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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

Correct Bgs1 function is important for CAR Integrity and maintenance of Ags1 and Bgs4 in the septum membrane.(A) Fluorescence micrographs of CW stained cps1-191 cells carrying Rlc1-RFP (ring) and GFP-Psy1 (plasma membrane). cps1-191 cells growing in YES+S (S = 1.3M sorbitol) at 25°C were shifted to 37°C for 6 h and imaged. Medial z slide (upper panels) and maximum-intensity projections of 28 z slides at 0.3 μm intervals (lower panels) of the same cells are shown. (B) Three-dimensional reconstructions (28 z slides at 0.3 μm intervals) of cells grown as in A. Left, equatorial plane; right, longitudinal plane. (C-E) Time-lapses (one medial z slide, 5 min intervals, although in some cases only is shown 10 min interval as indicated) of CW-stained wild-type (C) and cps1-191 (D and E) cells carrying Rlc1-RFP and Ags1-GFP or GFP-Bgs4. Wild type and cps1-191 cells growing in YES+S at 25°C were shifted to 37°C for 1.5 h and filmed to capture ring formation, and Ags1 or Bgs4 arrival to the division site. Two cps1-191 cells showing a sliding ring which does not constrict (left in D and E) and two cps1-191 cells with a slow ring constriction and septum deposition (right in D and E) are shown. Arrowheads indicate synthesis of the septum wall in the absence of ring constriction. A dashed line is drawn as reference for the ring position. (F and G) Fluorescence micrographs of CW-stained wild-type (F) and cps1-191 (G) cells carrying Rlc1-RFP, and GFP-Bgs4 or Ags1-GFP. Cells were grown as in A. Maximum-intensity projections of 28 z slides at 0.3 μm intervals (upper panels) and the corresponding three-dimensional reconstructions of the equatorial plane including the septum area (lower panels) of the complete cell are shown. In all the three-dimensional reconstructions, from the 28 slides acquired it was only selected those slides that cover the entire cell diameter. Scale bars, 5 μm.
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pgen.1005358.g002: Correct Bgs1 function is important for CAR Integrity and maintenance of Ags1 and Bgs4 in the septum membrane.(A) Fluorescence micrographs of CW stained cps1-191 cells carrying Rlc1-RFP (ring) and GFP-Psy1 (plasma membrane). cps1-191 cells growing in YES+S (S = 1.3M sorbitol) at 25°C were shifted to 37°C for 6 h and imaged. Medial z slide (upper panels) and maximum-intensity projections of 28 z slides at 0.3 μm intervals (lower panels) of the same cells are shown. (B) Three-dimensional reconstructions (28 z slides at 0.3 μm intervals) of cells grown as in A. Left, equatorial plane; right, longitudinal plane. (C-E) Time-lapses (one medial z slide, 5 min intervals, although in some cases only is shown 10 min interval as indicated) of CW-stained wild-type (C) and cps1-191 (D and E) cells carrying Rlc1-RFP and Ags1-GFP or GFP-Bgs4. Wild type and cps1-191 cells growing in YES+S at 25°C were shifted to 37°C for 1.5 h and filmed to capture ring formation, and Ags1 or Bgs4 arrival to the division site. Two cps1-191 cells showing a sliding ring which does not constrict (left in D and E) and two cps1-191 cells with a slow ring constriction and septum deposition (right in D and E) are shown. Arrowheads indicate synthesis of the septum wall in the absence of ring constriction. A dashed line is drawn as reference for the ring position. (F and G) Fluorescence micrographs of CW-stained wild-type (F) and cps1-191 (G) cells carrying Rlc1-RFP, and GFP-Bgs4 or Ags1-GFP. Cells were grown as in A. Maximum-intensity projections of 28 z slides at 0.3 μm intervals (upper panels) and the corresponding three-dimensional reconstructions of the equatorial plane including the septum area (lower panels) of the complete cell are shown. In all the three-dimensional reconstructions, from the 28 slides acquired it was only selected those slides that cover the entire cell diameter. Scale bars, 5 μm.

