmic reticulum to the plasma membrane via the Golgi apparatus measured by the VSV-G protein is severely hampered by manipulation of RhoD or WHAMM. In summary, our studies demonstrate a novel role for this member of the Rho GTPases in the regulation of Golgi function.
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| 42. |
- Blom, Magdalena, et al.
(författare)
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The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration
- 2017
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 352:2, s. 255-264
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Tidskriftsartikel (refereegranskat)abstract
- RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration.
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| 43. |
- Bonanini, Flavio, et al.
(författare)
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A comparison between different human hepatocyte models reveals profound differences in net glucose production, lipid composition and metabolism in vitro
- 2024
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 437:1
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Tidskriftsartikel (refereegranskat)abstract
- Hepatocytes are responsible for maintaining a stable blood glucose concentration during periods of nutrient scarcity. The breakdown of glycogen and de novo synthesis of glucose are crucial metabolic pathways deeply interlinked with lipid metabolism. Alterations in these pathways are often associated with metabolic diseases with serious clinical implications. Studying energy metabolism in human cells is challenging. Primary hepatocytes are still considered the golden standard for in vitro studies and have been instrumental in elucidating key aspects of energy metabolism found in vivo. As a result of several limitations posed by using primary cells, a multitude of alternative hepatocyte cellular models emerged as potential substitutes. Yet, there remains a lack of clarity regarding the precise applications for which these models accurately reflect the metabolic competence of primary hepatocytes. In this study, we compared primary hepatocytes, stem cell-derived hepatocytes, adult donor-derived liver organoids, immortalized Upcyte-hepatocytes and the hepatoma cell line HepG2s in their response to a glucose production challenge. We observed the highest net glucose production in primary hepatocytes, followed by organoids, stem-cell derived hepatocytes, Upcyte-hepatocytes and HepG2s. Glucogenic gene induction was observed in all tested models, as indicated by an increase in G6PC and PCK1 expression. Lipidomic analysis revealed considerable differences across the models, with organoids showing the closest similarity to primary hepatocytes in the common lipidome, comprising 347 lipid species across 19 classes. Changes in lipid profiles as a result of the glucose production challenge showed a variety of, and in some cases opposite, trends when compared to primary hepatocytes.
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| 44. |
- Borgenström, M, et al.
(författare)
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Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate
- 2001
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 264:2, s. 307-314
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Tidskriftsartikel (refereegranskat)abstract
- The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.
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| 45. |
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| 46. |
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| 47. |
- Cacci, Emanuele, et al.
(författare)
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Generation of human cortical neurons from a new immortal fetal neural stem cell line.
- 2007
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 313:3, s. 588-601
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Tidskriftsartikel (refereegranskat)abstract
- Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia. and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology. (c) 2006 Elsevier Inc. All rights reserved.
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| 48. |
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| 49. |
- Claesson-Welsh, Lena, et al.
(författare)
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Introduction to the ECR special angiogenesis issue
- 2013
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 319:9, s. 1239-1239
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 50. |
- Cracknell, Tobias, et al.
(författare)
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Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL
- 2020
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Ingår i: Experimental Cell Research. - : ELSEVIER INC. - 0014-4827 .- 1090-2422. ; 395:2
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Tidskriftsartikel (refereegranskat)abstract
- The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion.
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