Protein aggregation in live cells: N&B analysis of Huntingtin

Abstract

The aggregation of the huntingtin (Htt) protein is thought to be responsible for Huntington's disease. One problem is the detection of the different size of aggregates and the stages of aggregation directly in live cells. Recently we develop a method (N&B) to detect the size of aggregates and the number of particles in every pixel of a confocal image. N&B can be performed on the entire cells simultaneously so it is possible to follow the kinetics of aggregation. This method is based on the variance of intensity fluctuations of particles as they diffuse through the excitation volume. We performed experiments in transfected COS7 using different lengths of the polyglutamine sequence (Httex1 97QP-GFP, Httex1 46QP-GFP and Httex1 25QP-GFP). We can determine the presence of units, small aggregates and inclusion bodies in different parts of cell and their time evolution.. We observed the presence of Htt throughout the cell and that oligomer formation starts in the cytoplasm. At higher protein concentration aggregation occurs in the nucleus. When an inclusion body forms in the cytoplasm, all Htt including that in the nucleus, is recruited by the inclusion body. We found aggregates, which have a size of 5-10 protein units, diffusing in membrane tubules. We suggest that these aggregates my represent the precursors for protein nucleation events. We estimated that the inclusion body is formed by hundred of protein units. In different cell types the aggregation follows a similar dynamic. Our studies have shown the protein aggregation size distribution in live and how the nucleation events progress in real time. Support by U54 GM064346 CMC (MD, EG), NIH-P41-RRO3155 (EG), P50-GM076516 (EG,GO).