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Title: Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast}
Article:
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.
Introduction:
Cytokinesis is the final stage of the eukaryotic cell cycle, when a mother cell separates into two daughter cells. Cytokinesis is mediated by a contractile actomyosin ring( CAR) that is conserved between fungal and animal cells[1]. In addition to CAR contraction, fungal cells assemble a division septum wall which is essential for cell integrity[2]. Recent work proposed that the pulling force from CAR contraction is not sufficient to accomplish cytokinesis and that a pushing force is also necessary[3], and we showed that support of the lateral cell wall is crucial for proper cytokinesis[4]. Fission yeast CAR is composed of many proteins besides F-actin and heavy and light chains of myosin II[5, 6]. Significant progress in identifying and characterizing the proteins that participate in CAR positioning, assembly, stabilization, and integrity has already been made[1, 7, 8]. The septum of fission yeast is a three-layered polysaccharide structure made of a middle primary septum( PS) flanked by two secondary septa( SS), one on each side. Both the PS and SS are formed by essential β-glucans. The enzyme involved in their formation is the β( 1, 3) glucan synthase, composed of at least a regulatory and a catalytic subunit. The former is the GTPase Rho1[9, 10]. Fission yeast contains four different catalytic subunits named Bgs1 to Bgs4. Bgs1 is responsible for the linear β( 1, 3) glucan necessary for PS formation[11]; Bgs4 builds branched β( 1, 3) glucan[12], which is the most abundant polymer in the septum and cell wall. During cytokinesis this polymer is required for connecting the CAR to the extracellular cell wall, for SS formation, for the correct PS structure, and to maintain the cell integrity during cell separation[4]. Ags1 synthesizes α-glucan, which is also a major cell wall polymer[13, 14]. During cytokinesis α-glucan is essential for the PS adhesion strength needed to withstand the internal turgor pressure during cell abscission, for the SS structure, and for cell integrity[15]. While much is known about the protein components of the CAR, how these proteins coordinate and interact with the septum synthesis enzymes to form the cleavage furrow remains unclear. A key protein for CAR positioning and function is the F-BAR domain-containing phosphoprotein Cdc15[16]. Cdc15 is a membrane-anchored scaffold for CAR assembly and links the ring to the plasma membrane through its essential F-BAR domain[17]. Dephosphorylation of Cdc15 at mitotic entry, mediated by Clp1, induces a conformational switch in the protein that promotes oligomerization[18]. Cdc15 also participates in endocytosis[19], and it has recently been shown that it localizes to the Golgi where it helps in the delivery of Bgs1 to the plasma membrane during cytokinesis[20]. Additionally, Cdc15 collaborates with the F-BAR domain-containing Imp2[21] in CAR attachment to the membrane through their C-terminal SH3 domains, which mediate interactions with the paxillin homolog, Pxl1, and the C2-domain-containing protein Fic1[22]. Another F-BAR domain protein, Rga7, associates with Imp2 in a protein complex that coordinates the late stages of cytokinesis[23]. Animal cell paxillin is a multi-domain scaffold protein that localizes to the intracellular surface of the plasma membrane in sites of cell adhesion of extracellular matrix[24]. Fission yeast Pxl1 is a component of the CAR that shares with other paxillins the Lin-11, Isl-1, Mec-3( LIM) domains in its C-terminal half but presents a different N-terminal region, which is required for binding to the Cdc15 and Imp2 SH3 domains[22]. Pxl1 also binds to the type II myosin Myo2 and is necessary for CAR integrity and proper constriction[25, 26]. In this work, we show that the functions of Pxl1 in the CAR and Bgs1 in the membrane are necessary for stable CAR anchorage. Additionally, cooperation between Pxl1 and Bgs1 is shown to be essential for CAR maintenance and septum formation, since cells without Bgs1 display CAR disassembly and cannot form septa when Pxl1 is absent or when the Pxl1 levels in the ring are reduced, as in the case of cells carrying the defective cdc15ΔSH3 allele.
