Figure 1. Long myosin light chain kinase splice variant 1 (MLCK1) is specifically recruited to the perijunctional actomyosin ring (PAMR) in response to inflammatory stimuli.
(a) Protein domain structure of MYLK gene products. IgCAM domains are numbered from the amino terminus. Long MLCK is expressed as two splice variants, MLCK1 and MLCK2, in intestinal epithelial cells. Short MLCK is expressed in smooth muscle. (b) Normal human jejunum stained for long MLCK1 (green), total MLCK (red), and nuclei (blue). Bar, 50 μm. Inset of the boxed region is shown on the bottom right and includes an arrow indicating the PAMR. Bar, 10 μm. Images are representative of more than 10 independent experiments. (c) Caco-2BBe monolayers expressing MLCK1-EGFP or MLCK2-EGFP were primed with IFNγ followed by treatment with TNF for 4 hrs. Images are representative of more than 12 independent experiments. Bar, 10 μm. (d) MLCK1-EGFP or MLCK2-EGFP colocalization with F-actin at the PAMR was determined. For this experiment, which is representative of 4 independent studies, n=8 biologically independent samples with 3 – 5 fields analyzed for each condition. *, P<0.05; **, P<0.01 by Kruskal-Wallis test with Dunn’s multiple comparison test. (e) Monolayers were treated with IFNγ and/or TNF, as indicated prior to immunostaining for endogenous MLCK1 and F-actin. PAMR localization of endogenous MLCK1 was determined. Data show the fraction of total MLCK1 localized to the PAMR and are therefore independent of absolute MLCK1 expression, which increases in response to TNF. For this experiment, which is representative of 4 independent studies, n=3 biologically independent samples with 3 – 4 fields analyzed for each condition. **, P<0.01 by ANOVA with Dunn’s multiple comparison test. Mean ± SEM is shown. (f) Colon sections from healthy mice (control) or those with T cell transfer colitis (colitis) were stained for MLCK1 (green) and nuclei (blue). Intensity of MLCK1 staining in colonocytes from healthy control mice is enhanced to allow direct comparison with diseased colonocytes, which have increased MLCK1 expression. Arrows indicate the position of the PAMR, where distinct line of MLCK1 can be detected in colonocytes from colitic, but not control, mice. Bar, 5 μm. Images representative of more than 6 independent experiments are shown.