FRAP Analysis Reveals Stabilization of Adhesion Structures in the Epidermis Compared to Cultured Keratinocytes

Abstract

Proper development and tissue maintenance requires cell-cell adhesion structures, which serve diverse and crucial roles in tissue morphogenesis. Epithelial tissues have three main types of cell-cell junctions: tight junctions, which play a major role in barrier formation, and adherens junctions and desmosomes, which provide mechanical stability and organize the underlying cytoskeleton. Our current understanding of adhesion function is hindered by a lack of tools and methods to image junctions in mammals. To better understand the dynamics of adhesion in tissues we have created a knock-in ZO-1-GFP mouse and a BAC-transgenic mouse expressing desmoplakin I-GFP. We performed fluorescence recovery after photobleaching (FRAP) experiments to quantify the turnover rates of the tight junction protein ZO-1, the adherens junction protein E-cadherin, and the desmosomal protein desmoplakin in the epidermis. Proteins at each type of junction are remarkably stable in the epidermis, in contrast to the high observed mobility of E-cadherin and ZO-1 at adherens junctions and tight junctions, respectively, in cultured cells. Our data demonstrate that there are additional mechanisms for stabilizing junctions in tissues that are not modeled by cell culture.

Cell Adhesion in Epidermal Development and Barrier Formation

“…Clearly, changes in protein levels are an easy readout, but large changes do not necessarily correlate with perturbed adhesion. Similarly, AJ turnover can be used to look at stability (Delva & Kowalczyk, 2009; Foote, Sumigray, & Lechler, 2013; Georgiou, Marinari, Burden, & Baum, 2008), but this suffers from the same problems. As discussed later, tight-junction defects appear to be a more sensitive readout of AJ function, but there can be many other causes of these defects.…”

A transgenic toolkit for visualizing and perturbing microtubules in mice reveals unexpected functions for non-centrosomal arrays in epidermal morphogenesis

“…Primary keratinocytes begin to stratify when cultured, forming a proliferative basal layer and a single, post-mitotic, "suprabasal" layer, which recapitulates some of the features of spinous cell differentiation (Muroyama et al, 2016). Absolute polymerization rates were dramatically increased in isolated cells, suggesting that microtubule dynamics are altered as primary cells initiate a woundhealing response in culture, as has been noted for cell-cell junctions ( Figure 1D-G) (Foote et al, 2013). Interestingly, although the absolute polymerization rate was greatly increased, the overall trends across all three measured parameters consistently mirrored the trends seen at the basal to spinous transition in vivo ( Figure 1E-G).…”

Differential expression and homotypic enrichment of a classic Cadherin directs tissue-level contractile asymmetry during neural tube closure

“…Here we show that the same rules operate for Cad2 within an intact embryonic epithelium. The underlying mechanism(s) remain unclear, but likely involve multiple forms of feedback downstream of trans-homophillic engagement, including local stabilization of clustered Cadherins at cell-cell contacts (Cavey et al, 2008;Foote et al, 2013;Nose et al, 1988), and local inhibition of endocytosis (Izumi et al, 2004).…”