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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

A joint reduction of Cdc15 and Pxl1 functions induces severe CAR sliding and causes septum material deposition along the plasma membrane.(A) Fluorescence micrographs of Pnmt81-pxl1+ cells stained with CW and carrying hypomorphic Cdc15-GFP. Early log-phase cells growing at 28°C in EMM+S (time 0 h) were shifted to EMM+S+T (time 48 h +T) to repress pxl1+. Arrow: open septa without a Cdc15 ring and with lower CW staining. (B) Percentage of septa in cdc15-GFP (n = 840), Pnmt81-pxl1+ (at least 878 cells were quantified for each time) and cdc15-GFP Pnmt81-pxl1+ (at least 383 cells were quantified for each time). (C) Percentage of open septa without a stable Cdc15 ring (left) and open septa with a weak CW staining (right) in the strain Pnmt81-pxl1+cdc15-GFP at the indicated times of pxl1+ repression (at least 70 open septa were quantified for each time). (D) Histogram showing the indicated intervals of positions of the septa measured as the percent of septum offset from the cell center: white bars, cdc15-GFP cells (n = 42); light grey bars, Pnmt81-pxl1+ cells (time 0 h, pxl1+ induced) (n = 51); middle grey bars, Pnmt81-pxl1+ cells (time 48 h + T, pxl1+ repressed) (n = 60); dark grey bars, Pnmt81-pxl1+cdc15-GFP cells (time 0 h, pxl1+ induced) (n = 52); and black bars, Pnmt81-pxl1+cdc15-GFP cells (time 48 h + T, pxl1+ repressed) (n = 66). The percentages of septum offset were measured from CW staining images and calculated as is described for the Fig 1B. (E) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP. Cells were grown at 28°C in EMM+S+T for 48 h (pxl1+ repressed) and imaged. Arrow: Ring sliding and longitudinal synthesis of septum wall material along the cell cortex. For a better observation of longitudinal septum wall material synthesis, a high contrast has been applied to the CW images. (F) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP and RFP-Bgs4 or Ags1-RFP. Cells were grown at 28°C in EMM+S in the absence of T (left, time 0 h, pxl1+ induced) or shifted to EMM+S+T (right, time 48 h + T, pxl1+ repressed) and imaged. Dashed line: reference for the ring position. Scale bars, 5 μm.
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pgen.1005358.g007: A joint reduction of Cdc15 and Pxl1 functions induces severe CAR sliding and causes septum material deposition along the plasma membrane.(A) Fluorescence micrographs of Pnmt81-pxl1+ cells stained with CW and carrying hypomorphic Cdc15-GFP. Early log-phase cells growing at 28°C in EMM+S (time 0 h) were shifted to EMM+S+T (time 48 h +T) to repress pxl1+. Arrow: open septa without a Cdc15 ring and with lower CW staining. (B) Percentage of septa in cdc15-GFP (n = 840), Pnmt81-pxl1+ (at least 878 cells were quantified for each time) and cdc15-GFP Pnmt81-pxl1+ (at least 383 cells were quantified for each time). (C) Percentage of open septa without a stable Cdc15 ring (left) and open septa with a weak CW staining (right) in the strain Pnmt81-pxl1+cdc15-GFP at the indicated times of pxl1+ repression (at least 70 open septa were quantified for each time). (D) Histogram showing the indicated intervals of positions of the septa measured as the percent of septum offset from the cell center: white bars, cdc15-GFP cells (n = 42); light grey bars, Pnmt81-pxl1+ cells (time 0 h, pxl1+ induced) (n = 51); middle grey bars, Pnmt81-pxl1+ cells (time 48 h + T, pxl1+ repressed) (n = 60); dark grey bars, Pnmt81-pxl1+cdc15-GFP cells (time 0 h, pxl1+ induced) (n = 52); and black bars, Pnmt81-pxl1+cdc15-GFP cells (time 48 h + T, pxl1+ repressed) (n = 66). The percentages of septum offset were measured from CW staining images and calculated as is described for the Fig 1B. (E) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP. Cells were grown at 28°C in EMM+S+T for 48 h (pxl1+ repressed) and imaged. Arrow: Ring sliding and longitudinal synthesis of septum wall material along the cell cortex. For a better observation of longitudinal septum wall material synthesis, a high contrast has been applied to the CW images. (F) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP and RFP-Bgs4 or Ags1-RFP. Cells were grown at 28°C in EMM+S in the absence of T (left, time 0 h, pxl1+ induced) or shifted to EMM+S+T (right, time 48 h + T, pxl1+ repressed) and imaged. Dashed line: reference for the ring position. Scale bars, 5 μm.

