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First published online 25 April 2006
doi: 10.1242/jcs.02915

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Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure

¹ Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 1089,Chicago, IL 60637, USA
² GI Cell Biology, Combined Program in Pediatric Gastroenterology and Nutrition, and Departments of Pediatrics, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
³ Departments of Animal Sciences and Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA
⁴ Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA

View larger version (47K): [in a new window]   Fig. 1. Inducible tMLCK expression causes MLC phosphorylation and actin reorganization in differentiated Caco-2 monolayers. (A) tMLCK mRNA is readily detected by RT-PCR in Caco-2 cell monolayers after, but not before, induction. Digestion with NsiI results in a doublet, verifying the identity of the PCR product. Results are representative of three or more experiments. (B) Increased tMLCK transcription is accompanied by a 4.7±0.4-fold increase in MLC kinase activity, assayed using an in vitro kinase assay. In the assay shown, which was performed with exogenous calmodulin and Ca2+, the activity in lysates of cells without tMLCK expression represents endogenous MLC kinase. Results are representative of three or more independent experiments with this clone and at least two each with five independently generated clones. (C) Immunoblots using antisera specific for phosphorylated MLC show that induction of tMLCK expression causes progressive increases in endogenous MLC phosphorylation. Control immunoblots show that total MLC content did not change. Data from densitometric analysis of samples from the same experiment are shown in the graph. Data shown are means ± s.e.m. of triplicate samples and are representative of three or more independent experiments. Similar results were obtained with five independently generated clones. (D) tMLCK induction (open circles) caused increases in phalloidin binding that were not seen in uninduced (closed circles) monolayers. Total actin content, as assessed on western blots, was not changed. Data shown are means ± s.e.m. of quadruplicate samples and are representative of four independent experiments. (E) F-actin distribution was assessed by labeling with fluorescent phalloidin conjugates. The three-dimensional projection demonstrates typical perijunctional actin rings with focal mild intensifications within the ring and prominent apical microvillus cores. (F) Four hours after tMLCK induction, phalloidin-labeled F-actin shows perijunctional rings to be irregularly intensified, with brighter foci alternating with areas of reduced or unchanged labeling intensity. Bars, 10 µm.

View larger version (14K): [in a new window]   Fig. 2. tMLCK induction reduces barrier function. (A) TER decreases progressively after tMLCK induction. TER, normalized to control monolayers handled in parallel, fell progressively after doxycycline washout. TER 8-18 hours after tMLCK induction was similar to that seen as much as 48 hours after doxycycline washout (data not shown). Data shown are means ± s.e.m. of triplicate samples and are representative of more than ten independent experiments. Similar results were obtained with five independently generated clones. (B) Inhibition of myosin light chain kinase by addition of 10 µM ML-7 to control monolayers (closed bars) and monolayers expressing tMLCK (open bars) causes TER of the latter to increase to that of control monolayers not expressing tMLCK. tMLCK expression was induced for 18 hours and ML-7 added for 1 hour before TER measurement. Data shown are means ± s.e.m. of triplicate samples and are representative of four independent experiments. (C) tMLCK expression increases transepithelial flux of the small paracellular tracer mannitol, but not inulin, demonstrating size-selective increases in paracellular permeability as a consequence of tMLCK-induced MLC phosphorylation. Data shown are means ± s.e.m. of triplicate samples and are representative of three independent experiments.

View larger version (51K): [in a new window]   Fig. 3. TJ membranes are modified by tMLCK expression. Detergent-insoluble low-density membranes were isolated from control monolayers without tMLCK induction (closed circles on graphs) or 8 hours after induction of tMLCK expression (open circles on graphs). Fractions were analyzed by SDS-PAGE immunoblot for occludin, claudin-1, and claudin-2. GM1 ganglioside was detected with peroxidase-conjugated cholera toxin B subunit. Densitometric analysis (right) shows that tMLCK expression causes occludin to shift to fractions of higher density, whereas claudin-1, claudin-2 and GM1 distributions were not significantly changed. Results are typical of six independent experiments.

View larger version (66K): [in a new window]   Fig. 4. TJ ultrastructure and strand number are not altered by tMLCK expression. (A,B) Representative images of TJ from monolayers not induced (A) or induced (B) to express tMLCK. Magnification, 62,500x. (C) The number of strands in each TJ replica was counted at 160 nm intervals over a total distance of 195 µm and 185 µm in monolayers induced (open circles) or not induced (closed circles) to express tMLCK, respectively. This resulted in 56 and 54 strand counts in induced and uninduced monolayers, respectively. The histogram shows the frequency with which each strand number was observed. There was no significant difference between induced and uninduced monolayers.

