K8/K18 mutants have altered growth rates.

<p>Cells were grown on 24-well cell culture plates. After a week in culture cells were trypsinized and counted. As shown, the K8 mutants grow at a much higher rate than the K8 WT cell line, while the K18 S230T cells grow very slow, having only a quarter of the growth rate of K18 WT cells. All cell lines (mutant and wild type) have a normal cell cycle profile (not shown).</p

Effect of heat-stress on K8/K18 mutants.

<p>Cells were grown on coverslips until they formed smaller patches of cells, after which they were exposed to heat-stress for a brief period of time. At 0 time, 1 hour, 2 hours, 4 hours and 16 hours of recovery, cells were fixed and immmunofluorescently labelled for either K8 or K18 protein. Images shown are representative for the 1 hour time point of recovery, when the biggest differences were observed. In panel A are the K18 wild type control cells and these appear unaffected by heat stress. Panels B, C and D are the K18 S230T, K8 G62C and K8 K464N mutants respectively. Arrows mark border regions where cell sheets have detached from the surface and curled up against it. Images were acquired with a 20x objective lens.</p

K18 S230T may form an additional hydrogen bond within the K18 chain in the L12 linker.

<p>Molecular dynamics experiments were performed on the K8/K18 L12 linker model with the duration of 1 ns. This was repeated 4 times for the wild type sequence and 5 times for the mutant. The resulting data was used to calculate: (A) the average distances between the hydroxyl group of SER/THR230 and the backbone oxygen of ALA226 in K18, which in the case of the mutant (THR230) falls within the range of a moderately strong hydrogen bond (2.5–3.2 Å); (B) the relative root mean square (RMS) along the run, a measure of dimer stability. Both parameters indicate that the K18 S230T mutation may be forming an additional hydrogen bond within the K18 chain, which would be expected to increase the rigidity of this part of protein to additional conformational pressures.</p

K8/K18 mutants have higher paracellular permeability. Isogenic K8/K18 cell lines were grown on cell culture inserts with a 0.4 µm pore size.

<p>Lucifer yellow was used to test the permeability of tight junctions after cells reached confluence and matured over a period of 2 weeks in culture. (A) Permeability coefficient (Papp) of K8 cell lines. Both mutants have a much higher permeability than the K8 control cell line. (B) Papp of K18 cell lines, where the K18 S230T mutant (similar to K8 mutants) displays a 30% higher permeability from the K18 control cell line.</p

Western blot analysis of tight junction components in protein extracts from isogenic K8 and K18 cell lines.

<p>Tracks: (1) K8 WT cells; (2) K8 G62C cells; (3) K8 K464N cells; (4) K18 WT cells; (5) K18 S230T cells. Total protein extracts (10 µg) from selected clones were run on a 4–12% denaturing polyacrylamide gel, transferred to a PVDF membrane, and incubated with antibodies against ZO-1 (N-term) and claudin-4 (3E2C1). While ZO-1 expression levels are similar, claudin-4 levels are elevated in the two K8 mutants (tracks 2 and 3) and down-regulated in the K18 S230T cells (track 5).</p

Western blot analysis of protein extracts from isogenic K8 and K18 cell lines.

<p>Tracks: (1) K8 WT cells; (2) K8 G62C cells; (3) K8 K464N cells; (4) K18 WT cells; (5) K18 S230T cells. Total protein extracts (10 µg) from selected clones were run on a 5% denaturing polyacrylamide gel, transferred to a PVDF membrane, and incubated with antibodies against K8 (M20), K18 (LDK-18) and phosphorylated keratin (LJ4) protein. As shown, K8 and K18 expression levels are very similar between the selected clones, while phosphorylated keratin levels are altered in the mutants, up-regulated in the K8 G62C (track 2) and K8 K464N (track 3) cells and down-regulated in the K18 S230T cells (track 5).</p

The distribution of ZO-1 and claudin-4 is altered in the K18 S230T mutant.

<p>Colonocytes stably expressing the K18 S230T mutation were immunofluorescently labelled for ZO-1 (upper panels, A and B) and claudin-4 (bottom panels, C and D), important components of tight junctions. In both cases the K18 control cells (panels A and C) show a more regular pattern of staining that traces the cell membrane. In contrast, the K18 S230T mutant (panels B and D) has a very diffuse pattern of staining for ZO-1 and claudin-4, and smaller protein aggregates are also visible (see arrows). Images were acquired with a 60x objective lens.</p