So far, we have made plasmid constructs of mutant Huntingtin displaying varying lengths of polyglutamine repeats (25Q, 72Q, and 103Q) with and without a proline rich region (PRR) tagged with green fluorescent protein (GFP). The 25Q is our control because the strain does not exhibit toxicity. We have found that the 72Q and the 103Q strains with and without the PRR exhibit toxicity by observing large inclusion bodies (IBs) of Huntingtin aggregates through fluorescent microscopy.
Next we observed the effects of the molecular chaperone, Hsp42, on mutant Huntingtin and have found that in Wild-Type cells, the IBs were less intense than in Hsp42null cells indicating that Hsp42 is necessary in clearing mutant Huntingtin aggregates.
We then tagged Hsp42 with another red fluorescent protein (mCherry) and found that the molecular chaperone localizes with the mutant Huntingtin aggregates.
We are currently in the process of tagging the spindle pole body with mCherry in the 72Q strains in order to determine whether the mutant Huntingtin aggregates localize by means of the spindle pole body while forming an aggresome. Results should be within a week or so.
This week we are constructing a new plasmid which is inducible so that we can essentially “turn on and off” the amount and degree of mutant aggregates formation. We hope this will allow us to monitor how cells process mutant aggregates through the ubiquitin-proteasome system.