| 1. |
- Babazadeh, Roja, et al.
(författare)
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Rewiring yeast osmostress signalling through the MAPK network reveals essential and non-essential roles of Hog1 in osmoadaptation
- 2014
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data.
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| 2. |
- Furukawa, Kentaro, et al.
(författare)
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Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae.
- 2011
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 6:10
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Tidskriftsartikel (refereegranskat)abstract
- Yeast cells undergo diploid-specific developments such as spore formation via meiosis and pseudohyphal development under certain nutrient-limited conditions. Studies on these aspects require homozygous diploid mutants, which are generally constructed by crossing strains of opposite mating-type with the same genetic mutation. So far, there has been no direct way to generate and select diploids from haploid cells. Here, we developed a method for efficient construction of homozygous diploids using a PGAL1-HO gene (galactose-inducible mating-type switch) and a PSTE18-URA3 gene (counter selection marker for diploids). Diploids are generated by transient induction of the HO endonuclease, which is followed by mating of part of the haploid population. Since the STE18 promoter is repressed in diploids, diploids carrying PSTE18-URA3 can be selected on 5-fluoroorotic acid (5-FOA) plates where the uracil prototrophic haploids cannot grow. To demonstrate that this method is useful for genetic studies, we screened suppressor mutations of the complex colony morphology, strong agar invasion and/or hyper-filamentous growth caused by lack of the Hog1 MAPK in the diploid Σ1278b strain background. Following this approach, we identified 49 suppressor mutations. Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes. Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.
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| 3. |
- Furukawa, Kentaro, et al.
(författare)
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A fungicide-responsive kinase as a tool for synthetic cell fate regulation
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:14, s. 7162-70
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Tidskriftsartikel (refereegranskat)abstract
- Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic ‘suicide attack’ system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications.
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| 4. |
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| 5. |
- Furukawa, Kentaro, et al.
(författare)
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Expression of the yeast aquaporin Aqy2 affects cell surface properties under the control of osmoregulatory and morphogenic signalling pathways.
- 2009
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 74:5, s. 1272-86
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins mediate rapid and selective water transport across biological membranes. The yeast Saccharomyces cerevisiae possesses two aquaporins, Aqy1 and Aqy2. Here, we show that Aqy2 is involved in controlling cell surface properties and that its expression is controlled by osmoregulatory and morphogenic signalling pathways. Deletion of AQY2 results in diminished fluffy colony morphology while overexpression of AQY2 causes strong agar invasion and adherence to plastic surfaces. Hyper-osmotic stress inhibits morphological developments including the above characteristics as well as AQY2 expression through the osmoregulatory Hog1 mitogen-activated protein kinase. Moreover, two pathways known to control morphological developments are involved in regulation of AQY2 expression: the protein kinase A pathway derepresses AQY2 expression through the Sfl1 repressor, and the filamentous growth Kss1 mitogen-activated protein kinase pathway represses AQY2 expression in a Kss1 activity-independent manner. The AQY2 expression pattern resembles in many ways that of MUC1/FLO11, which encodes a cell surface glycoprotein required for morphological developments. Our observations suggest a potential link between aquaporins and cell surface properties, and relate to the proposed role of mammalian aquaporins in tumour cell migration and invasion.
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| 6. |
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| 7. |
- Furukawa, Kentaro, et al.
(författare)
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Fungal fludioxonil sensitivity is diminished by a constitutively active form of the group III histidine kinase
- 2012
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 586:16, s. 2417-2422
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Tidskriftsartikel (refereegranskat)abstract
- The fungicide fludioxonil is used to control plant-pathogenic fungi by causing improper activation of the Hog1-type MAPK. However, the appearance of fludioxonil resistant mutants, mostly caused by mutations in the group III histidine kinases, poses a serious problem. Moreover, such mutations cause also hyperosmotic sensitivity and the underlying mechanism has been elusive for a long time. Using Saccharomyces cerevisiae as an experimental host, we show that those phenotypes are conferred by a constitutively active form of the group III histidine kinase. Our results explain the different reasons for fludioxonil resistance conferred by its deletion and missense mutation.
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| 8. |
- Furukawa, Kentaro, et al.
