One of the key requirements of this project is to have a functional parM. This is because parM is the ATPase which provides the force for the DNA-DNA separation. There could be multiple reasons it may not work in mammals, eg: misfolding, misfolding + aggregation, undesired post-translational modifications or it could sequestered by some endogenous protein.
I tested the parM from 3 different prokaryotic systems.
Here are my findings:
The parM from pR1 in E. coli does not produce filaments as a GFP fusion (despite previous papers showing that this fusion is compatible with filament formation in prokaryotes):
The parM from pSK41 in S. aureus produces filaments! but… it is too strong! In fact, for this parM I could not recover any cells to image if I add an NLS to the construct (like I did with the other parMs) as this just causes cell death. (this is an interesting side project to cause mechanical nuclear bursting perhaps??)
Finally, the Goldilocks parM was the one from pO26 also from E. coli which produced nice filaments in the nucleus and cytoplasm and in a concentration dependent manner:
van Steensel lab Open Science blog 