View larger version (74K): [in a new window]   Fig. 5. Distributions of ZO-1 and perijunctional F-actin are altered by tMLCK expression. (A) ZO-1 profiles in monolayers not expressing tMLCK are smooth and arc-like. Only occasional mild undulations are present (arrow). (B) tMLCK expression causes ZO-1 profiles to be reorganized into irregularly undulating patterns (image shown is 4 hours after induction). These undulations are present throughout the monolayer (arrows). In addition, occasional cells are extremely affected (asterisk). Although relatively uncommon in induced cultures, such cells were never seen in monolayers without tMLCK expression. (C) Quantitative analysis of ZO-1 profile length shows that it is increased significantly (n=40 per condition; P<0.001) by tMLCK expression. MLCK inhibition using 250 µM PIK (Zolotarevsky et al., 2002) decreased profile length to levels observed in cells not expressing tMLCK (n=40 per condition; P>0.1). (D) Smooth F-actin and ZO-1 profiles are reorganized into irregular undulations 8 hours after tMLCK induction. Addition of 250 µM PIK (Zolotarevsky et al., 2002) during this tMLCK induction prevented the formation of undulations in both F-actin and ZO-1 distributions. Representative images are shown. Bars, 10 µm.

View larger version (56K): [in a new window]   Fig. 6. Distribution of occludin, but not claudin-1 and claudin-2, is altered by tMLCK expression. (A) Like ZO-1, arc-like occludin profiles are also remodeled to include irregular undulations 4 hours after induction of tMLCK expression. Although redistributed, occludin remains colocalized with ZO-1 (see merged images). Representative images are shown. (B) Rare claudin-1 undulations can be found before and 4 hours after induction of tMLCK expression. However, overall, tMLCK expression did not cause significant changes in claudin-1 localization. This is confirmed by quantitative analysis (Fig. 7). Representative images are shown. (C) Like claudin-1, focal claudin-2 undulations can be found before and 4 hours after induction of tMLCK expression. Also like claudin-1, the overall distribution of claudin-2 was not changed after induction of tMLCK expression. This is confirmed by quantitative analysis (Fig. 7). Representative images are shown. (D) Triple-label image of claudin-1 (green), ZO-1 (red) and F-actin (blue) in cells expressing tMLCK. Although undulations of the ZO-1 profile can be easily seen (arrows), no such undulations are apparent in the claudin-1 image. F-actin shows some minimal undulations, but to a far lesser degree than ZO-1. The absence of claudin-1 at sites of undulations can also be appreciated in the merged image. (E) High-magnification analysis of undulations in monolayers expressing tMLCK shows that, at these sites, F-actin (green) staining intensity is reduced (arrows). Claudin continues directly across the site of F-actin undulation, without deviating from the arc of the junction. However, claudin-1 labeling is decreased at this site (also seen in panel D). Bars, 10 µm (A-C); 5 µm (D); 2.5 µm (E).

View larger version (17K): [in a new window]   Fig. 7. Quantitative analysis of tight junction protein profile length. Profile length was assessed in monolayers stained for the indicated TJ protein using well-oriented en face images at the plane of the TJ. Actual profile length was normalized to linear length (mean ± s.e.m.), as described in the Materials and Methods. tMLCK expression caused marked increases in ZO-1 and occludin profile length, consistent with the presence of many undulations. By contrast, claudin-1, claudin-2 and F-actin profile length were unchanged by tMLCK expression.

View larger version (124K): [in a new window]   Fig. 8. Perijunctional actin and TJ proteins are focally separated by tMLCK expression. High-magnification analysis of undulations in monolayers expressing tMLCK shows that, at these sites, F-actin staining intensity is reduced (arrows). In cells labeled for either ZO-1 or occludin and F-actin this results in the undulated region staining red (ZO-1 or occludin) in the merged image, rather than yellow, owing to loss of F-actin staining (green) at these sites (arrows). By contrast, analysis of cells double labeled for ZO-1 and occludin shows that these two proteins remain precisely colocalized at sites of undulations. Bar, 2.5 µm.