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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

Pxl1 is required for stable CAR and septum positioning in the middle of the cell.(A) A Calcofluor White (CW) staining image of pxl1Δ cells with off-centered septa. (B) Histogram showing the indicated intervals of septum position measured as the percent of septum offset from the cell center: white bars, wild-type cells (n = 40); black bars, pxl1Δ cells (n = 142); dark grey bars, pxl1Δ ags1+-GFP cells (n = 131); and light grey bars, pxl1Δ GFP-bgs1+ cells (n = 59). The position of the septum was measured with Image J software as described in the Materials and Methods section. (C) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell with a centrally located ring that began to constrict at +24 min. The second panel shows a pxl1Δ cell with a ring that moved toward the upper pole at +15 min. Spindle microtubules appear at time 0. (D) Time courses of appearance of cortical nodes tracked with Rlc1-RFP (circle), completion of ring (square), onset of ring constriction (diamond), and ring sliding (triangle). Filled symbols are wild-type cells (circle n = 16; square n = 15; diamond n = 15), and open symbols are pxl1Δ cells (circle n = 40; square n = 50; diamond n = 45; triangle n = 27). (E) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Bgs1 was detected in the cell middle at +15 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min and where Bgs1 was detected in the cell middle at +15 min. Spindle microtubules appear at time 0. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Ags1-GFP, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Ags1 was detected in the cell middle at +12 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min where Ags1 was detected at +15 min. Spindle microtubules appear at time 0. Dashed line: reference for the ring position. Elapsed time is shown in minutes. Scale bars, 5 μm.
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pgen.1005358.g001: Pxl1 is required for stable CAR and septum positioning in the middle of the cell.(A) A Calcofluor White (CW) staining image of pxl1Δ cells with off-centered septa. (B) Histogram showing the indicated intervals of septum position measured as the percent of septum offset from the cell center: white bars, wild-type cells (n = 40); black bars, pxl1Δ cells (n = 142); dark grey bars, pxl1Δ ags1+-GFP cells (n = 131); and light grey bars, pxl1Δ GFP-bgs1+ cells (n = 59). The position of the septum was measured with Image J software as described in the Materials and Methods section. (C) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell with a centrally located ring that began to constrict at +24 min. The second panel shows a pxl1Δ cell with a ring that moved toward the upper pole at +15 min. Spindle microtubules appear at time 0. (D) Time courses of appearance of cortical nodes tracked with Rlc1-RFP (circle), completion of ring (square), onset of ring constriction (diamond), and ring sliding (triangle). Filled symbols are wild-type cells (circle n = 16; square n = 15; diamond n = 15), and open symbols are pxl1Δ cells (circle n = 40; square n = 50; diamond n = 45; triangle n = 27). (E) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Bgs1 was detected in the cell middle at +15 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min and where Bgs1 was detected in the cell middle at +15 min. Spindle microtubules appear at time 0. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Ags1-GFP, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Ags1 was detected in the cell middle at +12 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min where Ags1 was detected at +15 min. Spindle microtubules appear at time 0. Dashed line: reference for the ring position. Elapsed time is shown in minutes. Scale bars, 5 μm.

Mentions: Schizosaccharomyces pombe paxillin, Pxl1, is a Rho1 negative regulator that contributes to the maintenance of CAR integrity [25,26]. Cells lacking Pxl1 present a higher proportion of septated cells (45 to 50%) and the CAR constriction rate is slower than in wild type cells. These phenotypes are likely due to a defective actomyosin ring that occasionally splits into two [25,26]. A detailed observation of cells lacking Pxl1 labeled with Calcofluor White (CW) so the primary septum (PS) could be seen, revealed some cells with off-center septa (Fig 1A). While all wild type cells formed septa within 10% offset from the cell center, only 70% of the cells lacking Pxl1 formed septa in this section of the cell (Fig 1B). This phenotype was further analyzed by following CAR assembly in time-lapse experiments. Wild type and pxl1Δ cells carrying GFP-Atb2 (tubulin) and Rlc1-RFP (CAR) were adjusted to a timescale where zero corresponds to the spindle formation and spindle pole body separation. CARs from pxl1Δ cells assembled from condensed nodes as they do in wild type cells. However, once formed, pxl1Δ CARs delayed contraction and moved slightly along the longitudinal axis, suggesting that ring anchorage to the membrane was not steady (Fig 1C and 1D, dashed line). CAR sliding occurred until the septum was visible with CW in pxl1Δ cells (S1A Fig). No CAR sliding was observed in wild type cells (Fig 1C and 1D).


