| 1. |
- Bergqvist, Cecilia, et al.
(författare)
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Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1
- 2019
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:9
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Tidskriftsartikel (refereegranskat)abstract
- In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.
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| 2. |
- Dryselius, Rikard, et al.
(författare)
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Variable coordination of cotranscribed genes in Escherichia coli following antisense repression
- 2006
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 6, s. 97-
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Tidskriftsartikel (refereegranskat)abstract
- Background: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.
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| 3. |
- 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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| 4. |
- Kulytè, Agnè
(författare)
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New aspects on retinoic acid induced HL-60 granulocytic differentiation
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The underlying mechanisms of myeloid cell differentiation are still not entirely elucidated. It is therefore an urgent task to extent the basis for a comprehensive model of granulocytes differentiation. A primary goal for studies has been the identification of differentiation markers. These often correspond to genes that are expressed at defined steps during the differentiation process or in the fully differentiated phenotype. Here is demonstrated that the program of phenotype formation entails dramatic changes in long-term tyrosine phosphorylation of some cytoplasmic proteins during retinoic acid (RA)-induced HL-60 cell differentiation to granulocytes. A significant increase in the phosphotyrosine content of a kinase (Erk2) in the commitment and terminal stages of HL-60 cell differentiation (48-120 h) was related to promotion of cell survival and triggering of apoptosis. In addition, some cytosolic proteins of apoptotic origin were delineated in the differentiating population. The pattern of tyrosine phosphorylations of specific cytosolic proteins in maturing and apoptotic granulocytes should be a powerful tool for further elucidation of differentiation mechanisms. We developed a new approach based on cytosolic protein tagging and nuclear import analysis combined with assessment of specific protein tyrosine phosphorylations by two-dimensional gel electrophoresis. Many of the genes involved in the differentiation of multipotent stem cells to specific cellular lineages are still unknown. Accordingly, we searched for additional tyrosine phosphorylated, RA-induced transcription regulators that are translocated into the nucleus during commitment of HL-60 cells to become granulocyte-like cells. It is shown, that transcription factors required for lineage commitment of differentiating (C/EBPß, c-myb) and for survival of differentiated cells (STATs, NFKB) co-operatively promoted signaling in the HL-60 cell line in response to RA. Identification of the tyrosine-phosphorylated proteins being translocated into the nucleus during HL-60 cell differentiation was our further goaL This way, we show for the first time that human gamma~dystrobrevin is expressed in promyelocytic HL-60 cell line. We detected gamma-dystrobrevin in the cytosol and the nuclei of these cells, and, in the latter location; it was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of gamma-dystrobrevin could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and hwnan neutrophils. Moreover, we found gamma-dystrobrevin in association with actin and myosin light chain. Our results provide new information about potential involvement of gamma-dystrobrevin in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells. In addition, our study confirms and extends knowledge about the genes involved in granulocytic differentiation and growth arrest. This shuiy corroborates the value of cDNA RDA to isolate factors involved in myeloid maturation. In conclusion, the more detailed analysis of granulocytic differentiation of HL-60 cells presented here provides new data that should allow further refinement of models of myeloid differentiation.
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| 5. |
- Kulytè, Agnè, et al.
(författare)
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Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins
- 2001
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Ingår i: BioTechniques. - 0736-6205 .- 1940-9818. ; 31:3, s. 508-517
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Tidskriftsartikel (refereegranskat)abstract
- Phosphotyrosine signaling plays a vital role in cell regulation - from receptor activation, through stimulation of signal networks and nuclear targeting, to final cellular responses. Here, we propose a new approach to monitor the spatial and temporal aspects of tyrosine phosphorylation and dephosphorylation. The method can be used to determine whether protein tyrosine phosphorylations and dephosphorylations occur in the cytosol or the nucleus and to ascertain whether such modifications are associated with nuclear traffic. Promyelocytic leukemia (HL-60) cells are used as the experimental model. Biotinylated cytosolic proteins from donor cells are used to trace nuclear transport in permeabilized recipient cells. Thereafter, 2-D gel electrophoresis is applied to fractionate the cytosolic and nuclear proteins of the recipient cells, which are subsequently blotted onto polyvinylidene difluoride membranes. The membranes are developed with streptavidin and then reprobed with anti-phosphotyrosine antibodies. The major advantages of the protocol are that it is simple to perform, and reproducible results are obtained by overlaying the patterns of biotinylated and/or tyrosine-phosphorylated proteins. Moreover, several hundred cytosolic and nuclear proteins can be analyzed in parallel. Thus, by comparing the 2-D gel electrophoresis maps of biotinylated and tyrosine-phosphorylated proteins, it is possible to determine the involvement of trafficking of the latter proteins in cell signaling.
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| 6. |
- Navakauskiene, Ruta, et al.
(författare)
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Assessment of transcription regulators and translocation of these proteins into the nucleus during granulocyte lineage commitment of haematopoietis HL-60 cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Expression of transcription factors required for lineage commitment of differentiating cells (C/EBPß and c-myb) and for survival of differentiated cells (STATs and NFκB) was examined in the HL-60 cell line. Differentiation was induced by treating the cells with retinoic acid (RA). c-Myb expression in the nucleus restored at the precommitment stage (18 h) what concurred with the highest nuclear level of C/EBPß, which suggests a combinatorial interaction of these transcription factors in granulocytic signalling pathway. Expression of STAT5a and STAT5b varied during differentiation; there were no significant changes in STAT3 levels. Increased cytosolic level of NFκB p65 during precommitment and commitment stages of granulocytic differentiation coincides with augmentation of STAT5a protein level what could be an evidence of their possible co-operation during granulocyticlineage commitment of HL-60 cells. Our results suggest that the studied proteins cooperatively promote signalling in differentiating promyelocytic HL-60 cell line in response to retinoic acid.
