Kinetochore localized Mad2 and Cdc20 is itself insufficient for triggering the mitotic checkpoint when Mps1 is low inDrosophila melanogasterneuroblasts

Abstract

The relationships between the kinetochore and checkpoint control remain unresolved. Here, we report the characterization of the in vivo behavior of Cdc20 and Mad2 and the relevant spindle assembly checkpoint (SAC) functions in the neuroblasts of a Drosophila Mps1 weak allele (aldB4–2). aldB4–2 third instar larvae brain samples contain only around 16% endogenous Mps1 protein, and the SAC function is abolished. However, this does not lead to rapid anaphase onset and mitotic exit, in contrast to the loss of Mad2 alone in a mad2EY mutant. The level of GFP-Cdc20 recruitment to the kinetochore is unaffected in aldB4–2 neuroblasts, while the level of GFP-Mad2 is reduced to just about 20%. Cdc20 and Mad2 display only monophasic exponential kinetics at the kinetochores. The aldB4–2 heterozygotes expressed approximately 65% of normal Mps1 protein levels, and this is enough to restore the SAC function. The kinetochore recruitment of GFP-Mad2 in response to SAC activation increases by around 80% in heterozygotes, compared with just about 20% in aldB4–2 mutant. This suggests a correlation between Mps1 levels and Mad2 kinetochore localization and perhaps the existence of a threshold level at which Mps1 is fully functional. The failure to arrest the mitotic progression in aldB4–2 neuroblasts in response to colchicine treatment suggests that when Mps1 levels are low, approximately 20% of normal GFP-Mad2, alongside normal levels of GFP-Cdc20 kinetochore recruitments, is insufficient for triggering SAC signal propagation.

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

“…As in human and budding yeast ( Vázquez-Novelle and Petronczki, 2010 ; Aravamudhan et al, 2015 ), Drosophila kinetochores retain a fraction of Mps1 even after the formation of end-on attachments that exert proper kinetochore tension. Intriguingly, although low levels of Mps1 were shown to be sufficient to delay anaphase onset ( Herriott et al, 2012 ; Foijer et al, 2014 ), the residual pool of Mps1 at metaphase kinetochores does not prevent SAC silencing. Work in budding yeast proposes that this is due to changes on kinetochore architecture imposed by end-on attachments.…”

The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

“…6). In flies there appears to be only a fast pool of Cdc20, which might reflect a difference in how BubR1 interacts with kinetochores 34,37 .…”

Mps1 is SUMO-modified during the cell cycle

“…Mps1 ( mono-polar spindle 1 ) is an evolutionarily conserved protein that functions as a key component of SAC [ 9 , 10 ]. Specifically, Mps1 plays an essential role in recruiting Mad1 and Mad2 to unattached kinetochores, thus mediating proper chromosome congression and accurate chromosome segregation [ 11 – 17 ]. Given its essential role in SAC functions, Mps1 undergoes a dynamic distribution during mitosis [ 10 , 18 ].…”