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Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast.

G Cortés JC, Pujol N, Sato M, Pinar M, Ramos M, Moreno B, Osumi M, Ribas JC, Pérez P - PLoS Genet. (2015)

Bottom Line: In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1.On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins.In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC) / Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.

No MeSH data available.


Related in: MedlinePlus

Cooperation between Bgs1 and the SH3 Domain of Cdc15 is essential for CAR maintenance and septum formation.(A) Fluorescence micrographs of cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Cdc15ΔSH3-GFP. Cells were grown to early log-phase in EMM+S (time 0 h), shifted to the same medium plus thiamine, EMM+S+T (times 15, 24 and 40 h, bgs1+ repressed) and imaged at the indicated times. Arrow: Cell with an open septum without the ring of Cdc15. Arrowhead: Cell with a disorganized ring of Cdc15. (B) Histograms showing the indicated percentages of septa and Cdc15 structures in the strains Pnmt81-bgs1+ (n = 150 cells or hypha units were quantified for each time) and cdc15ΔSH3-GFP Pnmt81-bgs1+ (n = 100 cells or hypha units were quantified for each time). Note that Pnmt81-bgs1+ strains after 24 h of bgs1+ repression appear as hypha units, each being equivalent to several single cells. (C) Fluorescence micrographs of Pnmt81-bgs1+ and cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Ags1-RFP or RFP-Bgs4 after the indicated times of repression of bgs1+. (D) Fluorescence micrographs of Pnmt81-bgs1+, Pnmt81-bgs1+pxl1Δ and Pnmt81-bgs1+cdc15ΔSH3 cells carrying the hypomorphic version of Bgs1 GFP-Cps1-191, and wild-type cells carrying GFP-Bgs1 used as Bgs1 localization control. Since cps1-191 allele is lethal in pxl1Δ and cdc15ΔSH3 backgrounds, strains are maintained alive with an inducible version of bgs1+ (Pnmt81-bgs1+). Cells were grown at 25°C (permissive temperature for cps1-191 cells) in EMM+S (time 0 h, bgs1+ induced) and shifted to EMM+S+T (time 24 h +T, bgs1+ repressed) to visualize cell phenotype and GFP-Cps1-191 localization. Scale bars, 5 μm.
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pgen.1005358.g006: Cooperation between Bgs1 and the SH3 Domain of Cdc15 is essential for CAR maintenance and septum formation.(A) Fluorescence micrographs of cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Cdc15ΔSH3-GFP. Cells were grown to early log-phase in EMM+S (time 0 h), shifted to the same medium plus thiamine, EMM+S+T (times 15, 24 and 40 h, bgs1+ repressed) and imaged at the indicated times. Arrow: Cell with an open septum without the ring of Cdc15. Arrowhead: Cell with a disorganized ring of Cdc15. (B) Histograms showing the indicated percentages of septa and Cdc15 structures in the strains Pnmt81-bgs1+ (n = 150 cells or hypha units were quantified for each time) and cdc15ΔSH3-GFP Pnmt81-bgs1+ (n = 100 cells or hypha units were quantified for each time). Note that Pnmt81-bgs1+ strains after 24 h of bgs1+ repression appear as hypha units, each being equivalent to several single cells. (C) Fluorescence micrographs of Pnmt81-bgs1+ and cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Ags1-RFP or RFP-Bgs4 after the indicated times of repression of bgs1+. (D) Fluorescence micrographs of Pnmt81-bgs1+, Pnmt81-bgs1+pxl1Δ and Pnmt81-bgs1+cdc15ΔSH3 cells carrying the hypomorphic version of Bgs1 GFP-Cps1-191, and wild-type cells carrying GFP-Bgs1 used as Bgs1 localization control. Since cps1-191 allele is lethal in pxl1Δ and cdc15ΔSH3 backgrounds, strains are maintained alive with an inducible version of bgs1+ (Pnmt81-bgs1+). Cells were grown at 25°C (permissive temperature for cps1-191 cells) in EMM+S (time 0 h, bgs1+ induced) and shifted to EMM+S+T (time 24 h +T, bgs1+ repressed) to visualize cell phenotype and GFP-Cps1-191 localization. Scale bars, 5 μm.

Mentions: As described above, Pxl1 cooperates with Bgs1 in the formation of the septum; therefore we investigated if Cdc15, through the SH3 domain, also cooperates with Bgs1 in septum formation. We made and analyzed the Pnmt81-bgs1+cdc15ΔSH3-GFP strain grown in EMM+S+T during different times (Fig 6A and 6B). The absence of the SH3 domain of Cdc15 mimicked the phenotype observed in the absence of Pxl1 during the repression of bgs1+ (see Fig 3). An increase of cells with open septa without a CAR (analyzed as Cdc15ΔSH3-GFP ring) was observed during growth in the presence of thiamine (Fig 6A, arrow, and 6B), and after 48 h cells showed wide cell wall invaginations and no septum synthesis as detected by CW staining. The CAR of these cells was disorganized and did not constrict (Fig 6A and 6B). The localization of Ags1 and Bgs4 synthases was analyzed in Pnmt81-bgs1+cdc15ΔSH3-GFP cells carrying Ags1-RFP and RFP-Bgs4 upon bgs1+ repression. As described in pxl1Δ Pnmt81-bgs1+ cells, after 24 h with thiamine, both synthases appeared in the cytoplasm and extended along the plasma membrane of Pnmt81-bgs1+cdc15ΔSH3-GFP cells (Fig 6C). Collectively, these results suggest that Cdc15, through the SH3 domain, and likely through Pxl1, cooperates with Bgs1 to confer stability to the CAR and to form the septum during cytokinesis.


Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast.