Mentions: To further analyze the relationship between Pxl1 and Cdc15 we tried to construct a pxl1Δ cdc15ΔSH3 double mutant strain but it was not viable [34], supporting that cdc15ΔSH3 cytokinetic defects are not just caused by the lack of Pxl1, and that Cdc15 and Pxl1 might have additional functions that together are essential. A Pnmt41-pxl1+cdc15ΔSH3 mutant strain was not viable either, even in the absence of thiamine when the nmt41 promoter is active. Probably pxl1+ expression level is critical in cdc15ΔSH3 mutant cells and Pnmt41-pxl1+ expression is not regulated by the transcription factor Ace2 during septation as it is the expression of the endogenous pxl1+ [35]. GFP-tagging of Cdc15 at the C-terminus makes the protein partially nonfunctional, generating a hypomorphic allele also referred as cdc15-gc1 [36]. Indeed, deletion of Pxl1 is lethal in cdc15-GFP cells [26]. Therefore, we made a Pnmt81-pxl1+cdc15-GFP double mutant strain that was viable in the absence of thiamine (Fig 7A). Repression of pxl1+ in these cells induced the accumulation of septa, and 30% of the cells became multiseptated after 48 h of pxl1+ repression (Fig 7B). We also observed a considerable increase (5x) of open septa without a Cdc15-GFP ring and septa with a weak CW staining (60% and 30% after 48 h of pxl1+ repression respectively; Fig 7A, arrow and 7C). In addition, pxl1+ repression induced a significant increase in the number of cdc15-GFP cells with misplaced septa (Fig 7D), probably as a consequence of severe CAR sliding from the middle of the cell. This phenotype was also observed by time-lapse microscopy. While the CAR sliding in pxl1Δ cells stopped with septum synthesis, in Pnmt81-pxl1+cdc15-GFP cells some CARs continued sliding even after the onset of septum synthesis, causing a longitudinal deposition along the plasma membrane of linear β-glucan as detected by CW staining until septum ingression started (Fig 7E, arrow).


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)

