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1.
Fig. 4

Fig. 4. Relative MLCK1 mRNA levels increase during Caco-2 differentiation.. From: A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability.

Monolayers of Caco-2 cells were harvested at indicated points after reaching confluence. MLCK isoforms 1 and 2 were detected using primers 3 and 4 flanking the MLCK1 splice site. Thus, a single PCR allowed detection of both isoforms (see Fig. 3). As Caco-2 cells differentiated the amount of MLCK1 mRNA (white circles) as a percentage of total MLCK mRNA increased. The TER response of these monolayers to activation of Na+-glucose cotransport (as in Fig. 1) is shown for comparison (black circles). Development of the characteristic response of TER to Na+-glucose cotransport correlates strongly with increases in relative MLCK1 mRNA content (R2 = 0.92).

Daniel R. Clayburgh, et al. J Biol Chem. ;279(53):55506-55513.
2.
Fig. 7

Fig. 7. Reduction of MLCK1 mRNA via RNAi causes increased TER.. From: A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability.

A, Caco-2 monolayers were transfected with either nonspecific siRNA or a pool of 4 siRNAs targeting MLCK1. Semi-quantitative RT-PCR analysis of these transfectants demonstrates a specific decrease in MLCK1 mRNA in monolayers transfected with MLCK1-specific siRNA. B, densitometric analysis of semi-quantitative RT-PCR gels shows that MLCK1, as a percentage of total MLCK, fell from 52 ± 3% in nonspecific siRNA-transfected monolayers to 27 ± 2% in monolayers treated with MLCK1 siRNA. This corresponds to a 47 ± 3% drop in MLCK1 mRNA. C, Caco-2 monolayers were transfected with either nonspecific siRNA or a pool of four siRNAs targeting MLCK1. Lysates of these monolayers were separated by SDS-PAGE and immunoblotted for MLCK1, as in Fig. 6. There was a specific decrease in MLCK1 protein expression in monolayers transfected with MLCK1-specific siRNA. D, the TER of monolayers transfected with MLCK1-specific siRNA is increased by 25 ± 7% over control monolayers transfected with nonspecific siRNA.

Daniel R. Clayburgh, et al. J Biol Chem. ;279(53):55506-55513.
3.
Fig. 5

Fig. 5. Phosphorylated MLC and MLCK1 mRNA are concentrated at the villus tip.. From: A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability.

A, long MLCK1 RNA expression is greatest at the villus tip, whereas MLCK2 expression is constant along the crypt-villus axis as detected by RT-PCR. As controls, mRNA content of BRK and keratin 8 (K8) are also shown. BRK is known to be expressed preferentially at the villus tip, and keratin 8 is known to be expressed uniformly along the crypt-villus axis (28). B, densitometric analysis demonstrates MLCK1 enhancement at the villus tip. MLCK1 (blue), MLCK2 (red), and BRK (black) mRNA content was normalized to keratin 8 content. These ratios were then normalized to crypt mRNA content. Both BRK and MLCK1 show a gradient of increasing expression from crypt to villus, although MLCK1 mRNA is more restricted to the villus tip. C, phosphorylated MLC (red) is predominantly seen in villus enterocytes. The inset shows that phosphorylated MLC is specifically enhanced at areas of intercellular junctions. The scale bar is 20 μm (5 μm in the inset). D, when merged with F-actin (green), phosphorylated MLC colocalizes with the perijunctional actomyosin ring, producing a yellow color. The inset shows that phosphorylated MLC is specifically enhanced at areas of intercellular junctions within the perijunctional actomyosin ring. The scale bar is 20 μm (5 μm in the inset).

Daniel R. Clayburgh, et al. J Biol Chem. ;279(53):55506-55513.
4.
Fig. 3

Fig. 3. mRNA transcripts of long MLCK isoforms 1 and 2 are present in Caco-2 cells.. From: A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability.

A, a single 5.8-kb full-length PCR product corresponding to long MLCK is obtained from Caco-2 cDNA. B, PCR using primers to assess alternative splicing of long MLCK shows only a single product in lanes I, III, IV, and V, confirming that alternative splicing of these regions of long MLCK does not occur in intestinal epithelia. In contrast, three products are detected in lane II, showing that alternative splicing occurs in this region. Sequence analysis of these products confirmed that the upper 1.4-kb band is MLCK1 and the lower 1.1-kb band is MLCK2, whereas a faint, unknown band is located directly below MLCK1. C, diagram of the long MLCK gene and the primer pairs used for amplification and sequencing of Caco-2 cDNA. The shaded region marked splice site designates the location of the 207-base pair region that is present in MLCK1 and absent from MLCK2.

