From the beginning of the project in September 2021 until its completion in February 2025, the research has successfully achieved its primary objectives and contributed important findings to the field of mitotic regulation and cancer biology. The work has been focused on characterizing the role of hSpindly protein in the regulation of the Spindly Assembly Checkpoint (SAC) and evaluating its impact on the response of tumor cells to anti-mitotic drugs.
The project began with the identification of phosphorylation sites on hSpindly during mitosis using mass spectrometry. This was followed by the generation of inducible RPE-1 cell lines expressing wild-type and phospho-mutant forms of hSpindly fused to GFP, using the Flp-In T-REx system. These lines allowed precise control over protein expression and were central to all subsequent experiments.
Functional assays confirmed that hSpindly is a positive regulator of SAC activity, capable of recruiting Mad2 to kinetochores even in the absence of canonical scaffold proteins like Bub1 and KNL1. This revealed a novel RZZ-dependent pathway for SAC activation, previously poorly understood. Overexpression of hSpindly was shown to enhance checkpoint activity and sensitize cells to anti-mitotic drugs.
Among several candidate phosphorylation sites, threonine 552 (T552) emerged as critical for SAC function. Mutation of this residue (T552A) revealed major alterations in hSpindly's behavior: reduced kinetochore mobility, loss of oligomerization capacity (confirmed via N&B analysis), and increased resistance to anti-mitotic drugs. These findings strongly suggest that T552 phosphorylation is essential for hSpindly's function in SAC regulation and drug sensitivity.
Therefore, the key achievements and results were:
• hSpindly defined as a novel component of SAC, with the ability to regulate checkpoint signaling independently of the Bub1/KNL1 axis and the cellular response to anti-mitotic drugs.
• Threonine 552 identified as a critical phosphorylation site regulating hSpindly dynamics and SAC functionality.
• Integration of advanced microscopy techniques (RISC, N&B, confocal live-cell imaging) to study protein dynamics in real time.
• The relevance of hSpindly in the cellular response to anti-mitotic drugs.
The project´s results have been disseminated through presentations at international conferences, departmental seminars and sessions with Master’s students at the University of Seville, formal meeting with clinical research groups and several outreach activities.
The results obtained during this project provide a solid foundation for future translational research and commercial applications. The identification of hSpindly as a novel regulator of the Spindle Assembly Checkpoint (SAC) and the discovery that phosphorylation at threonine 552 is critical for its function open new opportunities for the development of diagnostic tools (such as a phospho-specific antibody) and targeted therapies for cancer.
This exploitation strategy aligns with the EU’s objectives to foster innovation in the health sector, promote personalized medicine, and reinforce the competitiveness of the European research and biotech landscape.
