Abstract
Occludin is a component of tight junctions, which are essential structural components of the blood–brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N‐ and C‐terminal occludin was used to pull‐down proteins from 1321N1 cell lysates and protein‐binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell‐types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states.
Highlights
Tight junctions formed by brain endothelial cells are a key component of the blood–brain barrier (BBB), limiting paracellular and intramembranous diffusion and giving the cell polarity (Chow & Gu, 2015; Zlokovic, 2008)
We examined the expression of occludin in human astrocytes in vitro and in human tissue in vivo
The subcellular localisation pattern, combined with the protein-binding partners identified by proteomic analysis, suggest non-tight junctionrelated functions in these neuroepithelial cells
Summary
Tight junctions formed by brain endothelial cells are a key component of the blood–brain barrier (BBB), limiting paracellular and intramembranous diffusion and giving the cell polarity (Chow & Gu, 2015; Zlokovic, 2008). Occludin knockout mice have a complex phenotype, with considerable growth retardation and alterations in their sexual behaviour but they exhibit no morphological abnormalities in their tight junctions and barrier function appears normal (Saitou et al, 2000; Schulzke et al, 2005) This contrasts with claudin-deficient mice in which barrier function is disturbed; for example claudin-1 null mice do not survive post-natally due to a compromised epidermal barrier (Furuse et al, 2002) while claudin-5 deficient mice have a “leaky” BBB which results in death post-natally (Nitta et al, 2003). To investigate possible additional functions, we used mass spectrometry (MS) to identify candidate binding partners of occludin in neuroepithelial cells
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