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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

Cdc15 SH3 domain is necessary for proper concentration of Pxl1 at the CAR.(A) Box plot showing the total fluorescence of GFP-Pxl1 in the cell middle of wild-type (n = 90) and cdc15ΔSH3 cells (n = 214). GFP-Pxl1 fluorescence was measured in cells stained with CW, and divided into four categories depending on the length of the septum in the cell: 1) no septum 2) early septum (less than 0.6 μm); 3) middle septum (0.6 to 1.2 μm); and 4) advanced septum (more than 1.2 μm). Total fluorescence was quantified by using Image J software as described in the Materials and Methods section. (B) Fluorescence micrographs showing representative septated wild-type and cdc15ΔSH3 cells carrying GFP-Pxl1, and used to measure the total fluorescence of GFP-Pxl1 in A. (C) Kymographs of fluorescence time series (one middle z slide, 2 min intervals) of wild-type and cdc15ΔSH3 cells stained with CW and carrying GFP-Pxl1. (D) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of wild-type (upper panels) and cdc15ΔSH3 (lower panels) cells carrying GFP-Pxl1 and RFP-Atb2. Spindle microtubules appear at time 0. (E) Total fluorescence of GFP-Pxl1 in the cell middle of the time series shown in D. Fluorescence was quantified along the time as described in A. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1 and RFP-Atb2. The first panel shows a wild-type cell where the Bgs1 band was detected in the septum assembly site at +12 min. The second panel shows a cdc15ΔSH3 cell where the Bgs1 band was detected in the septum assembly site at +18 min. The third panel shows a pxl1Δ cell where the Bgs1 band was detected in the septum assembly site at +12 min. Spindle microtubules appear at time 0. Squares indicate the first detection of Bgs1 as a band in the septum assembly site in the wild-type. (G) Time courses of appearance of the GFP-Bgs1 band (diamond) and GFP-Bgs1 ring (square). Open symbols are wild-type cells, and filled symbols are cdc15ΔSH3 (upper graph) and pxl1Δ (lower graph) cells. The same wild-type cells were used in both graphs. Wild-type (diamond n = 19; square n = 19), cdc15ΔSH3 (diamond n = 24; square n = 24), and pxl1Δ cells (diamond n = 15; square n = 14). Elapsed time is shown in minutes. Scale bars, 5 μm.
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pgen.1005358.g005: Cdc15 SH3 domain is necessary for proper concentration of Pxl1 at the CAR.(A) Box plot showing the total fluorescence of GFP-Pxl1 in the cell middle of wild-type (n = 90) and cdc15ΔSH3 cells (n = 214). GFP-Pxl1 fluorescence was measured in cells stained with CW, and divided into four categories depending on the length of the septum in the cell: 1) no septum 2) early septum (less than 0.6 μm); 3) middle septum (0.6 to 1.2 μm); and 4) advanced septum (more than 1.2 μm). Total fluorescence was quantified by using Image J software as described in the Materials and Methods section. (B) Fluorescence micrographs showing representative septated wild-type and cdc15ΔSH3 cells carrying GFP-Pxl1, and used to measure the total fluorescence of GFP-Pxl1 in A. (C) Kymographs of fluorescence time series (one middle z slide, 2 min intervals) of wild-type and cdc15ΔSH3 cells stained with CW and carrying GFP-Pxl1. (D) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of wild-type (upper panels) and cdc15ΔSH3 (lower panels) cells carrying GFP-Pxl1 and RFP-Atb2. Spindle microtubules appear at time 0. (E) Total fluorescence of GFP-Pxl1 in the cell middle of the time series shown in D. Fluorescence was quantified along the time as described in A. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1 and RFP-Atb2. The first panel shows a wild-type cell where the Bgs1 band was detected in the septum assembly site at +12 min. The second panel shows a cdc15ΔSH3 cell where the Bgs1 band was detected in the septum assembly site at +18 min. The third panel shows a pxl1Δ cell where the Bgs1 band was detected in the septum assembly site at +12 min. Spindle microtubules appear at time 0. Squares indicate the first detection of Bgs1 as a band in the septum assembly site in the wild-type. (G) Time courses of appearance of the GFP-Bgs1 band (diamond) and GFP-Bgs1 ring (square). Open symbols are wild-type cells, and filled symbols are cdc15ΔSH3 (upper graph) and pxl1Δ (lower graph) cells. The same wild-type cells were used in both graphs. Wild-type (diamond n = 19; square n = 19), cdc15ΔSH3 (diamond n = 24; square n = 24), and pxl1Δ cells (diamond n = 15; square n = 14). Elapsed time is shown in minutes. Scale bars, 5 μm.

