New preprint (TbMad2, TbAUK3)

Here is my preprint reporting 9-year-old observations with lots of speculations about what they might mean. I would like to see how bioRxiv “publication” with post-publication peer review works, so any comments are welcome on the bioRxiv website.

Spindle checkpoint is a quality control system in eukaryotes that ensures that cells separate chromosomes only after all chromosomes have formed proper kinetochore-microtubule attachments. Since its discovery in 1991 in budding yeast, there are lots of studies in popular model eukaryotes such as yeasts and human but these organisms represent only a fraction of eukaryotic diversity (Opisthokonta). Not much is known about spindle checkpoint in evolutionary divergent eukaryotes. Trypanosoma brucei is an evolutionary divergent eukaryote that belongs to kinetoplastid, Euglenozoa, Discoba (previously called Excavata). With lots of tools established, it is a great organism to study conservation/divergence of various biological functions. Among various spindle checkpoint components identified in yeasts/human, only one protein is identifiable in T. brucei, TbMad2. Its protein sequence looks very much like Mad2 rather than other HORMA domain containing proteins such as Hop1 and Rev7.

So when I started working in Keith Gull’s lab to identify its kinetochore component in T. brucei (none was known at that time), I first tagged TbMad2 with YFP with a hope that its immunoprecipitation would co-purify with kinetochore proteins. However, YFP-TbMad2 localized at near basal bodies (which locate outside of the nucleus). Because it was the first protein I tagged in trypanosomes, I thought I screwed something so made TbMad2-YFP, which again showed only basal body signal. Instead I found another basal body protein (named TbMBP65 in this preprint) and several components of the 26S proteasomes. I didn’t know what’s going on but decided to take a different approach to identify kinetochore protein. To focus on the kinetochore identification project, I didn’t do anything more on these dataset, until I decided to write this preprint.

This year (2020), I was going through my random old data at home (due to covid-19), I looked at the TbMBP65 sequence more carefully with additional sequences of its homolog and found a possible Mad2-interacting motif found in Mad1/Cdc20. This could mean that TbMBP65 recruits TbMad2 onto basal bodies, just like the way Mad1 recruits Mad2 onto kinetochore in other eukaryotes. What might be the function of TbMad2/MBP65 at basal bodies? While trypanosomes do not appear to have a functional spindle checkpoint (i.e. cells undergo cytokinesis when spindle microtubules are depolymerized), there is a hypothesis that the onset of cytokinesis might be linked to the segregation of basal bodies, proposed by Keith Gull in 1999. So I speculate that TbMad2 might monitor the segregation status of basal bodies (rather than nuclear DNA). It was also interesting to find proteasome regulatory subunits co-purifying with TbMad2, rather than APC/C. Totally speculative, but TbMad2 might regulate proteasome activity.

Given that little is known about whether/how spindle checkpoint functions in divergent eukaryotes, I thought these observations are interesting and worth sharing as a preprint.

Any feedback would be appreciated!

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