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First published online 13 March 2007
doi: 10.1242/jcs.03423

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Synemin is expressed in reactive astrocytes in neurotrauma and interacts differentially with vimentin and GFAP intermediate filament networks

Runfeng Jing^1,*, Ulrika Wilhelmsson^2,*, William Goodwill¹, Lizhen Li², Yihang Pan¹, Milos Pekny^2, and Omar Skalli^1,,

¹ Department of Cellular Biology and Anatomy and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
² Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Göteborg University, Medicinaregatan 9A, 41390 Göteborg, Sweden

View larger version (75K): [in this window] [in a new window]   Fig. 1. Double immunofluorescence staining with anti-synemin (A,C,F,H) and anti-GFAP (B,D,G) of the molecular layer of the dentate gyrus 4 days after entorhinal cortex lesion. Nuclei are stained blue with TOPRO-3. In wild-type mice (A-E), low-magnification images (A,B) show strong synemin and GFAP staining in numerous large cells in the dentate gyrus; at high magnification (C-E) these cells appear to be reactive astrocytes. An overlay image (E) shows colocalization (yellow) of synemin and GFAP. In Gfap-/-Vim-/- mice (F-J), reactive astrocytes were negative for synemin and GFAP (F,G). Some perivascular cells, however, were positive for synemin (H,J); these were also positive for -smooth muscle actin (ASMA) (I,J). Bars, 50 µm (A,B,F,G); 25 µm (C-E,H-J).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Synemin expression levels in the cerebral hemisphere of 2-day-old wild-type (wt) and Gfap-/-Vim-/- (G-/V-) mice. (A) Western blots show that synemin protein levels are markedly lower in G-/V-than wild-type mice (upper band, -synemin; lower band, -synemin) and confirm the absence of GFAP and vimentin in G-/V-mice. Actin control blots demonstrate equal protein loading. (B) A 0.8% ethidium-bromide agarose gel loaded with RT-PCR products obtained after amplification with synemin primers shows two bands corresponding to - and -synemin mRNA in wild-type and G-/V-mice. Lane M shows molecular mass markers, numbers indicate kb. (C) TaqMan real-time PCR amplification curves show no difference in synemin mRNA levels between wild-type mice (circles) and G-/V-mice (triangles). Rn, log fluorescence emission intensity of the reporter dye normalized to that of a passive reference dye. Values are mean ± s.e.m. of four different samples. Horizontal line indicates background fluorescence level.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Synemin mRNA and protein levels in passage 1 astrocyte cultures from the brain of 2-day-old wild-type, Gfap-/-, Vim-/- and Gfap-/-Vim-/- mice. (A) RT-PCR synemin products from wild type (wt) (lane 2), Gfap-/-Vim-/- (G-/V-) (lane 3), Gfap-/- (G-/V+) (lane 4) and Vim-/- (G+/V-) mice (lane 5). In all genotypes, PCR amplified two bands corresponding to - and -synemin mRNA. Lane 1 shows molecular mass markers; numbers to the left indicate size in kb. (B) TaqMan real-time PCR amplification curves show no difference in synemin mRNA levels between wild type (full circles) and the different knockout genotypes (triangles). Rn, log fluorescence emission intensity of the reporter dye normalized to a passive reference dye. Values are mean ± s.e.m., n=4. Horizontal lines indicate a background fluorescence level. (C) Immunoblots showing synemin, GFAP, vimentin and actin protein levels in passage 1 astrocyte cultures. Synemin was present in astrocytes expressing vimentin, but was undetectable in the absence of vimentin, even when GFAP was present. Actin control blots demonstrate equal protein loading. (D) Immunoblots showing the distribution of synemin, GFAP and vimentin in the detergent-insoluble cytoskeletal fraction (P) and the cytosolic fraction (SN).

View larger version (48K): [in this window] [in a new window]   Fig. 4. Superimposed immunofluorescence images of cultured wild-type astrocytes stained with anti-synemin (red) and anti-GFAP (green); in yellow or orange are areas where synemin and GFAP colocalize. Note that these cells also contain vimentin. A and B show that synemin colocalizes with IF in perinuclear (A), intermediate and peripheral cellular regions (B). In the latter, color variations occurring within individual fibers indicate variable ratio of vimentin and synemin. Bars, 5 µm (A); 0.5 µm (B).

