Ndc80 central kinetochore component is a tetrameric elongated complex composed of Ndc80, Nuf2, Spc24 and Spc25 proteins (Fig. 1A) that connects the kinetochore to the spindle microtubules. Almost all the KT subunits are essential for viability in budding yeast, indeed the corresponding TS mutants are lethal at restrictive temperature (34°C). The involvement of Gcn5 in mitotic progression is due to its many roles, among others the acetylation of regulatory proteins and the expressions of several cell cycle-related genes was demonstrated. In an attempt to analyze additional effects of KAT Gcn5 on the kinetochore functions, we carried out a screening of S.cerevisiae TS mutants in kinetochore subunits where we deleted K-acetyltransferase Gcn5.
Loss of Gcn5 induces cell growth in Ndc80 complex conditionally lethal-mutants
Here we show that TS mutants ndc80-1, spc24-1 spc25-1 and spc105-15 of central kinetochore, involved in the interaction with spindle microtubules, grow normally at 28°C and lose viability at non permissive temperatures (34°C). After deletion of KAT-Gcn5 we surprisingly found that the double mutants ndc80-1-gcn5Δ, spc24-1-gcn5Δ and spc25-1-gcn5Δ and spc105-15-gcn5Δ strains were able to rescue the growth arrest even at 34°C (Fig. 1B). To confirm the direct role of Gcn5 in this observation, we re-induced the expression of GCN5 in those double mutants by introducing the centromeric plasmid pRS316 containing a functional copy of Gcn5 (pGcn5) and empty vector pRS316 (vector) was used as control. According to our previous findings, the re-introduction of a functional wild type Gcn5p abolished the growth recovery also at 34°C, while the empty vector control did not (Fig. 1C). These results provide a direct evidence that Gcn5 acts as a strict regulator of the cell cycle during the kinetochore-MT interaction.
Role of the tubulin chaperon subunit Gim3
The chaperon complex Gim, composed by five subunits (Gim1-5) in yeast is involved in the formation of functional α and γ-tubulins, needed for a correct assembly of MTs. In order to understand if the dynamics and the stability of spindle MTs can play a role in the cell growth recovery of defective kinetochore mutants in absence of Gcn5 we disrupted the Gim3 prefoldin, component of the tubulin chaperon Gim complex. Loss of Gim3 abolished the growth recovery in ndc80-1-gcn5Δ mutant, as shown by the growth assay of the triple mutant ndc80-1-gim3Δ-gcn5Δ (Fig. 1D). This result demonstrates that the recovery of aberrant Ndc80 kinetochore in absence of Gcn5 is related to kinetochore-MT interaction and the relevance of acetylation on mitotic spindle with stabilising effects caused by Gcn5 dependent ipoacetylation.
Role of SAC mitotic checkpoint
Next, we wanted to test the component of SAC mitotic checkpont devoted to control the recovery of aberrant or unattached kinetochores by deleting the checkpoint proteins Mad2 and Bub3 in the ndc80-1 mutant. Spot growth assay (Fig. 1E) shows that the absence of Mad2 does not abrogate the growth recovery observed in ndc80-1-gcn5Δ mutant, while the deletion of Bub3 subunit is unable to skip the cell cycle arrest under restrictive temperature conditions. These results suggest that the recovery of growth in absence of Gcn5 in the ndc80-1 mutant is dependent on Mad2 and not Bub3 in the cell. Next, we analyzed cell morphology of the different mutants by optical microscopy, in figure 1F shows the distribution of the different cell types observed: normal G1 and G2 cells, aberrant elongated cells, undivided cell chains and unhealthy cell clumps. Results show that Mad2 deletion increased the number of dividing G1 cells in ndc80-1, while in absence of Bub3 there was a drastic decrease of dividing G1 cells and an enhancement of inviable aberrant or undivided chains indicating defects in cytokinesis. Collectively, the number and type of cell morphologies observed were in full agreement with the growth assay shown above. The recovery obtained both in ndc80-1-mad2Δ and in ndc80-1-gcn5Δ is similar to the triple ndc80-1-mad2Δ-gcn5Δ strain suggesting that, in absence of Gcn5, cells may overcome the Mad2 dependent cell cycle arrest possibly for improved kinetochore-MT interactions.
Production of ndc80Δ-gcn5Δ strain
Based on our collected results showing that the depletion of GCN5 in the conditionally-lethal mutant ndc80-1 is viable, we wanted to push further our assay asking whether we may fully disrupt the essential NDC80 gene in absence of Gcn5 (gcn5Δ) and obtain a viable strain. We therefore transformed gcn5Δ strain with the integrative cassette NDC80::HIS. HIS3 trasformants obtained were analyzed by colony PCR analysis was performed to confirm the correct chromosomal integration of NDC80::HIS cassette and the concomitant presence of the GCN5::KAN.MX in the strain (Fig. 1G). According to our predictions, we obtained the double deleted mutant ndc80Δ-gcn5Δ viable both at permissive and restrictive temperatures (28°C and 34°C). This result reinforces the meaning of our findings thus confirming the role of Gcn5 in the control of the central KT Ndc80 complex and its interaction with spindle microtubules during chromosome segregation.