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. 2011 Oct 7;89(4):564–571. doi: 10.1016/j.ajhg.2011.09.001

Figure 3.

Figure 3

Loss of Cystatin A Protein Affects Cell-Cell Adhesion

(A) Electron microscopy of a skin biopsy of ridged skin from palm and sole, respectively, from an affected member of the Turkish family. The upper layers of the epidermis appear mostly normal (1), whereas there is prominent intercellular edema of the suprabasal and basal cell layers (2) and aggregates of tonofilaments in the basal keratinocytes (3, arrow), suggesting that the skin peeling initiates at the basal-suprabasal level in the affected individuals. In higher magnification, cell-cell adhesion looks normal with a regular structure of desmosomes in upper layers (4) but there is loosening of the keratinocyte structure with irregularities in desmosomal interaction in the basal layer (5, arrow). The following abbreviations are used: SG, stratum granulosum; BL, basal layer.

(B) Staining of normal skin shows that cystatin A is localized throughout the epidermis and has a diffuse cytoplasmic distribution.

(C) The efficiency of cystatin A knockdown (CSTA KD) compared to cells treated with a nontargeting pool (NTP) of siRNA was analyzed by immunocytochemistry and immunoblotting of cell lysates. An approximately 85% knockdown was achieved.

(D) In an in vitro model of exfoliative ichthyosis, monolayers of CSTA KD cells were stressed mechanically and then stained for keratin 14 (green). Nuclei are stained with DAPI (blue). Upon intense stretching (4 hr) the CSTA KD monolayer broke into many fragments, whereas the NTP monolayer of cells remained intact (20×). At higher magnification (100×), breakage of keratin filaments and widened intercellular spaces could be seen between the CSTA KD cells, in contrast to no obvious cell-cell adhesion defects in the stretched NTP cell monolayer. The scale bar represents 20μm.

(E) A dispase-based dissociation assay was utilized to assess intercellular adhesion by the degree of cell monolayer integrity upon mechanical stress. CSTA KD cells showed an increased number of monolayer fragments in contrast to the small number of monolayer fragments observed for the NTP cells. These observations show a statistically significant decrease in cell-cell adhesion in monolayers lacking cystatin A protein. Error bars show the standard error of the mean.