Daniels, RD; (2014) An investigation into the role of OCRL1 in polarised epithelial cells. Doctoral thesis , UCL (University College London).

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Abstract

Mutations in the phosphoinositide 5-phosphatase OCRL1 cause Lowe Syndrome and Dents-2 disease, both involving selective renal proximal tubulopathy. Epithelial cells lining renal proximal tubules are highly polarised with distinct apical and basolateral membranes separated by intercellular junctions. Using Madin Darby canine kidney (MDCK) cells as a model of the renal tubular epithelium, we found a pool of OCRL1 targeted intercellular junctions and was required for the correct organisation of apical and basolateral membranes. One of the first events when cells were depleted of OCRL1 was a block in apical recycling. Apical cargo accumulated in exaggerated Rab11 positive recycling compartments with ectopic accumulation of the OCRL1 substrate PI(4,5)P2. Among the apical cargo were Gp135 and key regulators of apical membrane formation, including Cdc42 and the Par6-aPKC polarity complex. Rescue of recycling required the 5-phosphatase domain of OCRL1, suggesting down-regulation of 5-phosphoinositides is necessary for cargo exit from apical recycling endosomes. Eventually, the Rab11 positive vesicles containing apical cargo were targeted to the plasma membrane to re-form the apical domain. However, instead of being targeted to the cell apex, the apical vesicles were trafficked towards the lateral membrane where lumens formed, resembling the bile canaliculi in hepatocytes. This process was co-ordinated with cell division. Lateral lumens formed close to the midbody formed during cytokinesis. During cytokinesis Rab11 normally regulates a trafficking pathway to the midbody. Therefore, in cells lacking OCRL1 apical cargo held up in Rab11 positive compartments may be aberrantly trafficked along this pathway when cells divide. We also found that the orientation and subsequent resolution of mitoses was altered in cells lacking OCRL1, which may contribute to improper positioning of the midbody and subsequent failure to polarise correctly. In summary, these results implicate OCRL1 in multiple steps of the process that co-ordinates apical recycling and cell division in polarised cells.

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