EC Accepts, published online ahead of print on 10 April 2009

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Eukaryotic Cell doi:10.1128/EC.00379-08
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Formation of Hirano Bodies After Inducible Expression of a Modified Form of an Actin-Crosslinking Protein

Juan Reyes, Karen Stone, Jeanie Ramos, and Andrew Maselli^*

Chicago State University, Department of Biological Sciences, 9501 S. King Drive, Chicago Illinois 60628

^* To whom correspondence should be addressed. Email: amaselli{at}csu.edu.

Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin associated proteins. The formation of Hirano bodies has been reported in various neurodegenerative disorders including Alzheimer's Disease and amyotrophic lateral sclerosis. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain is not known, expression of the C-terminal fragment (CT, aa 124-295) from the endogenous 34 kDa actin binding protein of Dictyostelium discoideum leads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano Body formation using an inducible promoter system (rnrB). By fusing the CT fragment to a fluorescent probe (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry and western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fuse to form a single large aggregate. Transmission electron microscopy (TEM) further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano Bodies observed in post-mortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model.