Discussion:
Fission yeast Pxl1 binds to the type II myosin Myo2 and the F-BAR protein Cdc15, and participates in maintaining CAR integrity[26]. We show in this work that Pxl1 also collaborates in the stable anchorage of the ring to the membrane since the absence of Pxl1 causes CAR sliding until the onset of septation. The extent of CAR sliding in pxl1Δ strain was similar to that described in cdc15ΔSH3 cells[20], with more than 10% offset septa in around 30% of the cells. It is possible that Pxl1 participates in the physical link between the CAR and the plasma membrane mediated through Cdc15, which has been shown to play a role in this linkage[22]. It has recently been proposed that Bgs1 protein, besides being a major enzyme for PS formation, is the physical anchor of the ring to the plasma membrane and that Cdc15 participates in the transfer of Bgs1 from the Golgi apparatus to the plasma membrane[20]. We show here that the septum synthases Bgs1 and Ags1, located at the ring edge membrane, slide together with the CAR in cells lacking Pxl1, indicating that these transmembrane enzymes are connected to the CAR but are not sufficient to anchor it stably. Instead, the coincidence between the appearance of CW staining and the cessation of ring sliding, which was also shown in cells depleted of Bgs4[4], suggest that the PS synthesized by Bgs1 is required for the CAR to be stably anchored. Corroborating this hypothesis, bgs1+ mutations, such as that of cps1-191 allele[37], permit CAR sliding[20]. In addition, our results indicate that defective PS synthesis and ingression induces CAR instability, and that collaboration of Bgs1 with Pxl1 and the SH3 domain of Cdc15 is required to maintain the CAR structure and to form the septum, globally pointing to the existence of a physical link between the CAR and the PS through Pxl1, Cdc15, and Bgs1. In the budding yeast S. cerevisiae CAR closure and formation of the PS are processes that depend on one another. Thus, interference with one function would also block the other[38]. Mutant cells lacking either myosin type II or the chitin synthase Chs2, which is responsible for making the PS in this yeast, have indistinguishable cytokinesis defects and the double mutant lacking both myosin type II and Chs2 hardly differs from the single ones. Similarly, in fission yeast defects in the CAR can alter the rate and symmetry of septum ingression[3, 39], and vice versa[37]. However, in contrast with budding yeast, the type II myosin Myo2 is essential in fission yeast. Additionally, we show here that simultaneous depletion of Pxl1 and alteration of Cdc15 function uncouples two tightly linked processes, CAR closure and PS formation, resulting in the synthesis of CW-stained material along the plasma membrane advancing with a sliding CAR that is unable to constrict. When Bgs1 is depleted, Pxl1 is required for the salvage route that leads to the abnormal but complete septa found in these cells. The remedial septa under Bgs1 depletion are formed by layers of the SS[11], which somehow are guided by the CAR. It is possible that Pxl1 participates in the transmission of the ring tension generated by myosin II to the membrane in order to concentrate the SS synthases and narrow the area of septum synthesis. In the absence of Bgs1 and Pxl1 there is no PS synthesis and the connection between the plasma membrane and the ring is lost. As a consequence, there is no concentration of the SS synthases and some inward growth of cell wall along the lateral plasma membrane occurs, but cytokinesis is never accomplished. At present it is not clear how Pxl1 contributes to Bgs synthases function. It is tempting to propose that Pxl1 is acting as a mechanosensor that helps to transform the CAR contraction into an activation signal for the biosynthetic enzymes that form the septum. In this sense Pxl1 interacts with Rho1 GTPase, the major activator of both β( 1, 3) glucan synthase catalytic subunits[9, 26]. F-BAR proteins present at the ring edge membrane such as Cdc15, Imp2, or the recently described Rga7 might be part of