Mentions: Pxl1 requires the SH3 domain of either Cdc15 or Imp2, two F-BAR proteins, to localize to the CAR [22]. Thus, Pxl1 localizes to the ring in cells carrying Cdc15ΔSH3 because it binds to Imp2. We observed that GFP-Pxl1 formed a ring in cdc15ΔSH3 cells but the fluorescence intensity was dimmer than in wild type cells (Fig 5A, 5B, 5C and 5D and S4A Fig, arrow). We quantified the GFP-Pxl1 ring fluorescence in wild type and cdc15ΔSH3 cells at four different times during cytokinesis: no septa, early septa (<0.6 μm), middle septa (0.6–1.2 μm), and advanced septa (>1.2 μm). This quantification showed that in wild type cells Pxl1 fluorescence increased from the onset of septation until the completion of the septum (Fig 5A and 5B). In contrast, no increase or even a reduction in GFP-Pxl1 fluorescence was observed in cdc15ΔSH3 cells (Fig 5A and 5B). Interestingly, reduction in Pxl1 fluorescence was coincident with a noticeably slower progression of the septum (Fig 5C). Time-lapse analysis performed in wild type and cdc15ΔSH3 cells carrying GFP-Pxl1 and RFP-Atb2 confirmed the results obtained in the quantification of septating cells (Fig 5D and 5E). In wild type cells the Pxl1 ring was detected at +12 min after the spindle appeared and began to constrict at +21 min while in cdc15ΔSH3 cells the Pxl1 ring was detected at +18 min and began to constrict at +39 min (Fig 5D). Moreover, GFP-Pxl1 intensity in cdc15ΔSH3 cells was never greater than 25% of the intensity in wild type cells (Fig 5E). These results suggest that an increase in Pxl1 might be necessary to start and complete septation efficiently.


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)

