Epithelial microfilament regulators show regional distribution in mouse conjunctiva

Abstract

Purpose: The conjunctival epithelium is a continuous sheet of cells with regional characteristics that appear to be similar. This study was designed to investigate the distribution and levels of expression of a subset of microfilament regulators in the forniceal, palpebral, and bulbar conjunctival epithelia.

Methods: Balb/C mice were used. The localizations of paxillin, focal adhesion kinase, vinculin, talin1, cofilin, profilin, gelsolin, integrin β1, and integrin α6 were studied with the use of cross-sectional immunofluorescent staining. For a detailed cellular analysis, positioning and ablation with the laser microbeam (PALM) Combi System was used to obtain forniceal, bulbar, and palpebral conjunctival epithelia for expression comparison with the use of western blot analysis and quantitative real-time polymerase chain reaction.

Results: Immunostaining showed that focal adhesion kinase, cofilin, profilin, gelsolin, talin1, and vinculin were expressed in all layers of the forniceal, palpebral, and bulbar conjunctival epithelia. Paxillin, integrin β1, and α6 was found to be located in the basal cell layer in all three of these areas. Quantitative real-time polymerase chain reaction showed that the transcript levels of these microfilament regulators in the forniceal conjunctivae were higher than those levels found in the bulbar and palpebral conjunctivae. Western blot analysis confirmed the differential expression levels of these microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae.

Conclusions: Differences in the levels of microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae suggest different modes of interaction with their microenvironment and within cell layers.

Figure 1

Distribution of microfilament regulators in the conjunctiva. bcj, bulbar conjunctiva; pcj, palpebral conjunctiva. A: Integrin β1 (green) and CK4 (Red); B: Integrin α6 (green) and CK4 (red); C: Talin1 (green); D: Vinculin (green); E: Profilin1 (green); F: Double staining of profilin1 (red) and phalloidin (green); G: Paxillin (green); H: Double staining of paxillin (red) and phalloidin (green); I: FAK (green); J: Double staining of FAK (red) and phalloidin (green); K: Cofilin1 (green); L: Double staining of cofilin1 (red) and phalloidin (green); M: Gelsolin (green color); N: Double staining of gelsolin (red) and phalloidin (green); P, Q: Integrin β1 (green); R, S: Paxillin (green); T, U: Profilin1 (green). Blue color is DAPI as a counterstain, staining nuclear.

Figure 2

Summary of the microfilament regulators distribution in the forniceal, palpebral and bulbar conjunctival epithelia.

Figure 3

PALM laser dissection. A, D, G: OCT-embedded mouse eye tissue was cut at 10 μm, fixed and stained with hematoxylin and Eosin. B, E, H: Palpebral, bulbar and forniceal conjunctival epithelium region were identified and circled. C, F, I: The selected region was cut from the surrounding cells, and the same region was captured leaving a clear margin of surrounding cells. All pictures were taken at 400×. bcj, bulbar conjunctiva; pcj, palpebral conjunctiva.

Figure 4

Relative real-time PCR results. Fold difference of each target gene expression among different samples in comparison with conjunctival forniceal epithelial cells. The calculation of the fold difference was described in Methods. The asterisk indicates a significant difference, p<0.05, compared to the transcript level in forniceal epithelial cells. bcj, bulbar conjunctiva; pcj, palpebral conjunctiva.

Figure 5

Western blot analysis. GAPDH was used as the loading control. A: The proteins identities are indicated on the right. B: Band intensity was quantified by densitometry and the fold difference of each microfilament regulator in the conjunctival bulbar and palpebral epithelia compared to the conjunctival forniceal epithelia was expressed graphically. bcj, bulbar conjunctiva; pcj, palpebral conjunctiva.