Mentions: Previous work identified a synthetic lethal interaction between pxl1Δ and different bgs1/cps1 thermosensitive mutants [26], suggesting that Pxl1 and Bgs1 collaborate in an essential process. To test this hypothesis, we first analyzed the septation defects of cells carrying the bgs1 thermosensitive allele cps1-191. These cells were grown at the restrictive temperature (37°C) in the presence of 1.3M sorbitol (S) to allow the deposition of the septum (Fig 2A and 2B). In cells carrying Rlc1-RFP and GFP-Psy1 (a syntaxin homolog that is a marker for the plasma membrane [29]) the processes of membrane invagination and septum formation were accompanied by a disorganized ring which appeared fragmented, with Rlc1 strands connected to the ring. These CAR defects were similar to those of pxl1Δ cells [25,26]. Therefore, a defective Bgs1 function affects the CAR position in the cell middle as was already described [20], and also affects the ring integrity during septum ingression. Because other glucan synthases, such as Ags1 and Bgs4, must collaborate in the formation of the aberrant septum structures observed in cps1-191 cells, we analyzed the localization of these synthases, before and during septation, in wild type and cps1-191 cells carrying Rlc1-RFP grown at the restrictive temperature for 1.5 h (Fig 2C, 2D, and 2E). At this time point, cps1-191 cells made new CARs that were maintained for a long time. Some cells formed rudimentary or partial depositions of CW-stained septa without a proper CAR constriction (Fig 2D and 2E, arrowheads). Time-lapse images of cps1-191 rlc1+-RFP cells carrying Ags1-GFP or GFP-Bgs4 showed that both synthases localized to the division area as in wild type cells (Fig 2C, 2D, and 2E). Ags1 concentrated and moved with the ring along the plasma membrane, and in cells with septum deposition it remained concentrated at the septum area (Fig 2D). In agreement with previous observations [12], Bgs4 did not concentrate in the cell membrane of cells without septum deposition (Fig 2E, left) but did appear concentrated in cells with a partial septum (Fig 2E, right). After longer times (6 h) at the restrictive temperature, both synthases spread along the septum membrane and were also detected in cytoplasmic vesicles. They did not form a homogeneously filled disk-like surface as they do in wild type cells, but an irregularly filled disk indistinguishable from the numerous cytoplasmic vesicles located in the middle (Fig 2F and 2G). Therefore, Bgs1 function is required for ring integrity, for the correct synthesis of the PS, and for the proper localization of Ags1 and Bgs4 as a homogeneous disk structure in the membrane during septum ingression.


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)

Correct Bgs1 function is important for CAR Integrity and maintenance of Ags1 and Bgs4 in the septum membrane.(A) Fluorescence micrographs of CW stained cps1-191 cells carrying Rlc1-RFP (ring) and GFP-Psy1 (plasma membrane). cps1-191 cells growing in YES+S (S = 1.3M sorbitol) at 25°C were shifted to 37°C for 6 h and imaged. Medial z slide (upper panels) and maximum-intensity projections of 28 z slides at 0.3 μm intervals (lower panels) of the same cells are shown. (B) Three-dimensional reconstructions (28 z slides at 0.3 μm intervals) of cells grown as in A. Left, equatorial plane; right, longitudinal plane. (C-E) Time-lapses (one medial z slide, 5 min intervals, although in some cases only is shown 10 min interval as indicated) of CW-stained wild-type (C) and cps1-191 (D and E) cells carrying Rlc1-RFP and Ags1-GFP or GFP-Bgs4. Wild type and cps1-191 cells growing in YES+S at 25°C were shifted to 37°C for 1.5 h and filmed to capture ring formation, and Ags1 or Bgs4 arrival to the division site. Two cps1-191 cells showing a sliding ring which does not constrict (left in D and E) and two cps1-191 cells with a slow ring constriction and septum deposition (right in D and E) are shown. Arrowheads indicate synthesis of the septum wall in the absence of ring constriction. A dashed line is drawn as reference for the ring position. (F and G) Fluorescence micrographs of CW-stained wild-type (F) and cps1-191 (G) cells carrying Rlc1-RFP, and GFP-Bgs4 or Ags1-GFP. Cells were grown as in A. Maximum-intensity projections of 28 z slides at 0.3 μm intervals (upper panels) and the corresponding three-dimensional reconstructions of the equatorial plane including the septum area (lower panels) of the complete cell are shown. In all the three-dimensional reconstructions, from the 28 slides acquired it was only selected those slides that cover the entire cell diameter. Scale bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4489101&req=5

