Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint

Aktar, K., Davies, T., Leontiou, I., Clark, I., Spanos, C., Wallace, E., Tuck, L., Jeyaprakash, A.A., and Hardwick, K.G.

By Eleanor Casey

Cryptococcus neoformans is an opportunistic human pathogen which can cause severe respiratory illness in people with compromised immune systems, and is responsible for the death of around 20% of AIDS patients. Identifying key proteins and processes in the C. neoformans cell cycle is essential to identify targets for novel therapeutics and drugs to treat this disease. Through conservation analysis Aktar and colleagues identify Mad1 and Mad2 and demonstrate their role in the C. neoformans cell cycle. These proteins have a well characterised function in the spindle assembly checkpoint (SAC) a mechanism which prevents chromosomes from separating in cell division before they are ready. This ensures that the correct number of chromosomes are inherited by the daughter cell.

Firstly, the scientists demonstrate that Mad1 and Mad2 are key components of the C. neoforms SAC, by treating cells in which these proteins are deleted with nocodazole. Nocodazole should prevent healthy cells from dividing, as mitosis cannot be completed in the presence of nocodazole. But the mad1 mad2 deletion cells were able to keep dividing in the presence of this drug, indicating that the SAC is functioning incorrectly. To demonstrate that the role of Mad1 is conserved from other fungal species, the scientists imaged fluorescent tagged Mad1 and show that it also moves from the nuclear periphery in interphase to kinetochores in mitotic metaphase. Co-immunoprecipitation mass spectrometry revealed that the interaction partners of Mad1 are also highly conserved, with it interacting with other SAC proteins and anaphase promoting proteins. Finally, in vitro Mps1 kinase assays were used to identify an Mps1 phosphorylation site on the C terminus of Mad1. When this Mad1 phosphorylation site is mutated, live cells are no longer able to activate their SAC and are susceptible to nocodazole. Overall, this demonstrates that Mps1 and Mad1 Mad2 are conserved between C. neoformans and other fungal species and this provides targets for future therapeutics.