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| BMC Cell Biology - Latest Articles | ||
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| Mise à jour réussie | 15/3/2010 20:00:00 | |
| Description | The latest research articles published by BMC Cell Biology | |
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| High-throughput and single-cell imaging of NF-kappabeta oscillations using monoclonal cell lines | ||
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| Description: | Background: The nuclear factor-kappaB (NF-kappaB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-kappaB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF kappaB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-kappaB-induced genes is regulated by differential dynamics of single NF kappaB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-kappaB activation have been observed in response to the cytokine tumor necrosis factor alpha (TNFalpha), little is known about the occurrence of oscillations in response to bacterial infections. Results: To quantitatively assess NF-kappaB dynamics we generated human and murine monoclonal cell lines that stably express the NF-kappaB subunit p65 fused to GFP. Furthermore, a high-throughput assay based on automated microscopy coupled to image analysis to quantify p65-nuclear translocation was established. Using this assay, we demonstrate a stimulus- and cell line-specific temporal control of p65 translocation, revealing, for the first time, oscillations of p65 translocation in response to bacterial infection. Oscillations were detected at the single-cell level using real-time microscopy as well as at the population level using high-throughput image analysis. In addition, mathematical modeling of NF-kappaB dynamics during bacterial infections predicted masking of oscillations on the population level in asynchronous activations, which was experimentally confirmed. Conclusions: Taken together, this simple and cost effective assay constitutes an integrated approach to infer the dynamics of NF-kappaB kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-kappaB can be identified and analyzed, providing new possibilities for a wide range of applications from therapeutic discovery and understanding of disease to host-pathogen interactions. |
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| A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism | ||
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| Description: | Background: Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results: In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions: In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function. |
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| Nuclear variants of bone morphogenetic proteins | ||
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| Description: | Background: Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. Results: In all three proteins, a bipartite nuclear localization signal (NLS) was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5) containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. Conclusions: The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell. |
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| Mechanical strain modulates age-related changes in the proliferation and differentiation of mouse adipose-derived stromal cells | ||
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| Description: | Background: Previous studies on the effects of aging in human and mouse mesenchymal stem cells suggest that a decline in the number and differentiation potential of stem cells may contribute to aging and aging-related diseases. In this report, we used stromal cells isolated from adipose tissue (ADSCs) of young (8-10 weeks), adult (5 months), and old (21 months) mice to test the hypothesis that mechanical loading modifies aging-related changes in the self-renewal and osteogenic and adipogenic differentiation potential of these cells. Results: We show that aging significantly reduced the proliferation and increased the adipogenesis of ADSCs, while the osteogenic potential is not significantly reduced by aging. Mechanical loading (10% cyclic stretching, 0.5 Hz, 48 h) increased the subsequent proliferation of ADSCs from mice of all ages. Although the number of osteogenic colonies with calcium deposition was increased in ADSCs subjected to pre-strain, it resulted from an increase in colony number rather than from an increase in osteogenic potential after strain. Pre-strain significantly reduced the number of oil droplets and the expression of adipogenic marker genes in adult and old ADSCs. Simultaneously subjecting ADSCs to mechanical loading and adipogenic induction resulted in a stronger inhibition of adipogenesis than that caused by pre-strain. The reduction of adipogenesis by mechanical strain was loading-magnitude dependent: loading with 2% strain only resulted in a partial inhibition, and loading with 0.5% strain could not inhibit adipogenesis in ADSCs. Conclusions: We demonstrate that mechanical stretching counteracts the loss of self-renewal in aging ADSCs by enhancing their proliferation and, at the same time, reduces the heightened adipogenesis of old cells. These findings are important for the further study of stem cell control and treatment for a variety of aging related diseases. |
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| Role of casein kinase 1 in the glucose sensor-mediated signaling pathway in yeast | ||
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| Description: | Background: In yeast, glucose-dependent degradation of the Mth1 protein, a corepressor of the glucose transporter gene (HXT) repressor Rgt1, is a crucial event enabling expression of several HXT. This event occurs through a signaling pathway that involves the Rgt2 and Snf3 glucose sensors and yeast casein kinase 1 and 2 (Yck1/2). In this study, we examined whether the glucose sensors directly couple with Yck1/2 to convert glucose binding into an intracellular signal that leads to the degradation of Mth1. Results: High levels of glucose induce degradation of Mth1 through the Rgt2/Snf3 glucose signaling pathway. Fluorescence microscopy analysis indicates that, under glucose-limited conditions, GFP-Mth1 is localized in the nucleus and does not shuttle between the nucleus and cytoplasm. If glucose-induced degradation is prevented due to disruption of the Rgt2/Snf3 pathway, GFP-Mth1 accumulates in the nucleus. When engineered to be localized to the cytoplasm, GFP-Mth1 is degraded regardless of the presence of glucose or the glucose sensors. In addition, removal of Grr1 from the nucleus prevents degradation of GFP-Mth1. These results suggest that glucose-induced, glucose sensor-dependent Mth1 degradation occurs in the nucleus. We also show that, like Yck2, Yck1 is localized to the plasma membrane via C-terminal palmitoylation mediated by the palmitoyl transferase Akr1. However, glucose-dependent degradation of Mth1 is not impaired in the absence of Akr1, suggesting that a direct interaction between the glucose sensors and Yck1/2 is not required for Mth1 degradation. Conclusion: Glucose-induced, glucose sensor-regulated degradation of Mth1 occurs in the nucleus and does not require direct interaction of the glucose sensors with Yck1/2. |
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