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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Molecular and Cellular Biology
Year Degree Awarded
Month Degree Awarded
The kinetochore protein Spc105R (DmSpc105R) is a large intrinsically disordered protein (IDP) that recruits spindle assembly checkpoint (SAC) proteins and is required for SAC signaling in a conserved manner. Chromosome biorientation satisfies the SAC and while it has been proposed that SAC satisfaction may require the establishment of stable kinetochore-MT (KT-MT) attachments and tension generation, the question of whether tension directly regulates SAC signaling is unresolved and controversial. Here we present data in support of Spc105R as a tension-sensing IDP that directly regulates checkpoint protein localization and signaling independent of the establishment of stable KT-MT attachments. The N-terminus of DmSpc105R binds directly to MTs via electrostatic interaction. The affinity of this interaction was measured in vitro (429 nM +/-190.9 nM) and required at least two distinct binding motifs. Pull-down experiments with purified components demonstrated that the checkpoint protein Bub3 directly binds to the central disordered region of DmSpc105 in the absence of phospho-regulation due to the presence of divergent, "phospho-mimetic" Bub3-interacting motifs in DmSpc105R. Deletion of the N-terminal MT-binding region of Spc105R (Spc105R DeltaN) resulted in a 104 +/-15-minute delay in metaphase compared to an 8.7 +/- 0.8-minute metaphase duration in control cells. Spc105R DeltaN-expressing cells retained 2-fold higher levels of Bub3 at bioriented KTs that were hyper-stretched relative to controls. Importantly Spc105R DeltaN-expressing cells established KT-MT attachments that were equally or more stable, as measured by PA-GFP-a-tubulin turnover, than KT-MT attachments in control cells. The metaphase delay was partially rescued upon introduction of either the MT-binding protein Tau or a protein phosphatase 1 (PP1) binding motif to the N-terminus of Spc105R N. The delay was fully rescued when the N-terminus of Spc105R was replaced with both the PP1 binding motif and Tau. Single molecule experiments are ongoing to directly test if physiological force application to DmSpc105R reduces its affinity for checkpoint proteins. We propose that Spc105R acts as a tension sensor to directly regulate SAC signaling independent of KT-MT attachment stability. More specically, we posit that the central IDP domain of DmSpc105R binds SAC proteins under low tension, and when its N-terminus associates with dynamic MTs, tension-generation reduces its affinity for checkpoint proteins. We see no reason why this mechanical mechanism would not be conserved beyond Drosophila in systems where the focus has been exclusively on the phospho-regulation of Spc105R.
Audett, Margaux, "MECHANISMS OF MITOTIC CHECKPOINT SILENCING BY THE DISORDERED KINETOCHORE PROTEIN SPC105" (2021). Doctoral Dissertations. 2266.
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