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| 7. |
- Navakauskiene, Ruta, et al.
(författare)
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Translocation of transcription regulators into the nucleus during granulocyte commitment of HL-60 cells
- 2003
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Ingår i: Biochemistry and Cell Biology. - : Canadian Science Publishing. - 0829-8211 .- 1208-6002. ; 81:4, s. 285-295
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Tidskriftsartikel (refereegranskat)abstract
- Expression of transcription factors required for lineage commitment of differentiating cells (C/EBPβ and c-Myb) and for survival of differentiated cells (STATs and NFκB) was examined in the HL-60 cell line. Differentiation was induced by treating the cells with retinoic acid. c-Myb expression in the nucleus restored at the precommitment stage (18 h) what concurred with the highest nuclear level of C/EBPβ, which suggests a combinatorial interaction of these transcription factors in the granulocytic signalling pathway. Expression of STAT5a and STAT5b varied during differentiation, whereas no significant changes were seen in STAT3 levels. Increased cytosolic level of NFκB p65 during precommitment and commitment stages of granulocytic differentiation coincided with augmentation of the STAT5a protein level, which could be evidence of their possible cooperation during granulocytic-lineage commitment of HL-60 cells. Our results suggest that the studied transcription factors cooperatively promote signalling in the differentiating promyelocytic HL-60 cell line in response to retinoic acid.
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| 8. |
- Perskvist, Nasrin, 1952-, et al.
(författare)
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Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils
- 2002
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Ingår i: Journal of Cell Science. - 0021-9533 .- 1477-9137. ; 115:6, s. 1321-1330
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Tidskriftsartikel (refereegranskat)abstract
- Biogenesis of phagolysosomes proceeds through a sequential series of interactions with endocytic organelles, a process known to be regulated by Rab and SNARE proteins. The molecular mechanisms underlying phagosome maturation in neutrophils are, however, not clearly understood. We investigated fusion between phagosomes containing the intracellular pathogen Mycobacterium tuberculosis versus the extracellular pathogen Staphylococcus aureus (designated MCP for mycobacteria-containing phagosome and SCP for S. aureus-containing phagosome) and cytoplasmic compartments in human neutrophils. Western blot analysis of phagosomes isolated after internalisation revealed that lactoferrin (a constituent of secondary granules) and LAMP-1 were incorporated into both SCP and MCP, whereas hck (marker of azurophil granules) interacted solely with SCP. The subcellular distribution of the proteins Rab5a and syntaxin-4 suggested a role in docking of granules and/or endosomes to the target membrane in the neutrophil. We observed that during phagocytosis, Rab5a in GTP-bound form interacted with syntaxin-4 on the membrane of MCP and were retained for up to 90 minutes, whereas the complex was recruited to the SCP within 5 minutes but was selectively depleted from these vacuoles after 30 minutes of phagocytosis. Downregulation of Rab5a by antisense oligonucleotides efficiently reduced the synthesis of Rab5a, the binding of syntaxin-4 to MCP and SCP and the capacity for fusion exhibited by the pathogen-containing phagosomes, but it had no effect on bacteria internalisation. These data indicate that the difference in granule fusion is correlated with a difference in the association of Rab5a and syntaxin-4 with the phagosomes. Intracellular pathogen-containing phagosomes retain Rab5a and syntaxin-4, whereas extracellular pathogen-containing phagosomes bind briefly to this complex. These results also identified Rab5a as a key regulator of phagolysosome maturation in human neutrophils.
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| 9. |
- Rohm, Maria, et al.
(författare)
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An AMP-activated protein kinase-stabilizing peptide ameliorates adipose tissue wasting in cancer cachexia in mice
- 2016
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 22:10, s. 1120-1130
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Tidskriftsartikel (refereegranskat)abstract
- Cachexia represents a fatal energy-wasting syndrome in a large number of patients with cancer that mostly results in a pathological loss of skeletal muscle and adipose tissue. Here we show that tumor cell exposure and tumor growth in mice triggered a futile energy-wasting cycle in cultured white adipocytes and white adipose tissue (WAT), respectively. Although uncoupling protein 1 (Ucp1)-dependent thermogenesis was dispensable for tumor-induced body wasting, WAT from cachectic mice and tumor-cell-supernatant-treated adipocytes were consistently characterized by the simultaneous induction of both lipolytic and lipogenic pathways. Paradoxically, this was accompanied by an inactivated AMP-activated protein kinase (Ampk), which is normally activated in peripheral tissues during states of low cellular energy. Ampk inactivation correlated with its degradation and with upregulation of the Ampk-interacting protein Cidea. Therefore, we developed an Ampk-stabilizing peptide, ACIP, which was able to ameliorate WAT wasting in vitro and in vivo by shielding the Cidea-targeted interaction surface on Ampk. Thus, our data establish the Ucp1-independent remodeling of adipocyte lipid homeostasis as a key event in tumor-induced WAT wasting, and we propose the ACIP-dependent preservation of Ampk integrity in the WAT as a concept in future therapies for cachexia.
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| 10. |
- Treigyté, G., et al.
(författare)
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Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils
- 2000
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - 1420-682X .- 1420-9071. ; 57:13-14, s. 1997-2008
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Tidskriftsartikel (refereegranskat)abstract
- Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6-24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48-120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.
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