this transmission[22, 23]. In fact, Cdc15ΔSH3 causes defects similar to those caused by the lack of paxillin in Bgs1-depleted cells, suggesting that they are part of the same signaling pathway. Paxillin might collaborate with Cdc15 in the transfer of Bgs1 from the Golgi to the plasma membrane[20]. However, no delay in the transport of this synthase to the division site membrane was observed in cells lacking Pxl1. On the other hand, GFP-tagged Cps1-191 localizes to the membrane in wild type cells but forms intracellular aggregates in both pxl1Δ and cdc15ΔSH3. Importantly, these aggregates are not observed in pxl1Δ or cdc15ΔSH3 cells carrying wild type Bgs1, suggesting that the correct function of Bgs1 helps its localization at the plasma membrane. The observed aggregates in pxl1Δ or cdc15ΔSH3 cells could be caused by a deficient transport and/or recycling of nonfunctional Cps1-191 from trans-Golgi or endosomes to the plasma membrane. The lack of paxillin is lethal in cells carrying cdc15ΔSH3 or even the hypomorphic allele cdc15-GFP[26, 34]. As we show here, Cdc15 is necessary to concentrate Pxl1 to the CAR but is also required to concentrate Bgs1 to the membrane[20]. This double function could explain the lethality of pxl1Δ cdc15ΔSH3 double mutation. It also could explain the observed phenotype in cdc15-GFP hypomorphic mutant cells depleted of Pxl1. CAR sliding in these double mutant cells is more pronounced and longitudinal synthesis of linear β( 1, 3) glucan without ring contraction is sometimes observed, as if a low concentration of active Bgs1 was not sufficient to anchor the defective CAR of Pxl1-depleted cells, nor to activate CAR contraction and septum ingression. The lack of paxillin is also lethal in the absence of other F-BAR proteins such as Rga7[23]. Moreover, Pxl1 is essential in the absence of the C2-domain containing protein Fic1 which also binds to Cdc15 and Imp2[22]. S. cerevisiae Inn1, which is the ortholog of Fic1, couples CAR constriction and plasma membrane ingression[22, 40]. It has been proposed that Inn1 acts in conjunction with Hof1, the Cdc15 ortholog of budding yeast, and Cyk3 to regulate the catalytic domain of the PS-forming enzyme Chs2[41]. Cyk3 and Chs2 orthologs exist in fission yeast and have a role in cytokinesis[42, 43]. However, the lack of Fic1, Cyk3, or Chs2 orthologs does not cause a drastic phenotype in S. pombe cells, which do not have chitin in the septum wall. It will be interesting to determine whether Fic1, like Inn1, is required for CAR closure during cytokinesis. In summary, our studies indicate that both CAR and PS collaborate in the cleavage furrow and septum formation, shedding some light on the importance of the connection between the ring and the septum during S. pombe cytokinesis.
Cytokinesis requires assembly of an actomyosin ring adjacent to the plasma membrane , which upon contraction pulls the membrane to form a cleavage furrow . In fungi ring closure is coordinated with the synthesis of a cell wall septum . Knowledge about the molecules anchoring the ring to the membrane is very limited . We have found that fission yeast paxillin , located at the ring , and Bgs1 , the enzyme responsible for primary septum formation , located at the membrane , cooperate during cytokinesis . Both are required to anchor the ring to the membrane and to maintain it during cytokinesis . Moreover , both proteins cooperate to form the septum . Accordingly , paxillin is essential when Bgs1 is depleted . When both proteins are missing , the contractile ring forms but the lateral cell wall overgrows inwards without a defined cleavage furrow and septum formation . During cytokinesis there is an increase of paxillin which depends on the SH3 domain of the F-BAR protein Cdc15 . Consequently the absence of this domain mimics the phenotype of paxillin absence in Bgs1-depleted cells . Interestingly , a decreased function of both Cdc15 and paxillin uncouples the septum synthesis from the ring contraction , indicating an essential cooperation between these proteins and Bgs1 for proper cytokinesis .