Characterization and expression of K18 S33A and its binding to 14-3-3ζ in transgenic mice. (A) Liver fragments were isolated from the following mouse lines: nontransgenic (N) (one mouse), TG2 (one mouse), S33A¹ and S33A² (two mice each). Fragments were homogenized in 1% Empigen followed by centrifugation. The clarified lysate was used to immunoprecipitate keratins with mAb L2A1 (which recognizes hK18). Immunoprecipitates were analyzed by Coomassie staining (a) and by immunoblotting with anti-hK18 Abs that recognize K18 pS52 (b), or K18 pS33 (c). (d–f) Alternatively, overall keratin expression level was examined by blotting of total liver protein homogenates with anti-mK8, anti-mK18 (which also crossreacts with hK18), and anti-hK18 (which partially crossreacts with mK18). (B) TG2 mice were injected i.p. with MLR in saline, or saline alone, followed by removal of the livers and then homogenization in Laemmli sample buffer. Equal amounts (10 μg) of the total liver lysates were separated by SDS/PAGE followed by blotting with anti-K18 pS52, anti-K18 pS33, or anti-K8 Ab. A blot of nearly equal amounts of K8/18 precipitates (determined by Coomassie staining) gave a similar blotting pattern (not shown). M1 and M2 correspond to separate mice. (C) TG2, S33A¹, S33A², and S52A mice were injected with MLR followed by removal of the livers, solubilization in 1% Nonidet P-40, then keratin immunoprecipitation with mAb L2A1. Precipitates were analyzed by blotting with antibodies to K18 pS33 and 14-3-3ζ. Note that in S33A livers, only the endogenous mK18 remains phosphorylated on S33, whereas 14-3-3ζ coimmunoprecipitates primarily with keratins that are isolated from TG2 and S52A mice.

Immunofluorescence staining and transmission electron microscopy of TG2 and S33A mouse pancreata. (a and b) Immunofluorescence staining was conducted on fresh-frozen tissues obtained from age- and sex-matched TG2 and S33A² mouse pancreata by using mAb L2A1 as described for Fig. 2. Arrows in b highlight the collapsed apicolateral keratin filament staining that is a consequence of the K18 S33A mutation. (Bar = 100 μm.) (c and d) Transmission electron microscopy was performed on the same pancreata used in a and b. Arrows indicate keratin filament bundles. Similarly appearing bundles were confirmed to be keratins by immune electron microscopy (25, 27). L, lumen; ZG, zymogen granule. (Bar = 0.33 μm.)

Distribution of 14-3-3ζ during mitosis in TG2 and S33A mice. Mitosis was induced in TG2 and S33A mouse livers by partial hepatectomy. Livers were removed 2 or 3 days after hepatectomy, fixed, and then triple-stained with Toto-1 (DNA stain), anti-14-3-3ζ, and anti-mK8 pS79 (pK8) antibody LJ4. DNA staining was informative, in terms of the mitosis stage, only when clearly delineated mitotic figures were noted and confirmed by staining with the LJ4 antibody that recognizes mitotic hepatocytes (26). Arrows point to the identical triply stained cell for each mitotic stage shown. Note that in contrast to mitotic TG2 hepatocytes, 14-3-3ζ manifests a punctate nuclear staining pattern in mitotic S33A hepatocytes. Similar findings were noted for the 14-3-3σ isoform, although the staining was fainter than for the 14-3-3ζ isoform (not shown).

Immunofluorescence staining of keratins in transgenic mouse tissues. Liver and pancreas were isolated from the indicated transgenic lines. Tissues were freshly frozen, sectioned, and then fixed in cold acetone. Staining was performed with mAb L2A1, which recognizes the hK18 transgene product (a–h) and anti-K18 pS33 (i–l). Note that the “tight” apicolateral filament distribution, particularly noted in i and l (pancreata of TG2 and S52A mice, respectively), are more dispersed in S33A mice pancreata (j and k; similar findings were noted with anti-hK18 pS52 Ab, not shown). (Bar = 100 μm.)

Comparison of keratin filament organization and the nature of mitotic progression in TG2 and S33A mouse livers after partial hepatectomy. (A) Livers from 3-day posthepatectomy mice were frozen, sectioned, and then stained with anti-hK18 Ab. Note the dramatic loss of cytoplasmic filaments in the S33A livers. (B) TG2 and S33A mice were subjected to partial hepatectomy. At the indicated days, livers were retrieved, fixed, sectioned, and then stained with hematoxylin/eosin. Note the significantly higher numbers of mitotic bodies in the S33A livers after hepatectomy (indicated by arrows). (C) High magnification images of posthepatectomy livers (after hematoxylin/eosin staining) from TG2 and S33A mice. (Insets) Additional examples of mitotic figures from the indicated mouse line livers. Arrow in histogram c (anaphase cell) highlights an aberrant bridge that is not seen in TG2 mouse livers after hepatectomy (e.g., the late-anaphase cell from TG2 liver in histogram a). (D) At the indicated days (D) after hepatectomy, the total number of mitotic figures was counted (from 20 microscopic ×20 fields), then tabulated by assessing hematoxylin/eosin-stained liver sections of TG2 and S33A mice. (E) The percent of total mitotic figures during prophase, or metaphase (Meta) + anaphase (Ana) + telophase (Telo) for TG2 and S33A mouse livers are indicated 2 and 3 days, or 7/10/13 days after partial hepatectomy. Also, the percent of abnormal mitotic figures (e.g., tripolar, 2-arm angulated, or those with bridges) is shown.