| 1. |
- Appelgren, Henrik, et al.
(författare)
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Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells
- 2003
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 116:19, s. 4035-4042
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Tidskriftsartikel (refereegranskat)abstract
- Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one Another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion defect. Thus, S. pombe centromeres have two distinguishable domains even during mitosis, and our functional analyses support the previous observations that the kinetochore/central core and the heterochromatin domains have distinct functions both in interphase and mitosis.
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| 2. |
- Arabi, Azadeh, et al.
(författare)
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Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels
- 2003
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 116:9, s. 1707-1717
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Tidskriftsartikel (refereegranskat)abstract
- c-Myc is a predominately nuclear transcription factor that is a substrate for rapid turnover by the proteasome system. Cancer-related mutations in c-Myc lead to defects in its degradation and thereby contribute to the increase in its cellular level that is associated with the disease. Little is known about the mechanisms that target c-Myc to the proteasomes. By using a GFP fusion protein and live analysis we show that c-Myc shuttles between the nucleus and cytoplasm and thus it could be degraded in either compartment. Strikingly, at elevated levels of expression c-Myc accumulates at nucleoli in some cells, consistent with saturation of a nucleolus-associated degradation system in these cells. This idea is further supported by the observation that proteasome inhibitor treatment causes accumulation of c-Myc at the nucleoli of essentially all cells. Under these conditions c-Myc is relatively stably associated with the nucleolus, as would be expected if the nucleolus functions as a sequestration/degradation site for excess c-Myc. Furthermore, during elevated c-Myc expression or proteasome inhibition, nucleoli that are associated with c-Myc also accumulate proteasomes. c-Myc and proteasomes co-localise in intranucleolar regions distinct from the dense fibrillar component of the nucleolus. Based on these results we propose a model for c-Myc downregulation where c-Myc is sequestered at the nucleoli. Sequestration of c-Myc is accompanied by recruitment of proteasomes and may lead to subsequent degradation.
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| 3. |
- Barg, Sebastian, et al.
(författare)
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Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification
- 2001
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Ingår i: Journal of Cell Science. - 0021-9533 .- 1477-9137. ; 114:Pt 11, s. 2145-54
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Tidskriftsartikel (refereegranskat)abstract
- ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.
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| 4. |
- Bjork, P, et al.
(författare)
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The Chironomus tentans translation initiation factor eIF4H is present in the nucleus but does not bind to mRNA until the mRNA reaches the cytoplasmic perinuclear region
- 2003
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Ingår i: Journal of cell science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 116:22Pt 22, s. 4521-4532
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Tidskriftsartikel (refereegranskat)abstract
- In the cell nucleus, precursors to mRNA, pre-mRNAs, associate with a large number of proteins and are processed to mRNA-protein complexes, mRNPs. The mRNPs are then exported to the cytoplasm and the mRNAs are translated into proteins. The mRNAs containing in-frame premature stop codons are recognized and degraded in the nonsense-mediated mRNA decay process. This mRNA surveillence may also occur in the nucleus and presumably involves components of the translation machinery. Several translation factors have been detected in the nucleus, but their functional relationship to the dynamic protein composition of pre-mRNPs and mRNPs in the nucleus is still unclear.Here, we have identified and characterized the translation initiation factor eIF4H in the dipteran Chironomus tentans. In the cytoplasm, Ct-eIF4H is associated with poly(A+) RNA in polysomes. We show that a minor fraction of Ct-eIF4H enters the nucleus. This fraction is independent on the level of transcription. CteIF4H could not be detected in gene-specific pre-mRNPs or mRNPs, nor in bulk mRNPs in the nucleus. Our immunoelectron microscopy data suggest that Ct-eIF4H associates with mRNP in the cytoplasmic perinuclear region, immediately as the mRNP exits from the nuclear pore complex.
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| 5. |
- Castelo-Branco, G, et al.
(författare)
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GSK-3beta inhibition/beta-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons
- 2004
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Ingår i: Journal of cell science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 117:24Pt 24, s. 5731-5737
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Tidskriftsartikel (refereegranskat)abstract
- Wnts are important regulators of dopamine (DA) neuron differentiation in the developing ventral mesencephalon and could thus serve as potential tools in the treatment of Parkinson's disease. In this study, we investigate whether established intracellular Wnt signalling components could modulate the development of DA neurons. Two chemical inhibitors of glycogen synthase kinase (GSK)-3β, indirubin-3-monoxime and kenpaullone, were found to increase neuronal differentiation in ventral mesencephalon precursor cultures. In addition, the GSK-3β-specific inhibitor kenpaullone increased the size of the DA neuron population through conversion of precursors expressing the orphan nuclear receptor-related factor 1 into tyrosine hydroxylase positive neurons, thereby mimicking an effect of Wnts. We show that GSK-3β inhibitors stabilized β-catenin and that overexpression of β-catenin in ventral mesencephalic precursors resulted in increased DA differentiation. The three- to fivefold increase in DA differentiation of precursor cells by GSK-3β inhibitors suggests that such compounds could be used to improve stem/precursor cell therapy approaches in Parkinson's disease.