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)

Pxl1 is required for stable CAR and septum positioning in the middle of the cell.(A) A Calcofluor White (CW) staining image of pxl1Δ cells with off-centered septa. (B) Histogram showing the indicated intervals of septum position measured as the percent of septum offset from the cell center: white bars, wild-type cells (n = 40); black bars, pxl1Δ cells (n = 142); dark grey bars, pxl1Δ ags1+-GFP cells (n = 131); and light grey bars, pxl1Δ GFP-bgs1+ cells (n = 59). The position of the septum was measured with Image J software as described in the Materials and Methods section. (C) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell with a centrally located ring that began to constrict at +24 min. The second panel shows a pxl1Δ cell with a ring that moved toward the upper pole at +15 min. Spindle microtubules appear at time 0. (D) Time courses of appearance of cortical nodes tracked with Rlc1-RFP (circle), completion of ring (square), onset of ring constriction (diamond), and ring sliding (triangle). Filled symbols are wild-type cells (circle n = 16; square n = 15; diamond n = 15), and open symbols are pxl1Δ cells (circle n = 40; square n = 50; diamond n = 45; triangle n = 27). (E) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Bgs1 was detected in the cell middle at +15 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min and where Bgs1 was detected in the cell middle at +15 min. Spindle microtubules appear at time 0. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Ags1-GFP, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Ags1 was detected in the cell middle at +12 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min where Ags1 was detected at +15 min. Spindle microtubules appear at time 0. Dashed line: reference for the ring position. Elapsed time is shown in minutes. Scale bars, 5 μm.
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pgen.1005358.g001: Pxl1 is required for stable CAR and septum positioning in the middle of the cell.(A) A Calcofluor White (CW) staining image of pxl1Δ cells with off-centered septa. (B) Histogram showing the indicated intervals of septum position measured as the percent of septum offset from the cell center: white bars, wild-type cells (n = 40); black bars, pxl1Δ cells (n = 142); dark grey bars, pxl1Δ ags1+-GFP cells (n = 131); and light grey bars, pxl1Δ GFP-bgs1+ cells (n = 59). The position of the septum was measured with Image J software as described in the Materials and Methods section. (C) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell with a centrally located ring that began to constrict at +24 min. The second panel shows a pxl1Δ cell with a ring that moved toward the upper pole at +15 min. Spindle microtubules appear at time 0. (D) Time courses of appearance of cortical nodes tracked with Rlc1-RFP (circle), completion of ring (square), onset of ring constriction (diamond), and ring sliding (triangle). Filled symbols are wild-type cells (circle n = 16; square n = 15; diamond n = 15), and open symbols are pxl1Δ cells (circle n = 40; square n = 50; diamond n = 45; triangle n = 27). (E) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Bgs1 was detected in the cell middle at +15 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min and where Bgs1 was detected in the cell middle at +15 min. Spindle microtubules appear at time 0. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying Ags1-GFP, Rlc1-RFP and GFP-Atb2. The first panel shows a wild-type cell where Ags1 was detected in the cell middle at +12 min. The second panel shows a pxl1Δ cell with a ring that moved toward the lower pole at +18 min where Ags1 was detected at +15 min. Spindle microtubules appear at time 0. Dashed line: reference for the ring position. Elapsed time is shown in minutes. Scale bars, 5 μm.
Mentions: Schizosaccharomyces pombe paxillin, Pxl1, is a Rho1 negative regulator that contributes to the maintenance of CAR integrity [25,26]. Cells lacking Pxl1 present a higher proportion of septated cells (45 to 50%) and the CAR constriction rate is slower than in wild type cells. These phenotypes are likely due to a defective actomyosin ring that occasionally splits into two [25,26]. A detailed observation of cells lacking Pxl1 labeled with Calcofluor White (CW) so the primary septum (PS) could be seen, revealed some cells with off-center septa (Fig 1A). While all wild type cells formed septa within 10% offset from the cell center, only 70% of the cells lacking Pxl1 formed septa in this section of the cell (Fig 1B). This phenotype was further analyzed by following CAR assembly in time-lapse experiments. Wild type and pxl1Δ cells carrying GFP-Atb2 (tubulin) and Rlc1-RFP (CAR) were adjusted to a timescale where zero corresponds to the spindle formation and spindle pole body separation. CARs from pxl1Δ cells assembled from condensed nodes as they do in wild type cells. However, once formed, pxl1Δ CARs delayed contraction and moved slightly along the longitudinal axis, suggesting that ring anchorage to the membrane was not steady (Fig 1C and 1D, dashed line). CAR sliding occurred until the septum was visible with CW in pxl1Δ cells (S1A Fig). No CAR sliding was observed in wild type cells (Fig 1C and 1D).

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