A joint reduction of Cdc15 and Pxl1 functions induces severe CAR sliding and causes septum material deposition along the plasma membrane.(A) Fluorescence micrographs of Pnmt81-pxl1+ cells stained with CW and carrying hypomorphic Cdc15-GFP. Early log-phase cells growing at 28°C in EMM+S (time 0 h) were shifted to EMM+S+T (time 48 h +T) to repress pxl1+. Arrow: open septa without a Cdc15 ring and with lower CW staining. (B) Percentage of septa in cdc15-GFP (n = 840), Pnmt81-pxl1+ (at least 878 cells were quantified for each time) and cdc15-GFP Pnmt81-pxl1+ (at least 383 cells were quantified for each time). (C) Percentage of open septa without a stable Cdc15 ring (left) and open septa with a weak CW staining (right) in the strain Pnmt81-pxl1+cdc15-GFP at the indicated times of pxl1+ repression (at least 70 open septa were quantified for each time). (D) Histogram showing the indicated intervals of positions of the septa measured as the percent of septum offset from the cell center: white bars, cdc15-GFP cells (n = 42); light grey bars, Pnmt81-pxl1+ cells (time 0 h, pxl1+ induced) (n = 51); middle grey bars, Pnmt81-pxl1+ cells (time 48 h + T, pxl1+ repressed) (n = 60); dark grey bars, Pnmt81-pxl1+cdc15-GFP cells (time 0 h, pxl1+ induced) (n = 52); and black bars, Pnmt81-pxl1+cdc15-GFP cells (time 48 h + T, pxl1+ repressed) (n = 66). The percentages of septum offset were measured from CW staining images and calculated as is described for the Fig 1B. (E) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP. Cells were grown at 28°C in EMM+S+T for 48 h (pxl1+ repressed) and imaged. Arrow: Ring sliding and longitudinal synthesis of septum wall material along the cell cortex. For a better observation of longitudinal septum wall material synthesis, a high contrast has been applied to the CW images. (F) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP and RFP-Bgs4 or Ags1-RFP. Cells were grown at 28°C in EMM+S in the absence of T (left, time 0 h, pxl1+ induced) or shifted to EMM+S+T (right, time 48 h + T, pxl1+ repressed) and imaged. Dashed line: reference for the ring position. Scale bars, 5 μm.
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pgen.1005358.g007: A joint reduction of Cdc15 and Pxl1 functions induces severe CAR sliding and causes septum material deposition along the plasma membrane.(A) Fluorescence micrographs of Pnmt81-pxl1+ cells stained with CW and carrying hypomorphic Cdc15-GFP. Early log-phase cells growing at 28°C in EMM+S (time 0 h) were shifted to EMM+S+T (time 48 h +T) to repress pxl1+. Arrow: open septa without a Cdc15 ring and with lower CW staining. (B) Percentage of septa in cdc15-GFP (n = 840), Pnmt81-pxl1+ (at least 878 cells were quantified for each time) and cdc15-GFP Pnmt81-pxl1+ (at least 383 cells were quantified for each time). (C) Percentage of open septa without a stable Cdc15 ring (left) and open septa with a weak CW staining (right) in the strain Pnmt81-pxl1+cdc15-GFP at the indicated times of pxl1+ repression (at least 70 open septa were quantified for each time). (D) Histogram showing the indicated intervals of positions of the septa measured as the percent of septum offset from the cell center: white bars, cdc15-GFP cells (n = 42); light grey bars, Pnmt81-pxl1+ cells (time 0 h, pxl1+ induced) (n = 51); middle grey bars, Pnmt81-pxl1+ cells (time 48 h + T, pxl1+ repressed) (n = 60); dark grey bars, Pnmt81-pxl1+cdc15-GFP cells (time 0 h, pxl1+ induced) (n = 52); and black bars, Pnmt81-pxl1+cdc15-GFP cells (time 48 h + T, pxl1+ repressed) (n = 66). The percentages of septum offset were measured from CW staining images and calculated as is described for the Fig 1B. (E) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP. Cells were grown at 28°C in EMM+S+T for 48 h (pxl1+ repressed) and imaged. Arrow: Ring sliding and longitudinal synthesis of septum wall material along the cell cortex. For a better observation of longitudinal septum wall material synthesis, a high contrast has been applied to the CW images. (F) Time series of fluorescence micrographs (one middle z slide, 3 min intervals) of Pnmt81-pxl1+ cells carrying hypomorphic Cdc15-GFP and RFP-Bgs4 or Ags1-RFP. Cells were grown at 28°C in EMM+S in the absence of T (left, time 0 h, pxl1+ induced) or shifted to EMM+S+T (right, time 48 h + T, pxl1+ repressed) and imaged. Dashed line: reference for the ring position. Scale bars, 5 μm.
Mentions: To further analyze the relationship between Pxl1 and Cdc15 we tried to construct a pxl1Δ cdc15ΔSH3 double mutant strain but it was not viable [34], supporting that cdc15ΔSH3 cytokinetic defects are not just caused by the lack of Pxl1, and that Cdc15 and Pxl1 might have additional functions that together are essential. A Pnmt41-pxl1+cdc15ΔSH3 mutant strain was not viable either, even in the absence of thiamine when the nmt41 promoter is active. Probably pxl1+ expression level is critical in cdc15ΔSH3 mutant cells and Pnmt41-pxl1+ expression is not regulated by the transcription factor Ace2 during septation as it is the expression of the endogenous pxl1+ [35]. GFP-tagging of Cdc15 at the C-terminus makes the protein partially nonfunctional, generating a hypomorphic allele also referred as cdc15-gc1 [36]. Indeed, deletion of Pxl1 is lethal in cdc15-GFP cells [26]. Therefore, we made a Pnmt81-pxl1+cdc15-GFP double mutant strain that was viable in the absence of thiamine (Fig 7A). Repression of pxl1+ in these cells induced the accumulation of septa, and 30% of the cells became multiseptated after 48 h of pxl1+ repression (Fig 7B). We also observed a considerable increase (5x) of open septa without a Cdc15-GFP ring and septa with a weak CW staining (60% and 30% after 48 h of pxl1+ repression respectively; Fig 7A, arrow and 7C). In addition, pxl1+ repression induced a significant increase in the number of cdc15-GFP cells with misplaced septa (Fig 7D), probably as a consequence of severe CAR sliding from the middle of the cell. This phenotype was also observed by time-lapse microscopy. While the CAR sliding in pxl1Δ cells stopped with septum synthesis, in Pnmt81-pxl1+cdc15-GFP cells some CARs continued sliding even after the onset of septum synthesis, causing a longitudinal deposition along the plasma membrane of linear β-glucan as detected by CW staining until septum ingression started (Fig 7E, arrow).

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