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First published online 19 April 2005
doi: 10.1242/jcs.02339

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Alexander-disease mutation of GFAP causes filament disorganization and decreased solubility of GFAP

¹ Department of Pathology and the Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA
² School of Biological and Medical Sciences, University of Durham, Durham, DH1 3LE, UK
³ Department of Neurobiology, and The Civitan International Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA

View larger version (79K): [in a new window]   Fig. 1. Filament and aggregate patterns after the production of wild-type or mutant GFAP in primary rat astrocytes (A-C) and Cos-7 cells (D-F). (A) Filamentous pattern, 1 day after transfection, wild-type GFAP. (B) Aggregate, 1 day after transfection, wild-type GFAP. (C) Aggregate, 1 day after transfection, mutant GFAP. (D) Filamentous (arrow) and aggregate (arrowhead) patterns formed by wild-type GFAP at 3 days after transfection. (E) Filamentous pattern formed by mutant GFAP at 1 day after transfection. (F) Aggregates formed by mutant GFAP at 3 days after transfection. In all images, transfected GFAP was visualized with SMI21 and a FITC-conjugated secondary antibody. Magnification 900x for all images.

View larger version (125K): [in a new window]   Fig. 2. Filamentous and irregular patterns after expression of wild-type (A,B) and mutant GFAP (C,D) in SW13Vim– cells. (A) Filamentous pattern, 1 day after transfection with wild-type. (B) Filamentous pattern with `diffuse' background 1 day after transfection with wild-type GFAP, visualized with ALD-10, an anti-panGFAP antibody, and a FITC-conjugated secondary antibody. (C) Irregular pattern, 1 day after transfection with mutant GFAP. (D) 3 days after transfection with mutant. GFAP was visualized with SMI21 and a FITC-conjugated secondary antibody. All images represent 1 µm confocal optical sections. Magnification 900x for all images.

View larger version (136K): [in a new window]   Fig. 3. Filamentous, `irregular' and `diffuse' patterns 1 day after co-transfection of wild-type and mutant GFAP in SW13Vim– cells. Filamentous (A), `diffuse' (B) and irregular with a `diffuse' background (C,D) patterns are shown. All images represent 1 µm confocal optical sections. GFAP was visualized with ALD-10 and a FITC-conjugated secondary antibody. Magnification 900x for all images.

View larger version (25K): [in a new window]   Fig. 4. Distributions of patterns after transfection of wild-type and mutant GFAPs into SW13Vim– cells. (A) 1 day after transfection. (B) 3 days after transfection. For each plasmid and time point, cells on three or four coverslips were counted and assigned to a category based on their appearance with standard fluorescence microscopy, using a 100x oil objective. The mean proportions of cells with each pattern type are shown, along with error bars representing one standard deviation. R239C, mutant; wt, wild type; wt + m, co-transfection of wild-type and mutant GFAP plasmids. The difference between the proportion of cells with an exclusively `diffuse' pattern 1 day after transfection of mutant GFAP alone (57.8%) versus after co-transfection of wild-type and mutant GFAP (14.4%) is significant with a P value of 2.6 x 10–4 by Student's t test. After 3 days, the difference remains significant with a P value of 4.5 x 10–4 by Student's t test.

View larger version (37K): [in a new window]   Fig. 5. Solubility properties of wild-type and mutant GFAPs produced in SW13Vim– cells and extracted with Triton X-100 buffer with increasing salt concentrations. Transfected SW13Vim– cells were extracted with a 0.5% Triton X-100 buffer without KCl (A,B) or with buffer also containing either 0.5 M KCl (C,D) or 1.0 M KCl (E,F). Nitrocellulose transfers were probed with an anti-GFAP antibody (Chemicon) (A,C,E) and then stripped and reprobed with an anti-actin antibody (B,D,F). Lanes: S, supernatant fraction; P, pellet fraction after centrifugation at 16,000 g; untxft, untransfected cells; wt, wild-type GFAP; mut, mutant GFAP; c, Triton-X-100-insoluble fraction from primary rat astrocytes, as positive control. Mr, molecular weight markers (60 kDa and 40 kDa).

View larger version (138K): [in a new window]   Fig. 6. Electron microscopy of in-vitro-assembled wild-type and mutant GFAP. Recombinant wild-type (A) and mutant (B) GFAP were assembled in vitro as described. Notice that both samples contain intermediate sized filaments (10 nm) that are morphologically similar. Scale bars, 100 nm.

View larger version (63K): [in a new window]   Fig. 7. Sedimentation assays. Recombinant human wild-type GFAP (WT) and mutant GFAP (R239C) were purified and assembled individually or as a 1:1 (WT+R239C) mixture at 0.5 mg ml–1. After assembly, the samples were centrifuged and the supernatant (S) and pellet (P) fractions analysed by SDS-PAGE.

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