Cdc15 SH3 domain is necessary for proper concentration of Pxl1 at the CAR.(A) Box plot showing the total fluorescence of GFP-Pxl1 in the cell middle of wild-type (n = 90) and cdc15ΔSH3 cells (n = 214). GFP-Pxl1 fluorescence was measured in cells stained with CW, and divided into four categories depending on the length of the septum in the cell: 1) no septum 2) early septum (less than 0.6 μm); 3) middle septum (0.6 to 1.2 μm); and 4) advanced septum (more than 1.2 μm). Total fluorescence was quantified by using Image J software as described in the Materials and Methods section. (B) Fluorescence micrographs showing representative septated wild-type and cdc15ΔSH3 cells carrying GFP-Pxl1, and used to measure the total fluorescence of GFP-Pxl1 in A. (C) Kymographs of fluorescence time series (one middle z slide, 2 min intervals) of wild-type and cdc15ΔSH3 cells stained with CW and carrying GFP-Pxl1. (D) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of wild-type (upper panels) and cdc15ΔSH3 (lower panels) cells carrying GFP-Pxl1 and RFP-Atb2. Spindle microtubules appear at time 0. (E) Total fluorescence of GFP-Pxl1 in the cell middle of the time series shown in D. Fluorescence was quantified along the time as described in A. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1 and RFP-Atb2. The first panel shows a wild-type cell where the Bgs1 band was detected in the septum assembly site at +12 min. The second panel shows a cdc15ΔSH3 cell where the Bgs1 band was detected in the septum assembly site at +18 min. The third panel shows a pxl1Δ cell where the Bgs1 band was detected in the septum assembly site at +12 min. Spindle microtubules appear at time 0. Squares indicate the first detection of Bgs1 as a band in the septum assembly site in the wild-type. (G) Time courses of appearance of the GFP-Bgs1 band (diamond) and GFP-Bgs1 ring (square). Open symbols are wild-type cells, and filled symbols are cdc15ΔSH3 (upper graph) and pxl1Δ (lower graph) cells. The same wild-type cells were used in both graphs. Wild-type (diamond n = 19; square n = 19), cdc15ΔSH3 (diamond n = 24; square n = 24), and pxl1Δ cells (diamond n = 15; square n = 14). Elapsed time is shown in minutes. Scale bars, 5 μm.
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pgen.1005358.g005: Cdc15 SH3 domain is necessary for proper concentration of Pxl1 at the CAR.(A) Box plot showing the total fluorescence of GFP-Pxl1 in the cell middle of wild-type (n = 90) and cdc15ΔSH3 cells (n = 214). GFP-Pxl1 fluorescence was measured in cells stained with CW, and divided into four categories depending on the length of the septum in the cell: 1) no septum 2) early septum (less than 0.6 μm); 3) middle septum (0.6 to 1.2 μm); and 4) advanced septum (more than 1.2 μm). Total fluorescence was quantified by using Image J software as described in the Materials and Methods section. (B) Fluorescence micrographs showing representative septated wild-type and cdc15ΔSH3 cells carrying GFP-Pxl1, and used to measure the total fluorescence of GFP-Pxl1 in A. (C) Kymographs of fluorescence time series (one middle z slide, 2 min intervals) of wild-type and cdc15ΔSH3 cells stained with CW and carrying GFP-Pxl1. (D) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of wild-type (upper panels) and cdc15ΔSH3 (lower panels) cells carrying GFP-Pxl1 and RFP-Atb2. Spindle microtubules appear at time 0. (E) Total fluorescence of GFP-Pxl1 in the cell middle of the time series shown in D. Fluorescence was quantified along the time as described in A. (F) Time series of fluorescence micrographs (one medial z slide, 3 min intervals) of cells carrying GFP-Bgs1 and RFP-Atb2. The first panel shows a wild-type cell where the Bgs1 band was detected in the septum assembly site at +12 min. The second panel shows a cdc15ΔSH3 cell where the Bgs1 band was detected in the septum assembly site at +18 min. The third panel shows a pxl1Δ cell where the Bgs1 band was detected in the septum assembly site at +12 min. Spindle microtubules appear at time 0. Squares indicate the first detection of Bgs1 as a band in the septum assembly site in the wild-type. (G) Time courses of appearance of the GFP-Bgs1 band (diamond) and GFP-Bgs1 ring (square). Open symbols are wild-type cells, and filled symbols are cdc15ΔSH3 (upper graph) and pxl1Δ (lower graph) cells. The same wild-type cells were used in both graphs. Wild-type (diamond n = 19; square n = 19), cdc15ΔSH3 (diamond n = 24; square n = 24), and pxl1Δ cells (diamond n = 15; square n = 14). Elapsed time is shown in minutes. Scale bars, 5 μm.
Mentions: Pxl1 requires the SH3 domain of either Cdc15 or Imp2, two F-BAR proteins, to localize to the CAR [22]. Thus, Pxl1 localizes to the ring in cells carrying Cdc15ΔSH3 because it binds to Imp2. We observed that GFP-Pxl1 formed a ring in cdc15ΔSH3 cells but the fluorescence intensity was dimmer than in wild type cells (Fig 5A, 5B, 5C and 5D and S4A Fig, arrow). We quantified the GFP-Pxl1 ring fluorescence in wild type and cdc15ΔSH3 cells at four different times during cytokinesis: no septa, early septa (<0.6 μm), middle septa (0.6–1.2 μm), and advanced septa (>1.2 μm). This quantification showed that in wild type cells Pxl1 fluorescence increased from the onset of septation until the completion of the septum (Fig 5A and 5B). In contrast, no increase or even a reduction in GFP-Pxl1 fluorescence was observed in cdc15ΔSH3 cells (Fig 5A and 5B). Interestingly, reduction in Pxl1 fluorescence was coincident with a noticeably slower progression of the septum (Fig 5C). Time-lapse analysis performed in wild type and cdc15ΔSH3 cells carrying GFP-Pxl1 and RFP-Atb2 confirmed the results obtained in the quantification of septating cells (Fig 5D and 5E). In wild type cells the Pxl1 ring was detected at +12 min after the spindle appeared and began to constrict at +21 min while in cdc15ΔSH3 cells the Pxl1 ring was detected at +18 min and began to constrict at +39 min (Fig 5D). Moreover, GFP-Pxl1 intensity in cdc15ΔSH3 cells was never greater than 25% of the intensity in wild type cells (Fig 5E). These results suggest that an increase in Pxl1 might be necessary to start and complete septation efficiently.

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