Haspin Modulates the G2/M Transition Delay in Response to Polarization Failures in Budding Yeast

Abstract

Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of Saccharomyces cerevisiae cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In alk1Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in Saccharomyces cerevisiae, where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition.

Keywords: Saccharomyces cerevisiae; actin cytoskeleton; cell cycle; mitosis; morphogenesis checkpoint; polarization.

Figure 1

Haspin mutants show altered response to polarity failures. (A) Serial dilutions of cultures of the indicated strains were spotted on either DMSO or LatA containing plates. After 24 h incubation at 28°C plates were imaged. (B) Cells of the indicated strains expressing Tub1-GFP were arrested in G1 with mating pheromone and then released in the presence of 100 μM LatA. After 4 h cells were fixed and analyzed for nuclear division by fluorescence microscopy. (C) Cells were arrested in G1 at the permissive temperature (25°C), shifted for 45' at the restrictive temperature (37°C) and then released at 37°C. After 2 h, samples were fixed and analyzed for nuclear division, as above. Error bars in (B,C) represent standard deviation, statistical significance was measured by T-test, ns: not significant, **p < 0.01, ***p < 0.005.

Figure 2

Alk1 regulates cell cycle progression through Mih1. Cells of the indicated strains were arrested in G1 at 25°C and held at 37°C for further 45' before being released at 37°C. After 2 h samples were taken and nuclear segregation was evaluated by fluorescence microscopy (A) For wt, alk1Δ, alk2Δ and alk1Δalk2Δ, the mean value was calculated on data from experiment in Figure 1C combined with new biological replicates performed together with the other indicated strains. (B) Cells of the indicated strains were synchronized in G1 and treated with LatA. Following release, samples were taken to monitor nuclear division by fluorescence microscopy. Representative images at the 120' time point are shown (C) Cells were treated as in A, taking samples at the indicated time points to monitor nuclear segregation or cell-cycle progression (Supplementary Figure 2). Error bars represent standard deviation, statistical significance was measured by T-test, ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 3

Alk1 does not modulate Mih1 post-translational modifications. (A) Cells of the indicated strains were synchronized in G1 at permissive temperature and held at 37°C for further 45' before being released into the cell cycle at restrictive temperature. Samples were taken every 15' to follow protein levels and modifications by western blot. The graph shows the relative Mih1-3HA abundance normalized on tubulin levels. Error bars represent standard deviation.

Figure 4

Alk1 is required for sustained Cdc28-Y19 phosphorylation. (A) Cells of the indicated strains were synchronized in G1 at permissive temperature and held at 37°C for further 45' before being released into the cell cycle at restrictive temperature. Samples were taken every 20' to follow protein levels by western blot using antibodies for total Cdc28 and phosphospecific antibodies for Cdc28-Y19p. The right panel reports the quantification of the phosphorylation signals normalized over the total Cdc28. Error bars represent standard deviation (B) Logarithmically growing cells, bearing the pGAL-GST or pGAL-GST-ALK1 constructs, were incubated in the presence of 2% galactose to induce protein overexpression. Samples were taken every hour to monitor protein levels. Western blotting was performed with antibodies for total Cdc28 and phosphospecific antibodies for Cdc28-Y19p. Expression levels of GST and GST-Alk1 were analyzed with anti GST antibodies. The graph shows the ratio between phosphorylated Cdc28-Y19 and total Cdc28, error bars represent standard deviation.