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Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

Frey S, Reschka EJ, Pöggeler S - PLoS ONE (2015)

Bottom Line: Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes.The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP).In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University Göttingen, Göttingen, Germany.

ABSTRACT
The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

No MeSH data available.


Related in: MedlinePlus

Localization of SmKIN3-eGFP and SmKIN24-eGFP in S. macrospora.SmKIN3-eGFP and SmKIN24-eGFP localize to septa. For visualization of cell walls and septa, hyphae were co-stained with Calcofluor white. DIC = differential interference contrast microscopy, scale bars as indicated.
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pone.0139163.g007: Localization of SmKIN3-eGFP and SmKIN24-eGFP in S. macrospora.SmKIN3-eGFP and SmKIN24-eGFP localize to septa. For visualization of cell walls and septa, hyphae were co-stained with Calcofluor white. DIC = differential interference contrast microscopy, scale bars as indicated.

Mentions: In order to determine the localization of SmKIN3 and SmKIN24 in vivo, fluorescence microscopy was performed. Since C-terminally tagged versions of SmKIN3 and SmKIN24 did not complement the mutant phenotype, genes encoding N-terminally eGFP-tagged full-length SmKIN3 or SmKIN24 were expressed in the respective S. macrospora deletion strains. These constructs complemented the mutants and revealed that both kinases were localized at the septa (Fig 7). However, SmKIN3 was localized mainly at the outer part of the septum, whereas SmKIN24 was localized at the septal pore (Fig 7), and this was verified by co-staining with Calcofluor white (Fig 7). These findings lead us to quantify septum formation in the ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 strains and their respective complementation strains by staining with Calcofluor white after 18 or 32 h of growth (Fig 8A). Furthermore, we quantified the distances between adjacent septa in wt and deletion mutants after 24 h of growth (Fig 8B). Septa were distributed in a uniform manner in wt with hyphal compartment length between 31–70 μm (Fig 8B). In contrast, ΔSmkin3 exhibited larger distances between adjacent septa (>71 μm), although this effect begun to revert after 24 h of growth (Fig 8A). Strikingly, ΔSmkin24 developed numerous closely-packed septal bundles of abnormal shape, with much smaller hyphal compartments of 0–30 μm (Fig 8B). ΔSmkin3 produced about half of the total number of septa within a distance of 18 mm than wt, while ΔSmkin24 produced 20% more than wt after 24 h of growth. With respect to septation, the double-deletion mutant ΔSmkin3/ΔSmkin24 displayed a similar phenotype to the ΔSmkin24 single-deletion strain. Ectopic integration of the respective wt gene into deletion mutants restored septal formation to wt level (Fig 8A and 8B). The septa of ΔSmkin3 and ΔSmkin24 were checked for functionality under the microscope directly after cutting of the hyphae. Both strains did not differ from wt; shortly after cutting, hyphal compartments of ΔSmkin3 and ΔSmkin24 were plugged by Woronin bodies.


Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

Frey S, Reschka EJ, Pöggeler S - PLoS ONE (2015)

Localization of SmKIN3-eGFP and SmKIN24-eGFP in S. macrospora.SmKIN3-eGFP and SmKIN24-eGFP localize to septa. For visualization of cell walls and septa, hyphae were co-stained with Calcofluor white. DIC = differential interference contrast microscopy, scale bars as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587736&req=5

pone.0139163.g007: Localization of SmKIN3-eGFP and SmKIN24-eGFP in S. macrospora.SmKIN3-eGFP and SmKIN24-eGFP localize to septa. For visualization of cell walls and septa, hyphae were co-stained with Calcofluor white. DIC = differential interference contrast microscopy, scale bars as indicated.
Mentions: In order to determine the localization of SmKIN3 and SmKIN24 in vivo, fluorescence microscopy was performed. Since C-terminally tagged versions of SmKIN3 and SmKIN24 did not complement the mutant phenotype, genes encoding N-terminally eGFP-tagged full-length SmKIN3 or SmKIN24 were expressed in the respective S. macrospora deletion strains. These constructs complemented the mutants and revealed that both kinases were localized at the septa (Fig 7). However, SmKIN3 was localized mainly at the outer part of the septum, whereas SmKIN24 was localized at the septal pore (Fig 7), and this was verified by co-staining with Calcofluor white (Fig 7). These findings lead us to quantify septum formation in the ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 strains and their respective complementation strains by staining with Calcofluor white after 18 or 32 h of growth (Fig 8A). Furthermore, we quantified the distances between adjacent septa in wt and deletion mutants after 24 h of growth (Fig 8B). Septa were distributed in a uniform manner in wt with hyphal compartment length between 31–70 μm (Fig 8B). In contrast, ΔSmkin3 exhibited larger distances between adjacent septa (>71 μm), although this effect begun to revert after 24 h of growth (Fig 8A). Strikingly, ΔSmkin24 developed numerous closely-packed septal bundles of abnormal shape, with much smaller hyphal compartments of 0–30 μm (Fig 8B). ΔSmkin3 produced about half of the total number of septa within a distance of 18 mm than wt, while ΔSmkin24 produced 20% more than wt after 24 h of growth. With respect to septation, the double-deletion mutant ΔSmkin3/ΔSmkin24 displayed a similar phenotype to the ΔSmkin24 single-deletion strain. Ectopic integration of the respective wt gene into deletion mutants restored septal formation to wt level (Fig 8A and 8B). The septa of ΔSmkin3 and ΔSmkin24 were checked for functionality under the microscope directly after cutting of the hyphae. Both strains did not differ from wt; shortly after cutting, hyphal compartments of ΔSmkin3 and ΔSmkin24 were plugged by Woronin bodies.

Bottom Line: Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes.The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP).In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University Göttingen, Göttingen, Germany.

ABSTRACT
The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

No MeSH data available.


Related in: MedlinePlus