RESUMO
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.
RESUMO
Acquisition of cell polarization is essential for the performance of crucial functions, like a successful secretion and appropriate cell signaling in many tissues, and it depends on the correct functioning of polarity proteins, including the Crumbs complex. The CRB proteins, CRB1, CRB2 and CRB3, identified in mammals, are expressed in epithelial-derived tissues like brain, kidney and retina. CRB2 has a ubiquitous expression and has been detected in embryonic brain tissue; but currently there is no data regarding its localization in the adult brain. In our study, we characterized the presence of CRB2 in adult mice brain, where it is particularly enriched in cortex, hippocampus, hypothalamus and cerebellum. Double immunofluorescence analysis confirmed that CRB2 is a neuron-specific protein, present in both soma and projections where colocalizes with certain populations of exocytic and endocytic vesicles and with other members of the Crumbs complex. Finally, in the cortex of CRB1rd8 mutant mice that contain a mutation in the Crb1 gene generating a truncated CRB1 protein, there is an abnormal increase in the expression levels of the CRB2 protein which suggests a possible compensatory mechanism for the malfunction of CRB1 in this mutant background.