Suppressors of Spindle Checkpoint Defect (such) Mutants Identify New mdf-1/MAD1 Interactors in Caenorhabditis elegans

Abstract

The spindle assembly checkpoint (SAC) governs the timing of metaphase-to-anaphase transition and is essential for genome stability. The Caenorhabditis elegans mutant strain gk2 carries a deletion within the mdf-1/MAD1 gene that results in death of the homozygous strain after two or three generations. Here we describe 11 suppressors of the mdf-1(gk2) lethality, 10 identified in an ethyl methanesulfonate (EMS) mutagenesis screen and 1 isolated using the dog-1(gk10) (deletions of guanine-rich DNA) mutator strain. Using time-lapse imaging of early embryonic cells and germline mitotic division, we demonstrate that there are two classes of suppressors. Eight suppressors compensate for the loss of the checkpoint by delaying mitotic progression, which coincides with securin (IFY-1/Pds1) accumulation; three suppressors have normal IFY-1/Pds1 levels and normal anaphase onset. Furthermore, in the class of suppressors with delayed mitotic progression, we have identified four alleles of known suppressors emb-30/APC4 and fzy-1/CDC20, which are components of the anaphase-promoting complex/cyclosome (APC/C). In addition, we have identified another APC/C component capable of bypassing the checkpoint requirement that has not previously been described in C. elegans. The such-1/APC5-like mutation, h1960, significantly delays anaphase onset both in germline and in early embryonic cells.

Functional Redundancy of Paralogs of an Anaphase Promoting Complex/Cyclosome Subunit in Caenorhabditis elegans Meiosis

“…All other model organisms contain only a single copy of APC5. Identity between each C. elegans paralog and the ortholog in human, Drosophila, and S. cerevisiae is comparable, indicating that both C. elegans paralogs are equally similar to APC5 (Tarailo et al 2007).…”

“…which is 38% identical and 71% similar to SUCH-1 at the amino acid level, including identity at the site that is mutant in av9gf (for an amino acid alignment, see Tarailo et al 2007). All other model organisms contain only a single copy of APC5.…”

“…Therefore, we examined the such-1 loss-of-function phenotype. Two loss-of-function alleles exist for such-1, a penetrant paternal-effect lethal mutation, such-1(t1668) (Gö nczy et al 1999, Bezler and Gö nczy 2010) and such-1(h1960ts), a point mutant that exhibits near-normal hatching at 20°but reduced hatching and brood size at 25° (Tarailo et al 2007). such-1(h1960ts) embryos that fail to hatch arrest at a range of embryonic stages, from rare one-cell embryos to multicellular embryos.…”

“…RNAi-mediated knockdown of individual APC/C subunits in late larval hermaphrodites also leads to one-cell meiotic arrest with the exception of two subunits, APC5 and APC10 (Davis et al 2002), which exhibit arrest at a multicellular stage when depleted. It recently has been established that there are two genes encoding each of these subunits in C. elegans (Tarailo et al 2007). Therefore, APC5 and APC10 may not exhibit an RNAi meiotic 1 arrest phenotype because the two paralogs of each subunit might function redundantly at the meiotic divisions.…”

“…The SAC mutants probably suppress mat-3(or180ts) by increasing the level of APC/C activity via a reduction of the strength of the spindle checkpoint. The interdependence of the regulation of the SAC and the APC/C is demonstrated by reciprocal studies in which a loss-of-function in the SAC can be suppressed by reduction-of-function APC/C mutants (Furuta et al 2000;Tarailo et al 2007). Further, Bezler and Gö nczy (2010, accompanying article in this issue) assert that proper mitotic timing is achieved through the negative regulation of the SAC by the APC/C.…”