A link between mitotic entry and membrane growth suggests a novel model for cell size control - PubMed (original) (raw)

A link between mitotic entry and membrane growth suggests a novel model for cell size control

Steph D Anastasia et al. J Cell Biol. 2012.

Abstract

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2A(Cdc55)). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Blocking membrane traffic triggers a checkpoint arrest. (A) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. Cleavage of Mcd1-6×HA was assayed by Western blotting. (B) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. DNA staining was used to determine the percentage of cells with multiple nuclei. (C) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 45 min after release. Levels of Clb2 were assayed by Western blotting. (D and E) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. The percentage of cells with short or long spindles was determined. Error bars represent SEMs for three biological replicates. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 2.

Figure 2.

Normal signaling to Mih1 and Swe1 fails to occur when membrane traffic is blocked. (A and B) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. The behavior of Mih1 and Swe1 were assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 3.

Figure 3.

Blocking membrane traffic triggers rapid signaling to Mih1. (A and B) Cells were released from a G1 arrest and were shifted to the restrictive temperature (34°C) at 70 min after release, when Mih1 dephosphorylation was being initiated. Mih1 phosphorylation was assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 4.

Figure 4.

The response to arrest of membrane traffic is not a consequence of indirect effects on actin. (A) Wild-type cells were released from a G1 arrest at room temperature. A sample was taken at 70 min after release (t = 0), and the cells were divided into two aliquots. Latrunculin A (LatA) was added to one aliquot, and solvent (DMSO) was added to the other as a control. Mih1 phosphorylation was assayed by Western blotting. (B) Cells were grown to log phase in YPD media and then shifted to the restrictive temperature (34°C) for 5 min. Cells were then fixed and stained with FITC-phalloidin. Bar, 5 µm. Numbers shown next to the Western blot indicate molecular mass in kilodaltons.

Figure 5.

Figure 5.

Overexpression of Zds1 drives cells through the checkpoint arrest. (A–C) Cells were grown in YEP with 2% raffinose and arrested in G1 with α-factor at 25°C. Cells were released from the arrest into YEP with 2% raffinose at 25°C. When 10% of cells had undergone bud emergence, they were shifted to the restrictive temperature (34°C) to induce the checkpoint arrest. After 30 min, galactose was added to induce expression of ZDS1. Cleavage of Mcd1-3×HA and phosphorylation of Mih1 and Swe1 were assayed by Western blotting. The asterisks denote background bands that appear with some batches of purified anti-Mih1 antibody. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 6.

Figure 6.

Pkc1 associates with PP2ACdc55 and is required for Mih1 dephosphorylation. (A) Anti-HA antibodies were used to immunoprecipitate PP2ACdc55-3×HA from wild-type, zds1Δ zds2Δ, and untagged control cells. Coprecipitation of Pkc1 was assayed by Western blotting with anti-Pkc1 antibodies. Crude extract samples were electrophoresed longer than the immunoprecipitated samples to resolve phosphorylation forms. (B) Cells were inoculated into YPD media at low density and grown at 30°C until they reached an OD of ∼1.7. Bar, 10 µm. (C) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 75 min after release. Mih1 phosphorylation and Clb2 levels were assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 7.

Figure 7.

Pkc1 signals to Mih1 via PP2ACdc55-Zds1/2. (A) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 30°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. (B) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 25 or 34°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. (C) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Cells were shifted to 34°C for 60 min to induce a checkpoint arrest, and galactose was then added. Mih1 phosphorylation was assayed by Western blotting. (D) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 30°C at t = 0. Zds1 phosphorylation was assayed by Western blotting. The asterisks denote background bands that appear with some batches of purified anti-Mih1 antibody. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 8.

Figure 8.

Rho1 signals to Mih1 via Pkc1. (A) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 35 min after release. Mih1 phosphorylation was assayed by Western blotting. The arrowhead marks a hyperphosphorylated form that appears in the rho1-2 cells. At 34°C, rho1-2 cells grow slowly but are viable. (B) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 45 min after release. Swe1 phosphorylation was assayed by Western blotting. (C) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 45 min after release. Clb2 levels were assayed by Western blotting. (D) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol. Galactose was added, and the cells were shifted to 34°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. The pkc1-21 allele was used because it was found to cause rapid inactivation of Pkc1. (E) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol. The cells were shifted to 34°C for 60 min to induce a checkpoint arrest, and galactose was then added. Mih1 phosphorylation was assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 9.

Figure 9.

Signaling to Pkc1 is dependent on membrane traffic. (A) Cells were released from a G1 arrest at room temperature. The behavior of Pkc1, Cln2-3×HA, and Clb2 were assayed by Western blotting. All blots are from the same samples, so timing of events may be directly compared. (B) Wild-type and sec6-4 cells were released from a G1 arrest. A sample was taken at 70 min (t = 0), and the cells were then shifted to the restrictive temperature (34°C). Pkc1 phosphorylation was assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Figure 10.

Figure 10.

A model for signals that link mitotic entry to membrane growth. (A) Dependency relationships in the Rho1–Pkc1 signaling axis. (B) Known binding interactions in the Rho1–Pkc1 signaling axis. (C) A hypothetical model for generation of a signal that is proportional to membrane growth. Rho1 is activated at the site of membrane growth by a guanine nucleotide exchange factor (GEF). As more Rho1-bearing vesicles are delivered to the site of growth, the amount of active Rho1 increases. Downstream components of the signaling axis read the signal and flip a switch to initiate mitosis when the signal reaches a threshold level.

Comment in

References

    1. Abe M., Qadota H., Hirata A., Ohya Y. 2003. Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae. J. Cell Biol. 162:85–97 10.1083/jcb.200301022 - DOI - PMC - PubMed
    1. Altman R., Kellogg D.R. 1997. Control of mitotic events by Nap1 and the Gin4 kinase. J. Cell Biol. 138:119–130 10.1083/jcb.138.1.119 - DOI - PMC - PubMed
    1. Amon A., Tyers M., Futcher B., Nasmyth K. 1993. Mechanisms that help the yeast cell cycle clock tick: G2 cyclins transcriptionally activate G2 cyclins and repress G1 cyclins. Cell. 74:993–1007 10.1016/0092-8674(93)90722-3 - DOI - PubMed
    1. Amon A., Irniger S., Nasmyth K. 1994. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 77:1037–1050 10.1016/0092-8674(94)90443-X - DOI - PubMed
    1. Anderson C.W., Baum P.R., Gesteland R.F. 1973. Processing of adenovirus 2-induced proteins. J. Virol. 12:241–252 - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources