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Hof1 and Rvs167 have redundant roles in actomyosin ring function during cytokinesis in budding yeast.

Nkosi PJ, Targosz BS, Labib K, Sanchez-Diaz A - PLoS ONE (2013)

Bottom Line: This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3.Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors.The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

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Assembly of the actin ring is defective in cells lacking both Hof1 and Rvs167.A similar range of strains to those described above for Figure 4 were grown as before, and assembly of the actin ring was monitored in time-course experiments by staining fixed cells with rhodamine phalloidin. (A) In control cells, actin patches accumulate in the new bud until mitosis (1), before depolarising throughout the cell during anaphase when the actin ring assembles (2; marked by arrow). Contraction of the actomyosin ring is associated with the accumulation of actin patches on either side of the bud-neck (3). The scale bars correspond to 2 µm. (B) The proportion of bi-nucleate cells was determined throughout two sets of time-course experiments involving inactivation of Hof1-td and Cyk3-td (i), or combination of Hof1-td with rvs167 or rvs167-SH3 (ii). Samples from the same time-course experiments were stained with rhodamine phalloidin and used to determine the proportion of cells with actin rings (C-D) and the percentage of cells that failed to complete cytokinesis (E). Images of hof1-td cyk3-td and hof1-td rvs167 cells that failed to complete cytokinesis are shown in panel (F), in which the scale bars correspond to 2 µm.
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pone-0057846-g005: Assembly of the actin ring is defective in cells lacking both Hof1 and Rvs167.A similar range of strains to those described above for Figure 4 were grown as before, and assembly of the actin ring was monitored in time-course experiments by staining fixed cells with rhodamine phalloidin. (A) In control cells, actin patches accumulate in the new bud until mitosis (1), before depolarising throughout the cell during anaphase when the actin ring assembles (2; marked by arrow). Contraction of the actomyosin ring is associated with the accumulation of actin patches on either side of the bud-neck (3). The scale bars correspond to 2 µm. (B) The proportion of bi-nucleate cells was determined throughout two sets of time-course experiments involving inactivation of Hof1-td and Cyk3-td (i), or combination of Hof1-td with rvs167 or rvs167-SH3 (ii). Samples from the same time-course experiments were stained with rhodamine phalloidin and used to determine the proportion of cells with actin rings (C-D) and the percentage of cells that failed to complete cytokinesis (E). Images of hof1-td cyk3-td and hof1-td rvs167 cells that failed to complete cytokinesis are shown in panel (F), in which the scale bars correspond to 2 µm.

Mentions: A previous study showed that recruitment of Inn1 to the cleavage site is blocked by treatment of hof1Δ cells with the drug Latrunculin A that depolymerises actin and prevents assembly of the actomyosin ring [23]. It thus seemed possible that a defect in assembling the actin ring could contribute to the defective recruitment of Inn1 in cells lacking Rvs167 and Hof1. In similar experiments to those described above, we used rhodamine-phalloidin staining to monitor the actin cytoskeleton as G1-phase cells passed synchronously through a complete cell cycle at 37°C. In control cells, assembly of the actin ring during mitosis was associated with delocalisation of actin throughout the whole cell, whereas disassembly of the actin ring was followed by the accumulation of actin patches at either side of the bud-neck (Figure 5A).


Hof1 and Rvs167 have redundant roles in actomyosin ring function during cytokinesis in budding yeast.

Nkosi PJ, Targosz BS, Labib K, Sanchez-Diaz A - PLoS ONE (2013)

Assembly of the actin ring is defective in cells lacking both Hof1 and Rvs167.A similar range of strains to those described above for Figure 4 were grown as before, and assembly of the actin ring was monitored in time-course experiments by staining fixed cells with rhodamine phalloidin. (A) In control cells, actin patches accumulate in the new bud until mitosis (1), before depolarising throughout the cell during anaphase when the actin ring assembles (2; marked by arrow). Contraction of the actomyosin ring is associated with the accumulation of actin patches on either side of the bud-neck (3). The scale bars correspond to 2 µm. (B) The proportion of bi-nucleate cells was determined throughout two sets of time-course experiments involving inactivation of Hof1-td and Cyk3-td (i), or combination of Hof1-td with rvs167 or rvs167-SH3 (ii). Samples from the same time-course experiments were stained with rhodamine phalloidin and used to determine the proportion of cells with actin rings (C-D) and the percentage of cells that failed to complete cytokinesis (E). Images of hof1-td cyk3-td and hof1-td rvs167 cells that failed to complete cytokinesis are shown in panel (F), in which the scale bars correspond to 2 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585203&req=5

pone-0057846-g005: Assembly of the actin ring is defective in cells lacking both Hof1 and Rvs167.A similar range of strains to those described above for Figure 4 were grown as before, and assembly of the actin ring was monitored in time-course experiments by staining fixed cells with rhodamine phalloidin. (A) In control cells, actin patches accumulate in the new bud until mitosis (1), before depolarising throughout the cell during anaphase when the actin ring assembles (2; marked by arrow). Contraction of the actomyosin ring is associated with the accumulation of actin patches on either side of the bud-neck (3). The scale bars correspond to 2 µm. (B) The proportion of bi-nucleate cells was determined throughout two sets of time-course experiments involving inactivation of Hof1-td and Cyk3-td (i), or combination of Hof1-td with rvs167 or rvs167-SH3 (ii). Samples from the same time-course experiments were stained with rhodamine phalloidin and used to determine the proportion of cells with actin rings (C-D) and the percentage of cells that failed to complete cytokinesis (E). Images of hof1-td cyk3-td and hof1-td rvs167 cells that failed to complete cytokinesis are shown in panel (F), in which the scale bars correspond to 2 µm.
Mentions: A previous study showed that recruitment of Inn1 to the cleavage site is blocked by treatment of hof1Δ cells with the drug Latrunculin A that depolymerises actin and prevents assembly of the actomyosin ring [23]. It thus seemed possible that a defect in assembling the actin ring could contribute to the defective recruitment of Inn1 in cells lacking Rvs167 and Hof1. In similar experiments to those described above, we used rhodamine-phalloidin staining to monitor the actin cytoskeleton as G1-phase cells passed synchronously through a complete cell cycle at 37°C. In control cells, assembly of the actin ring during mitosis was associated with delocalisation of actin throughout the whole cell, whereas disassembly of the actin ring was followed by the accumulation of actin patches at either side of the bud-neck (Figure 5A).

Bottom Line: This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3.Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors.The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

Show MeSH
Related in: MedlinePlus