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Re-evaluating the role of Tao1 in the spindle checkpoint
Frederick G Westhorpe1, Maria A Diez, Mark D J Gurden
1University of Manchester, UK.
Abstract:
The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.
Insights
Previous studies suggested Tao1 kinase is crucial for the spindle checkpoint. However, this research demonstrates that Tao1 is not a component of the spindle checkpoint, and prior findings were due to off-target effects on Mad2.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The spindle checkpoint ensures accurate chromosome segregation during cell division.
- Tao1 protein kinase was recently proposed as a novel component of this critical checkpoint.
Purpose of the Study:
- To re-evaluate the role of Tao1 protein kinase in the spindle checkpoint.
- To investigate the validity of previous findings implicating Tao1 in spindle checkpoint function.
Main Methods:
- Utilized multiple small interfering RNAs (siRNAs) to repress Tao1 protein levels.
- Performed co-immunoprecipitation assays to assess protein interactions.
- Analyzed protein degradation patterns post-mitosis.
- Conducted rescue experiments by expressing exogenous Mad2 and Tao1.
Main Results:
- Four new siRNAs efficiently repressed Tao1 but did not disrupt the spindle checkpoint.
- Tao1 does not interact with BubR1 and is not rapidly degraded after mitosis.
- The previously reported siRNA against Tao1 also significantly reduced Mad2 protein levels.
- Exogenous Mad2 expression, but not Tao1, rescued the observed spindle checkpoint defects.
Conclusions:
- The spindle checkpoint phenotype previously attributed to Tao1 inhibition is likely due to off-target effects on Mad2.
- Tao1 protein kinase is not a component of the spindle checkpoint.
- Revises the understanding of spindle checkpoint regulation and identifies potential siRNA off-target effects.
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