G Cortés JC, Pujol N, Sato M, Pinar M, Ramos M, Moreno B, Osumi M, Ribas JC, Pérez P - PLoS Genet. (2015)

Cooperation between Bgs1 and the SH3 Domain of Cdc15 is essential for CAR maintenance and septum formation.(A) Fluorescence micrographs of cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Cdc15ΔSH3-GFP. Cells were grown to early log-phase in EMM+S (time 0 h), shifted to the same medium plus thiamine, EMM+S+T (times 15, 24 and 40 h, bgs1+ repressed) and imaged at the indicated times. Arrow: Cell with an open septum without the ring of Cdc15. Arrowhead: Cell with a disorganized ring of Cdc15. (B) Histograms showing the indicated percentages of septa and Cdc15 structures in the strains Pnmt81-bgs1+ (n = 150 cells or hypha units were quantified for each time) and cdc15ΔSH3-GFP Pnmt81-bgs1+ (n = 100 cells or hypha units were quantified for each time). Note that Pnmt81-bgs1+ strains after 24 h of bgs1+ repression appear as hypha units, each being equivalent to several single cells. (C) Fluorescence micrographs of Pnmt81-bgs1+ and cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Ags1-RFP or RFP-Bgs4 after the indicated times of repression of bgs1+. (D) Fluorescence micrographs of Pnmt81-bgs1+, Pnmt81-bgs1+pxl1Δ and Pnmt81-bgs1+cdc15ΔSH3 cells carrying the hypomorphic version of Bgs1 GFP-Cps1-191, and wild-type cells carrying GFP-Bgs1 used as Bgs1 localization control. Since cps1-191 allele is lethal in pxl1Δ and cdc15ΔSH3 backgrounds, strains are maintained alive with an inducible version of bgs1+ (Pnmt81-bgs1+). Cells were grown at 25°C (permissive temperature for cps1-191 cells) in EMM+S (time 0 h, bgs1+ induced) and shifted to EMM+S+T (time 24 h +T, bgs1+ repressed) to visualize cell phenotype and GFP-Cps1-191 localization. Scale bars, 5 μm.
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pgen.1005358.g006: Cooperation between Bgs1 and the SH3 Domain of Cdc15 is essential for CAR maintenance and septum formation.(A) Fluorescence micrographs of cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Cdc15ΔSH3-GFP. Cells were grown to early log-phase in EMM+S (time 0 h), shifted to the same medium plus thiamine, EMM+S+T (times 15, 24 and 40 h, bgs1+ repressed) and imaged at the indicated times. Arrow: Cell with an open septum without the ring of Cdc15. Arrowhead: Cell with a disorganized ring of Cdc15. (B) Histograms showing the indicated percentages of septa and Cdc15 structures in the strains Pnmt81-bgs1+ (n = 150 cells or hypha units were quantified for each time) and cdc15ΔSH3-GFP Pnmt81-bgs1+ (n = 100 cells or hypha units were quantified for each time). Note that Pnmt81-bgs1+ strains after 24 h of bgs1+ repression appear as hypha units, each being equivalent to several single cells. (C) Fluorescence micrographs of Pnmt81-bgs1+ and cdc15ΔSH3-GFP Pnmt81-bgs1+ cells stained with CW and carrying Ags1-RFP or RFP-Bgs4 after the indicated times of repression of bgs1+. (D) Fluorescence micrographs of Pnmt81-bgs1+, Pnmt81-bgs1+pxl1Δ and Pnmt81-bgs1+cdc15ΔSH3 cells carrying the hypomorphic version of Bgs1 GFP-Cps1-191, and wild-type cells carrying GFP-Bgs1 used as Bgs1 localization control. Since cps1-191 allele is lethal in pxl1Δ and cdc15ΔSH3 backgrounds, strains are maintained alive with an inducible version of bgs1+ (Pnmt81-bgs1+). Cells were grown at 25°C (permissive temperature for cps1-191 cells) in EMM+S (time 0 h, bgs1+ induced) and shifted to EMM+S+T (time 24 h +T, bgs1+ repressed) to visualize cell phenotype and GFP-Cps1-191 localization. Scale bars, 5 μm.
Mentions: As described above, Pxl1 cooperates with Bgs1 in the formation of the septum; therefore we investigated if Cdc15, through the SH3 domain, also cooperates with Bgs1 in septum formation. We made and analyzed the Pnmt81-bgs1+cdc15ΔSH3-GFP strain grown in EMM+S+T during different times (Fig 6A and 6B). The absence of the SH3 domain of Cdc15 mimicked the phenotype observed in the absence of Pxl1 during the repression of bgs1+ (see Fig 3). An increase of cells with open septa without a CAR (analyzed as Cdc15ΔSH3-GFP ring) was observed during growth in the presence of thiamine (Fig 6A, arrow, and 6B), and after 48 h cells showed wide cell wall invaginations and no septum synthesis as detected by CW staining. The CAR of these cells was disorganized and did not constrict (Fig 6A and 6B). The localization of Ags1 and Bgs4 synthases was analyzed in Pnmt81-bgs1+cdc15ΔSH3-GFP cells carrying Ags1-RFP and RFP-Bgs4 upon bgs1+ repression. As described in pxl1Δ Pnmt81-bgs1+ cells, after 24 h with thiamine, both synthases appeared in the cytoplasm and extended along the plasma membrane of Pnmt81-bgs1+cdc15ΔSH3-GFP cells (Fig 6C). Collectively, these results suggest that Cdc15, through the SH3 domain, and likely through Pxl1, cooperates with Bgs1 to confer stability to the CAR and to form the septum during cytokinesis.

Bottom Line: In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1.On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins.In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC) / Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.

No MeSH data available.


Related in: MedlinePlus