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. 2009 Jun;8(6):852-7.
doi: 10.1128/EC.00379-08. Epub 2009 Apr 10.

Formation of Hirano bodies after inducible expression of a modified form of an actin-cross-linking protein

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Formation of Hirano bodies after inducible expression of a modified form of an actin-cross-linking protein

Juan F Reyes et al. Eukaryot Cell. 2009 Jun.

Abstract

Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 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 to a green fluorescent protein (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 fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem 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.

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Figures

FIG. 1.
FIG. 1.
Rhodamine phalloidin-stained cells. Row A, DXA-CT-GFP cells continuously produce large amounts of CT-GFP protein and form Hirano bodies. Row B, cells containing the pRNR-CT-GFP sequence show inclusions, which colocalize with F-actin and GFP. Row C, cell lines expressing the CT sequence alone from the RNR promoter show inclusions visible after phalloidin staining. UV light exposure did not have a discernible effect on AX2 cells. All cell lines, including AX2 (row D) wild-type populations, show the presence of F-actin in the cell cortex and other regions of dynamic actin activity. (Scale bar, 10 μm.) Images were taken at 24 h after promoter induction.
FIG. 2.
FIG. 2.
TEM of AX2 cells expressing CT-GFP. (A) Cell expressing CT-GFP from a constitutively active promoter. (B) RNR-CT-GFP cells at 24 h after UV exposure. Note the highly ordered structures present in the cytoplasm of both types of cells. Scale bar, 200 nm.
FIG. 3.
FIG. 3.
FACS analysis of the GFP signal intensity in RNR-CT-GFP cells after exposure to UV light. Both the percentage of cells with GFP signal above background (gray bars) and the mean fluorescence intensity of the population (black bars) increases over the first 6 hours after promoter induction. Each bar represents measurement of 10,000 cells at the time point listed on the x axis. The error bars represent the standard errors of the means.
FIG. 4.
FIG. 4.
Time-lapse images of live cells expressing pRNR-CT-GFP. Cells were induced with UV light and incubated for 3 h. A sub-area of a larger image was selected to keep the central cell in the center of the frame. Note the large inclusion (arrow) that forms in the central cell as smaller inclusions merge. Time is represented in seconds after the beginning of image collection.

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References

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