pgen.1005358.g002: Correct Bgs1 function is important for CAR Integrity and maintenance of Ags1 and Bgs4 in the septum membrane.(A) Fluorescence micrographs of CW stained cps1-191 cells carrying Rlc1-RFP (ring) and GFP-Psy1 (plasma membrane). cps1-191 cells growing in YES+S (S = 1.3M sorbitol) at 25°C were shifted to 37°C for 6 h and imaged. Medial z slide (upper panels) and maximum-intensity projections of 28 z slides at 0.3 μm intervals (lower panels) of the same cells are shown. (B) Three-dimensional reconstructions (28 z slides at 0.3 μm intervals) of cells grown as in A. Left, equatorial plane; right, longitudinal plane. (C-E) Time-lapses (one medial z slide, 5 min intervals, although in some cases only is shown 10 min interval as indicated) of CW-stained wild-type (C) and cps1-191 (D and E) cells carrying Rlc1-RFP and Ags1-GFP or GFP-Bgs4. Wild type and cps1-191 cells growing in YES+S at 25°C were shifted to 37°C for 1.5 h and filmed to capture ring formation, and Ags1 or Bgs4 arrival to the division site. Two cps1-191 cells showing a sliding ring which does not constrict (left in D and E) and two cps1-191 cells with a slow ring constriction and septum deposition (right in D and E) are shown. Arrowheads indicate synthesis of the septum wall in the absence of ring constriction. A dashed line is drawn as reference for the ring position. (F and G) Fluorescence micrographs of CW-stained wild-type (F) and cps1-191 (G) cells carrying Rlc1-RFP, and GFP-Bgs4 or Ags1-GFP. Cells were grown as in A. Maximum-intensity projections of 28 z slides at 0.3 μm intervals (upper panels) and the corresponding three-dimensional reconstructions of the equatorial plane including the septum area (lower panels) of the complete cell are shown. In all the three-dimensional reconstructions, from the 28 slides acquired it was only selected those slides that cover the entire cell diameter. Scale bars, 5 μm.
Mentions: Previous work identified a synthetic lethal interaction between pxl1Δ and different bgs1/cps1 thermosensitive mutants [26], suggesting that Pxl1 and Bgs1 collaborate in an essential process. To test this hypothesis, we first analyzed the septation defects of cells carrying the bgs1 thermosensitive allele cps1-191. These cells were grown at the restrictive temperature (37°C) in the presence of 1.3M sorbitol (S) to allow the deposition of the septum (Fig 2A and 2B). In cells carrying Rlc1-RFP and GFP-Psy1 (a syntaxin homolog that is a marker for the plasma membrane [29]) the processes of membrane invagination and septum formation were accompanied by a disorganized ring which appeared fragmented, with Rlc1 strands connected to the ring. These CAR defects were similar to those of pxl1Δ cells [25,26]. Therefore, a defective Bgs1 function affects the CAR position in the cell middle as was already described [20], and also affects the ring integrity during septum ingression. Because other glucan synthases, such as Ags1 and Bgs4, must collaborate in the formation of the aberrant septum structures observed in cps1-191 cells, we analyzed the localization of these synthases, before and during septation, in wild type and cps1-191 cells carrying Rlc1-RFP grown at the restrictive temperature for 1.5 h (Fig 2C, 2D, and 2E). At this time point, cps1-191 cells made new CARs that were maintained for a long time. Some cells formed rudimentary or partial depositions of CW-stained septa without a proper CAR constriction (Fig 2D and 2E, arrowheads). Time-lapse images of cps1-191 rlc1+-RFP cells carrying Ags1-GFP or GFP-Bgs4 showed that both synthases localized to the division area as in wild type cells (Fig 2C, 2D, and 2E). Ags1 concentrated and moved with the ring along the plasma membrane, and in cells with septum deposition it remained concentrated at the septum area (Fig 2D). In agreement with previous observations [12], Bgs4 did not concentrate in the cell membrane of cells without septum deposition (Fig 2E, left) but did appear concentrated in cells with a partial septum (Fig 2E, right). After longer times (6 h) at the restrictive temperature, both synthases spread along the septum membrane and were also detected in cytoplasmic vesicles. They did not form a homogeneously filled disk-like surface as they do in wild type cells, but an irregularly filled disk indistinguishable from the numerous cytoplasmic vesicles located in the middle (Fig 2F and 2G). Therefore, Bgs1 function is required for ring integrity, for the correct synthesis of the PS, and for the proper localization of Ags1 and Bgs4 as a homogeneous disk structure in the membrane during septum ingression.

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