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Formation of Hirano bodies in Dictyostelium and mammalian cells induced by expression of a modified form of an actin-crosslinking protein

Andrew G. Maselli, Richard Davis, Ruth Furukawa and Marcus Fechheimer*

Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA



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  		Fig. 1. Expression of full-length 34 kDa-myc and CT-myc proteins in Dictyostelium. (A,B) Lysates of CT-myc expressing cells (lane 1), wild-type AX-2 (lane 2), and cells expressing full-length 34 kDa-myc (lane 3) were resolved by SDS-PAGE, blotted to nitrocellulose, and probed with monoclonal antibody B2C, which is reactive to native 34 kDa protein (A), or monoclonal antibody 9E10, which is reactive to the myc epitope tag (B). Note that 34 kDa-myc and CT-myc proteins are expressed at levels roughly equivalent to and significantly less than the endogenous 34 kDa protein, respectively. (C) Nucleotide and amino acid sequence at the translational start site of the CT protein. The wild-type sequence (WT), the frame shift (FS) resulting from deletion of an adenosine (arrow), and the alternative ATG methionine codon presumed to initiate synthesis of the CT-myc protein are shown.

 


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  		Fig. 2. Intracellular localization of F-actin and myc epitope in wild-type cells, and cells expressing 34 kDa-myc and CT-myc proteins. Wild-type AX-2 (A), 34 kDa-myc- (B) and CT-myc (C)-expressing cells were observed by DIC (left), and stained with rhodamine-labelled phalloidin (middle) and antibody to the myc-epitope tag (right). There is extensive cortical staining of F-actin in the AX-2 and 34 kDa-myc cells (A,B). By contrast, the CT-myc cells reveal ellipsoids that label strongly for actin and for the myc epitope (C). The asterisks indicate the ellipsoids. Bar, 10 µm.

 


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  		Fig. 3. Intracellular localization of F-actin, 34 kDa protein, and myc epitope tag in CT-myc cells. CT-myc cells were viewed by DIC microscopy (A,B), stained with phalloidin to localize F-actin (C,D), and stained with antibody to localize the 34 kDa protein (F), or myc epitope tag (E). F-actin and the 34 kDa protein epitopes are present in both the ellipsoids and the cortical regions of the CT-myc cells. By contrast, the myc epitope is largely confined to the ellipsoids in CT-myc cells. Bar, 10 µm.

 


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  		Fig. 4. Localization of tubulin, myosin, cofilin, {alpha}-actinin, ABP 120 and EF 1-{alpha} in CT-myc cells. In each set of images, the cells were viewed by DIC microscopy (left), stained with phalloidin to localize F-actin (middle), and stained with antibody to localize specific cytoskeletal proteins (right). (A) {alpha}-tubulin; (B) myosin II; (C) cofilin; (D) {alpha}-actinin; (E) ABP 120; (F) EF 1-{alpha}. The CT-myc-induced ellipsoids are enriched for actin, myosin II, cofilin and {alpha}-actinin, but not for {alpha}-tubulin, ABP 120, and EF-1{alpha}. Bar, 10 µm.

 


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  		Fig. 5. Ultrastructure of the CT-myc induced ellipsoid.

(A) Micrograph of CT-myc-expressing cell, with a large inclusion with herringbone pattern (bar, 1 µm). (B,C) High magnification tilt images of ellipsoid inclusion at 0° tilt and 45° tilt for panels B and C, respectively (bar, 10 nm). An asterisk marks the region in A that is shown in B and C.

 


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  		Fig. 6. Transverse and longitudinal sections reveal the organization of the actin filaments in ellipsoids of CT-myc cells. (A) Note regular inclusion in cytoplasm revealing square packed and hexagonally packed organizations. (B) Higher magnification of the lower right region of the ellipsoid in A. (C) Longitudinal section of 10 nm filaments in an inclusion. (D) Cross-section of square packed filaments. Bar, 0.5 µm (A); 0.1 µm (B); 50 nm (C,D).

 


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  		Fig. 7. Rate of development of wild-type AX-2 (top), 34 kDa-myc (middle), and CT-myc (bottom) cells. Note that CT-myc-expressing cells are delayed in development by 3-6 hours compared with wild-type and 34 kDa-myc cells.

 


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  		Fig. 8. Formation of Hirano Bodies in L cells expressing the CT Fragment. The distribution of F-actin was determined by staining with oregon green 488-labelled phalloidin as described in Materials and Methods. (A) Cortical actin, stress fibers and few punctate foci characterize the distribution of actin filaments in control cells. (B) Cells transfected with CT fragment exhibit loss of stress fibers and accumulation of punctate F-actin foci. (C-E) Some cells transfected with CT exhibit large elliptical inclusions highly enriched in F-actin that resemble Hirano bodies (marked with arrowheads) (bar, 10 µm). (F) L cells were mock transfected (gray bars) and CT transfected (black bars) and scored for F-actin distribution as: normal (N), most of the actin in cortical arrays and stress fibers; multiple foci (M), most of the F-actin in multiple punctate foci; aggregate (A), contain a large elliptical aggregate resembling a Hirano body. Results are shown as the mean±s.e.m. of six counts from three independent transfections of mock and CT cells. The CT-transfected cells have fewer and less prominent stress fibers and accumulate multiple foci and large aggregates of F-actin.

 

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