Daniel R. Clayburgh, et al. J Biol Chem. ;279(53):55506-55513.
5.
Fig. 6

Fig. 6. MLCK1 is localized to the perijunctional actomyosin ring at the villus tip in human jejunum.. From: A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability.

A, recombinant MLCK1, MLCK2, or Caco-2 lysates were blotted for total MLCK (anti-MLCK) or with the MLCK1-specific antisera (anti-MLCK1). The total MLCK antibody detects both recombinant MLCK1 and recombinant MLCK2, as well as a corresponding band in the Caco-2 lysate. The affinity-purified anti-MLCK1 antiserum detects recombinant MLCK1 but not recombinant MLCK2. A 215-kDa band is detected in Caco-2 lysates. B, MLCK1 detected by the anti-MLCK1 antiserum (red) is present primarily within enterocytes at the villus tip (scale bar, 20 μm). C, MLCK1 (red) is found in a narrow band just subapical to the brush border and displays enhancement in the area of cell-cell junctions (scale bar, 5 μm). A Hoechst stain for DNA is shown for reference (blue). When merged with f-actin (green), it is apparent that MLCK1 localizes to the peri-junctional actomyosin ring. D, total MLCK (green) can be found in two intracellular pools. The major pool is concentrated in the cytoplasm near the nucleus, whereas a more limited pool of MLCK is seen as a faint line near the brush border. This second pool colocalizes with MLCK1 (red; scale bar, 5 μm).

Daniel R. Clayburgh, et al. J Biol Chem. ;279(53):55506-55513.
6.
Fig. 1

Fig. 1. Tight junction regulation in response to Na+-glucose cotransport is dependent on intestinal epithelial differentiation.. From: A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability.

At the indicated times after reaching confluence, Caco-2 monolayers were incubated in Hank’s balanced salt solution containing 25 mm glucose (black circles) or 5 mm glucose, 20 mm mannitol, and 2 mm phloridzin, a Na+-glucose cotransport inhibitor (white circles). TER was measured after 2 h of incubation, at which time steady-state TER was established. The data are normalized to TER in monolayers with inactive Na+-glucose cotransport (i.e. in Hank’s balanced salt solution with phloridzin). No significant responses were seen in monolayers less than 4 days post-confluence (p = 0.79). At 6 or more days post-confluence, a robust tight junction regulatory response was present (p = 0.002).

Daniel R. Clayburgh, et al. J Biol Chem. ;279(53):55506-55513.
7.
Fig. 2

Fig. 2. Long MLCK is found in intestinal epithelium and is primarily responsible for Na+-glucose cotransport-induced tight junction regulation.. From: A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability.

A, Caco-2 monolayers were incubated in Hank’s balanced salt solution with 25 mm glucose (active Na+-glucose cotransport) or 5 mm glucose, 20 mm mannitol, and 2 mm phloridzin (inactive Na+-glucose cotransport), as in Fig. 1, or with 25 mm glucose and the MLCK inhibitor PIK (250 μm), the Rho kinase inhibitor Y27632 (10 μm), or the phosphatase 1 and 2A inhibitor calyculin A (10 nm). TER after 2 h, at which time TER had stabilized, is shown. Activation of Na+-glucose cotransport reduced TER by 30 ± 4%. The addition of PIK elevated TER to that of monolayers with inactive Na+-glucose cotransport. Y27632 and calyculin A had no effect on TER, indicating that MLCK is primarily responsible for Na+-glucose cotransport-dependent tight junction regulation. B, analysis of Caco-2 cell lysates by SDS-PAGE and immunoblot with broadly reactive anti-MLCK monoclonal antibody detects a single band of ~215 kDa. C, immunodepletion of Caco-2 lysates using the same anti-MLCK monoclonal antibody removed 97 ± 4% of MLC kinase activity.

Daniel R. Clayburgh, et al. J Biol Chem. ;279(53):55506-55513.

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