View larger version (63K): [in this window] [in a new window]   Fig. 5. Double immunofluorescence with anti-vinculin (A,C) and antibodies against - and -synemin (B,D) on cultured wild-type astrocytes. In these cells, there is no apparent overlap between vinculin and synemin (A,B). In synemin-positive (A) and synemin-negative cells (C,D), the overall distribution of vinculin (C) is comparable. Bar, 5 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 6. (A) Coomassie Blue-stained 4-15% SDS-polyacrylamide gel shows bacterially expressed -synemin (lane 1), -synemin (lane 2), GFAP (lane 3) and vimentin (lane 4) after purification through polymerization/depolymerization cycles and gel filtration; numbers to the left indicate kDa. (B) Overlay assays to assess binding of -synemin to vimentin, GFAP and actin. Upper panel: Coomassie Blue-stained gel demonstrates equal protein loading. Middle panel: western blot incubated with -synemin and then with anti-synemin followed by a secondary antibody. -synemin bound equally well to monomeric GFAP and vimentin but did not bind to actin. Lower panel: similar western blot incubated with anti-synemin followed by a secondary antibody. (C) Dot-blot assay in which decreasing amounts of GFAP, vimentin and actin were adsorbed to nitrocellulose under nondenaturing, nonpolymerizing conditions. Synemin bound similarly to GFAP and vimentin but did not bind to actin.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Coomassie Blue-stained 4-15% SDS-polyacrylamide gels demonstrate changes in synemin sedimentation properties when allowed to copolymerize with vimentin or GFAP (A) or to bind to vimentin or GFAP IFs (B). Numbers indicate molecular mass in kDa. (A) Mixtures of -synemin and buffer, or of -synemin and either vimentin or GFAP were assembled in 8 M urea buffer and dialyzed under conditions that promote the assembly of vimentin and GFAP IFs. After dialysis, the mixtures were ultracentrifuged and proteins in the supernatant (SN) and pellet (P) were analyzed by SDS-PAGE. Most -synemin was recovered in the supernatant in the absence of vimentin or GFAP, but was mostly in the pellet in the presence of these proteins. (B) -synemin was added either to vimentin or GFAP IFs or to polymerization buffer. After a 1-hour incubation, the mixtures were ultracentrifuged, and proteins in the supernatant (SN) and pellet (P) were analyzed by SDS-PAGE. In the presence of vimentin IFs, -synemin was mostly recovered in the supernatant. By contrast, in the presence of GFAP IFs, most -synemin was recovered in the pellet.

View larger version (69K): [in this window] [in a new window]   Fig. 8. SW13-cl2 cells transiently transfected with GFAP (A,B) and cotransfected with GFAP and -synemin (C-H). Confocal microscopy revealed three GFAP staining patterns, consisting of a filamentous network with little evidence of bundling (cell on left in A), or a network with some parts displaying large bundles and coils (cell on right in A and E). In overexpressing cells, GFAP formed large rods or comma-like structures (B,G). In cells with little GFAP bundling, synemin did not associate with the GFAP network, but formed dot-shaped accumulations (D). Synemin did associate with large GFAP bundles (F) or overexpression aggregates (H). In cells where only part of the GFAP network formed large bundles, synemin only associated with those parts (compare E with F). Bar, 10 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 9. SW13-cl2 cells stably transfected with GFAP and transiently transfected with -synemin (A,B) or -synemin (C,D). Double immunofluorescence staining for GFAP (A,C) and synemin (B,D) was performed 48 hours after transfection. Confocal microscopy revealed a filamentous GFAP network in most cells. Neither -nor -synemin associated with this network, instead forming dot-shaped structures not stained by anti-GFAP. Bar, 10 µm.

View larger version (106K): [in this window] [in a new window]   Fig. 10. SW13-cl2 cells stably transfected with GFAP and transiently cotransfected with vimentin and -synemin. Double immunofluorescence staining for vimentin (A) and synemin (B) was performed 48 hours after transfection. Confocal microscopy revealed that synemin associated with the IF network in cells transfected with vimentin. (C,F) SW13-cl2 cells were stably transfected with vimentin and transiently cotransfected with GFAP and -synemin, and double immunofluorescence staining with anti-GFAP (C,E) and anti-synemin (D,F) was performed 48 hours after transfection. In vimentin-containing cells transiently cotransfected with synemin and GFAP, confocal microscopy showed that synemin incorporated into the entire GFAP network (D,F). (E,F) Synemin also incorporated into the most peripheral area of the GFAP network, where it is the least dense. Bars, 10 µm (A-D); 1 µm (E,F).

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