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| 6. |
- Dyachok, Oleg, et al.
(författare)
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Store-operated influx of Ca2+ in the pancreatic β-cells exhibits graded dependence on the filling of the endoplasmic reticulum
- 2001
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Ingår i: Journal of Cell Science. - 0021-9533 .- 1477-9137. ; 114:Pt 11, s. 2179-2186
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Tidskriftsartikel (refereegranskat)abstract
- The store-operated pathway for Ca2+ entry was studied in individual mouse pancreatic β-cells by measuring the cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mn2+ ([Mn2+]i) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca2+ sequestration. To avoid interference with voltage-dependent Ca2+ entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca2+ depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca2+]i or from the rate of increase in [Mn2+]i due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn2+ depended on store filling in a graded manner. The store-operated influx of Ca2+/Mn2+ was inhibited by Gd3+ and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the β-cell has direct implications for the control of membrane potential and insulin secretion.
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| 7. |
- Edlund, Sofia, et al.
(författare)
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Smad7 is required for TGF-ß-induced activation of the small GTPase Cdc42
- 2004
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 117:Pt 9, s. 1835-1847
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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. The inhibitory Smads, Smad6 and Smad7, are considered to function as negative regulators of the TGF-beta/Smad signaling cascade. In a previous study, we found that TGF-beta induces rearrangements of the actin filament system in human prostate carcinoma cells and that this response requires the small GTPases Cdc42 and RhoA. On the basis of the current view on the function of Smad7 in TGF-beta signaling, we hypothesized that Smad7 would function as a negative regulator of the TGF-beta-induced activation of Cdc42 and RhoA, but instead we found that the reverse is the case; Smad7 is required for the TGF-beta-induced activation of Cdc42 and the concomitant reorganization of the actin filament system. These observations propose a novel role for Smad7 in TGF-beta-dependent activation of Rho GTPases.
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| 8. |
- Filonova, LH, et al.
(författare)
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Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce
- 2000
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Ingår i: Journal of cell science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 113113 Pt 24:24, s. 4399-4411
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Tidskriftsartikel (refereegranskat)abstract
- In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts. Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny. During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce.
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| 9. |
- Fotin-Mleczek, Mariola, et al.
(författare)
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Apoptotic crosstalk of TNF receptors : TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-8
- 2002
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Ingår i: Journal of Cell Science. - 0021-9533 .- 1477-9137. ; 115:Pt 13, s. 2757-70
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Tidskriftsartikel (refereegranskat)abstract
- We have recently shown that stimulation of TNF-R2 selectively enhances apoptosis induction by the death receptor TNF-R1. Here, we demonstrate that stimulation of CD30 or CD40 also leads to selective enhancement of TNF-R1-induced cell death. Enhancement of apoptosis was correlated with the depletion of endogenous TRAF2 within 1 to 6 hours. Selective prestimulation of TNF-R2 for several hours inhibited TNF-R2-induced activation of the anti-apoptotic NF-kappaB pathway up to 90% and dramatically enhanced apoptosis induction by this receptor. When both TNF-receptors were stimulated simultaneously, TNF-R1-induced NF-kappaB activation remained unaffected but TNF-R1-induced apoptosis was still significantly enhanced. Compared with FasL-induced cell death TNF-R1-induced activation of caspase-8 was significantly weaker and delayed. Costimulation or prestimulation of TNF-R2 enhanced caspase-8 processing. Life cell imaging and confocal microscopy revealed that both TNF-R1 and TNF-R2 recruited the anti-apoptotic factor cIAP1 in a TRAF2-dependent manner. Thus, TNF-R2 may compete with TNF-R1 for the recruitment of newly synthesized TRAF2-bound anti-apoptotic factors, thereby promoting the formation of a caspase-8-activating TNF-R1 complex. Hence, TNF-R2 triggering can interfere with TNF-R1-induced apoptosis by inhibition of NF-kappaB-dependent production of anti-apoptotic factors and by blocking the action of anti-apoptotic factors at the post-transcriptional level.
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| 10. |
- Geberhiwot, T, et al.
(författare)
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Laminin-8 (alpha4beta1gamma1) is synthesized by lymphoid cells, promotes lymphocyte migration and costimulates T cell proliferation
- 2001
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Ingår i: Journal of cell science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 114:2Pt 2, s. 423-433
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Tidskriftsartikel (refereegranskat)abstract
- Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin alpha4, beta1 and gamma1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin beta1 and gamma1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(alpha5beta1gamma1/alpha5beta2gamma1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using alpha6beta1 integrin, but minimally to laminin-1 (alpha1beta1gamma1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and -10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology.
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