(författare)
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Genetic screening for suppressor mutations of the hyper- or non-filamentous growth of the yeast osmotic signalling mutants
- 2010
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Ingår i: FEBS JOURNAL. - 1742-464X .- 1742-4658. ; 277:Suppl.1, s. 142-143
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- In certain yeast Saccharomyces cerevisiae strains, diploid cells develop pseudohyphae under nitrogen starvation, while haploid cells produce invasive filaments that penetrate the agar in rich medium. We have recently reported that these morphological developments are strongly inhibited under hyper-osmotic condition through the high-osmolarity glycerol (HOG) response MAPK pathway (Furukawa et al., 2009, Mol Microbiol). Deletion of the HOG1 MAPK gene enhances morphological developments, while expression of active Hog1 inhibits those even in the absence of hyper-osmotic stress. Moreover, it has been reported that HOG1 affects morphology and pathogenicity also in other fungal species. Candida albicans hog1 mutant cells form (pseudo)hyphae and show related transcriptome changes even in the absence of morphogenetic signals. Cryptococcus neoformans hog1 mutant cells enhance production of capsule and melanin, which are crucial virulence factors. Hence, fungal Hog1 orthologues are thought to be central negative regulators of many aspects of morphological developments and virulence, and studies on the underlying mechanisms are relevant for the identification of novel targets for antifungal therapy. In order to more deeply understand the inhibitory role of Hog1 in morphological developments, the proteins or signalling pathways affected by Hog1 need to be identified. In this study, we attempted to screen suppressor mutations of the hyper- or non-filamentous growth which are caused by HOG1 deletion or active Hog1, respectively. On the basis of the screening results, the mechanism by which Hog1 negatively regulates morphological developments will be discussed.
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| 9. |
- Furukawa, Kentaro
(författare)
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Potential pitfalls of gene knockout
- 2013
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Ingår i: Seibutsu-kogaku Kaishi (= Journal of Biotechnology). ; 91:5
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Recension (övrigt vetenskapligt/konstnärligt)
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| 10. |
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| 11. |
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| 12. |
- Furukawa, Kentaro, et al.
(författare)
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Synthetic biology: lessons from engineering yeast MAPK signalling pathways.
- 2013
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 88:1, s. 5-19
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Tidskriftsartikel (refereegranskat)abstract
- All living cells respond to external stimuli and execute specific physiological responses through signal transduction pathways. Understanding the mechanisms controlling signalling pathways is important for diagnosing and treating diseases and for reprogramming cells with desired functions. Although many of the signalling components in the budding yeast Saccharomyces cerevisiae have been identified by genetic studies, many features concerning the dynamic control of pathway activity, cross-talk, cell-to-cell variability or robustness against perturbation are still incompletely understood. Comparing the behaviour of engineered and natural signalling pathways offers insight complementary to that achievable with standard genetic and molecular studies. Here, we review studies that aim at a deeper understanding of signalling design principles and generation of novel signalling properties by engineering the yeast mitogen-activated protein kinase (MAPK) pathways. The underlying approaches can be applied to other organisms including mammalian cells and offer opportunities for building synthetic pathways and functionalities useful in medicine and biotechnology.
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| 13. |
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| 14. |
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| 15. |
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| 16. |
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| 17. |
- Kaur, H., et al.
(författare)
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Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p
- 2014
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:29, s. 20245-20258
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Tidskriftsartikel (refereegranskat)abstract
- Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1-5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1-5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension similar to 365 angstrom. Placement of a sequence similarity based model of the HAMP1-5 protein inside experimental data-based models showed how two chains of HAMP1-5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1-5 structure mediate its function.
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| 18. |
- Padamsee, Mahajabeen, et al.
(författare)
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The genome of the xerotolerant mold Wallemia sebi reveals adaptations to osmotic stress and suggests cryptic sexual reproduction
- 2012
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Ingår i: Fungal Genetics and Biology. - : Elsevier BV. - 1087-1845 .- 1096-0937. ; 49:3, s. 217-226
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Tidskriftsartikel (refereegranskat)abstract
- Wallemia (Wallemiales, Wallemiomycetes) is a genus of xerophilic Fungi of uncertain phylogenetic position within Basidiomycota. Most commonly found as food contaminants, species of Wallemia have also been isolated from hypersaline environments. The ability to tolerate environments with reduced water activity is rare in Basidiomycota. We sequenced the genome of W. sebi in order to understand its adaptations for surviving in osmotically challenging environments, and we performed phylogenomic and ultrastructural analyses to address its systematic placement and reproductive biology. W. sebi has a compact genome (9.8Mb), with few repeats and the largest fraction of genes with functional domains compared with other Basidiomycota. We applied several approaches to searching for osmotic stress-related proteins. In silico analyses identified 93 putative osmotic stress proteins; homology searches showed the HOG (High Osmolarity Glycerol) pathway to be mostly conserved. Despite the seemingly reduced genome, several gene family expansions and a high number of transporters (549) were found that also provide clues to the ability of W. sebi to colonize harsh environments. Phylogenetic analyses of a 71-protein dataset support the position of Wallemia as the earliest diverging lineage of Agaricomycotina, which is confirmed by septal pore ultrastructure that shows the septal pore apparatus as a variant of the Tremella-type. Mating type gene homologs were identified although we found no evidence of meiosis during conidiogenesis, suggesting there may be aspects of the life cycle of W. sebi that remain cryptic.
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| 19. |
- Regot, Sergi, et al.
(författare)
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Distributed biological computation with multicellular engineered networks.
- 2011
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 469:7329, s. 207-11
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Tidskriftsartikel (refereegranskat)abstract
- Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.
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