Detection of presenilin-1 homodimer formation in intact cells using fluorescent lifetime imaging microscopy

Presenilin-1 (PS1) is a multipass transmembrane domain protein, which is believed to he the catalytic complex. The complex is comprised of four major components: PS 1. nicastrin, Aph- 1. and Pen-2. The exact stoichiometric relationship between the four components remains unclear. It has been shown that gamma-secretase exists is high molecular weight complexes. suggesting the possibility of dimer/multimer formation. We combined a biochemical approach with it novel morphological microscopy assay to analyze PS1 dimer formation and subcellular distribution in situ. in intact mammalian cells. Both coimmunoprecipitation and fluorescent lifetime imaging microscopy approaches showed that wildtype PS1 Molecules form dimers. Moreover, PS1 holoproteins containing the D257A mutation also come into close enough proximity to form a dimer, suggesting that cleavage within the tool is not necessary for dimer formation. Taken together these data suggest that PSI dimerization occurs during normal PS1 function, (c) 2005 Elsevier Inc. All rights reserved.