Abstract
Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells. We found that CRB2 plays a key role in tight junction maintenance as well as in cell cycle arrest. In addition, our studies in vivo show that the knockdown of CRB2 in the RPE affects to the distribution of different apical polarity proteins and results in perturbed retinal homeostasis, manifested by the invasion of activated microglial cells into the subretinal space. Together our results demonstrate that CRB2 is a key protein for the development and maintenance of a polarized epithelium.
Highlights
Cell polarity is a key feature of many types of cells, and consists of an asymmetrical distribution of molecules, organelles, and structures within the cell
To study the condition of the polarity protein complexes during epithelial polarization we developed a simplified protocol for differentiating human fetal retinal pigment epithelial (RPE) (Hf-RPE) cells, as described in the Methods
Most of the proteins that make up the Crb complex, namely CRB3, PALS1, and PATJ, are known to play a key role in apicobasal polarity and tight junctions establishment in epithelial cells (Roh et al, 2003; Lemmers et al, 2004; Straight et al, 2004; Shin et al, 2005), no such role had been identified for CRB2
Summary
Cell polarity is a key feature of many types of cells, and consists of an asymmetrical distribution of molecules, organelles, and structures within the cell. Epithelial cells exhibit a special category of polarity, named apicobasal polarity, in which distinct apical and basolateral surfaces are exposed to different environments (Rodriguez-Boulan and Macara, 2014). This organization is essential to the barrier function of an epithelium. It is the first to be established at the plasma membrane and initiates the polarization process by determining the apical domain of the cell. This event is followed by specification of the basolateral surface by the Scrib complex, consisting of SCRIB, DLG, and LGL proteins. The Crb complex, comprised of the CRB proteins and the PALS1 and PATJ proteins, is recruited more apically than the Par complex, and CRB2 Regulates Apicobasal Polarity Maintenance antagonizes Scrib function to establish apicobasal polarity (Assemat et al, 2008)
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