hSpindly’s dynamic controls SAC activity independently of the KBB pathway at unattached kinetochores

The Spindle Assembly Checkpoint (SAC) ensures that sister chromatids do not separate until all chromosomes are properly attached to spindle microtubules and correctly bi-oriented. SAC controls the metaphase-to-anaphase transition by inhibiting the Anaphase Promoting Complex/Cyclosome (APC/C) through the formation of the Mitotic Checkpoint Complex (MCC). A critical step in this process is the recruitment of the Mad1–Mad2 complex to unattached kinetochores. Two pathways are known to mediate this recruitment: the KBB pathway (KNL1–Bub3–Bub1) and the RZZ pathway (Rod– Zw10–Zwilch). Here, we demonstrate that hSpindly plays a central role in controlling the recruitment of the Mad1–Mad2 complex through the RZZ pathway, independently of the KBB pathway. We show that hSpindly is a dynamic protein that oligomerizes at unattached kinetochores. Importantly, we identify a specific residue, threonine 552, as critical for hSpindly’s function and mobility. A non-phosphorylatable mutant (T552A) stabilizes hSpindly at kinetochores, impairs SAC signaling, and increases cellular resistance to antimitotic drugs. Altogether, our findings identify hSpindly as a novel dynamic modulator of SAC functionality via the RZZ pathway and highlight it as a potential therapeutic target for overcoming resistance to mitotic inhibitors.