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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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Actin ring assembly still occurs following inactivation of Arp2/3.(A) The indicated strains were arrested in G2-M phase with nocodazole at 24°C, before expression of GAL-UBR1 and incubation at 37°C for 60’ to inactivate Hof1-td and Arp2-2. Cells were then released at 37°C into fresh medium lacking nocodazole, and processed as described above for Figure 5. (B) Images of cells with actin rings (marked by arrows), from the 30-minute timepoint in the experiment in (A). The scale bars correspond to 2 µm.
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pone-0057846-g006: Actin ring assembly still occurs following inactivation of Arp2/3.(A) The indicated strains were arrested in G2-M phase with nocodazole at 24°C, before expression of GAL-UBR1 and incubation at 37°C for 60’ to inactivate Hof1-td and Arp2-2. Cells were then released at 37°C into fresh medium lacking nocodazole, and processed as described above for Figure 5. (B) Images of cells with actin rings (marked by arrows), from the 30-minute timepoint in the experiment in (A). The scale bars correspond to 2 µm.

Mentions: We found that assembly of the actin ring was also abolished in hof1-td rvs167-ΔSH3 cells (Figure 5C, D), consistent with the fact that the SH3 domain of Rvs167 becomes essential in the absence of Hof1, and suggesting that the SH3 domain of Rvs167 might help to recruit some factor that contributes to assembly of the actomyosin ring. Previous work indicated that the SH3 domain of Rvs167 interacts with regulators of the Arp2/3 complex (Figure S1), though Arp2/3 generates branched actin fibres that are not thought to contribute directly to assembly of the actin ring, in contrast to the structures generated by formin and IQGAP [41]. To test directly whether Arp2/3 played a redundant role with Hof1 in actomyosin ring assembly, we performed similar experiments to those above, with control, hof1-td, the temperature sensitive arp2-2 allele and hof1-td arp2-2 cells. As Arp2/3 is required for the assembly of actin patches and thus for polarised growth, so that bud assembly is blocked at 37°C in arp2-2 cells (data not shown), we synchronised cells after bud formation in G2-M phase by addition of nocodazole. The cultures were then shifted to 37°C to inactivate Arp2-2 and deplete Hof1-td, before release into fresh medium lacking nocodazole. As shown in Figure 6A (i), inactivation of either Hof1 or Arp2 at 37°C blocked the completion of cell division. However, actin rings formed in all four strains (Figure 6A (ii) and 6B; the frequency of cells with actin rings was somewhat lower in hof1-td arp2-2), whereas the subsequent formation of actin patches at the bud-neck was blocked upon inactivation of Arp2 (Figure 6A (iii)). These data indicated that Arp2/3 was not essential for actin ring formation in the absence of Hof1, suggesting that another partner of the SH3 domain of Rvs167 becomes essential for actin ring formation in the absence of Hof1.


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)

Actin ring assembly still occurs following inactivation of Arp2/3.(A) The indicated strains were arrested in G2-M phase with nocodazole at 24°C, before expression of GAL-UBR1 and incubation at 37°C for 60’ to inactivate Hof1-td and Arp2-2. Cells were then released at 37°C into fresh medium lacking nocodazole, and processed as described above for Figure 5. (B) Images of cells with actin rings (marked by arrows), from the 30-minute timepoint in the experiment in (A). The scale bars correspond to 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057846-g006: Actin ring assembly still occurs following inactivation of Arp2/3.(A) The indicated strains were arrested in G2-M phase with nocodazole at 24°C, before expression of GAL-UBR1 and incubation at 37°C for 60’ to inactivate Hof1-td and Arp2-2. Cells were then released at 37°C into fresh medium lacking nocodazole, and processed as described above for Figure 5. (B) Images of cells with actin rings (marked by arrows), from the 30-minute timepoint in the experiment in (A). The scale bars correspond to 2 µm.
Mentions: We found that assembly of the actin ring was also abolished in hof1-td rvs167-ΔSH3 cells (Figure 5C, D), consistent with the fact that the SH3 domain of Rvs167 becomes essential in the absence of Hof1, and suggesting that the SH3 domain of Rvs167 might help to recruit some factor that contributes to assembly of the actomyosin ring. Previous work indicated that the SH3 domain of Rvs167 interacts with regulators of the Arp2/3 complex (Figure S1), though Arp2/3 generates branched actin fibres that are not thought to contribute directly to assembly of the actin ring, in contrast to the structures generated by formin and IQGAP [41]. To test directly whether Arp2/3 played a redundant role with Hof1 in actomyosin ring assembly, we performed similar experiments to those above, with control, hof1-td, the temperature sensitive arp2-2 allele and hof1-td arp2-2 cells. As Arp2/3 is required for the assembly of actin patches and thus for polarised growth, so that bud assembly is blocked at 37°C in arp2-2 cells (data not shown), we synchronised cells after bud formation in G2-M phase by addition of nocodazole. The cultures were then shifted to 37°C to inactivate Arp2-2 and deplete Hof1-td, before release into fresh medium lacking nocodazole. As shown in Figure 6A (i), inactivation of either Hof1 or Arp2 at 37°C blocked the completion of cell division. However, actin rings formed in all four strains (Figure 6A (ii) and 6B; the frequency of cells with actin rings was somewhat lower in hof1-td arp2-2), whereas the subsequent formation of actin patches at the bud-neck was blocked upon inactivation of Arp2 (Figure 6A (iii)). These data indicated that Arp2/3 was not essential for actin ring formation in the absence of Hof1, suggesting that another partner of the SH3 domain of Rvs167 becomes essential for actin ring formation in the absence of Hof1.

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