[Todos] Seminario Enrico Gratton viernes 27/11/09

[Elizabeth Jares-Erijman]

Seminario de Química Orgánica 2009
Viernes 27/11/09, 11 hs, aula de seminario del Departamento de Química  
Orgánica, FCEyN

Expositor: Enrico Gratton
Laboratory for Fluorescence Dynamics; University of California,  
Irvine, USA

Título: Aggregation of Proteins in cells:  the N&B method

Aggregation of misfolded proteins is a hallmark of several  
neurodegenerative diseases such as Huntington's disease.  Here we  
describe the utility of the recently developed Number and molecular  
Brightness method (N&B) to monitor the aggregation process of  
Hungtintin exon 1 (Httex1).  N&B measures the molecular brightness of  
the protein aggregates in the entire cell non-invasively based on the  
fluctuation dynamics at each pixel of an image.  Dynamic imaging by  
the N&B method allowed detection of monomers, oligomers and inclusions  
in different regions of the cell simultaneously at different time  
points. We observed the behavior of Httex1-EGFP, with polyglutamine  
(polyQ) repeats of different lengths (Httex1-97QP-EGFP, Httex1-46QP- 
EGFP, Httex1-25QP-EGFP in transfected COS-7 cells. We found that  
Httex1 is present throughout the cell and that the non-pathogenic  
protein (Httex1-25QP-EGFP) does not aggregate while the pathogenic  
proteins (Httex1-46Q-EGFP and Httex1-97QP-EGFP) form inclusions. We  
establish that the process of nucleation leading to inclusion  
formation has four phases: i) Initially only monomers are present; ii)  
Following an increase in protein concentration, due to protein  
accumulation, small oligomers (8-15 proteins) form throughout the  
cell; iii) At higher protein concentrations, an inclusion is formed in  
the cytoplasm; iv) The inclusion recruits most of the Httex1 protein  
in the cell, including those in the nucleus, leaving only monomers at  
very low concentration.  The protein with 46 glutamine repeats  
(Httex1-46QP-EGFP) aggregated after a longer lag phase (50 hours)  
compared to Httex1-97QP-EGFP (24 hours). In a different cell type,  
ST14A cells, the aggregation process followed similar dynamics.
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