Oculocerebrorenal syndrome of Lowe (OCRL) is caused by mutations in a
phosphatidylinositol 5-phosphatase, OCRL1, and is believed to lead to an
elevation of its preferred substrate, PI(4,5)P2. To date, much of the work on
OCRL1 has centred on its role at Golgi and endosomal membranes.
However, there is also evidence of plasma membrane activity for OCRL1,
where its PI(4,5)P2 substrate is known to be highly abundant. PI(4,5)P2
regulates a wide array of downstream cellular functions such as cytoskeletal
dynamics, membrane trafficking and signalling. The tight regulation of
PI(4,5)P2 levels and localisation, like other phosphoinositides, provides a
framework upon which many of these cellular processes work. In this thesis,
effects of OCRL1 loss have been tested through siRNA depletion of OCRL1,
focussing where possible on multiple PI(4,5)P2-dependent mechanisms, and
also focussing on cells forming polarised epithelia. Firstly, we have visualised
the localisation of PI(4,5)P2 in living HeLa cells lacking OCRL1 through
immunostaining for Annexin A2, which showed a marked translocation to the
plasma membrane. This change in distribution of Annexin A2 suggested that
OCRL1 depletion may have an effect on intracellular calcium dynamics as
well as PI(4,5)P2 localisation. We also used a GFP-chimera of the well
characterised PI(4,5)P2-binding pleckstrin homology domain of PLCδ1. This
showed no difference in localisation upon OCRL1 depletion. As OCRL1 is
highly enriched at the TGN, we fused the pleckstrin homology domain of
PLCδ1 to a mutated pleckstrin homology domain of OSBP known to bind ARF1 at the TGN, to act as a coincidence detector for PI(4,5)P2 at the TGN.
This construct also showed no reproducible effect of OCRL1 depletion.
Secondly we tested the effect of loss of OCRL1 on cytosolic calcium levels.
Using two phospholipase C (PLC) agonists, and a SERCA pump inhibitor, we
found no consistent differences in calcium handling upon depletion of OCRL1.
Thirdly, we have assessed the potential specialised role that OCRL1 has in
polarised epithelial cells, which might relate to the clinical picture in Lowe
Syndrome. We found that OCRL1 targets the tight junctions of immortalised
lines and primary cells. Through co-immunoprecipitation, we found OCRL1 in
complexes with the tight junction scaffold protein ZO-1. Most significantly, we
found that depletion of OCRL1 in human polarised epithelial cell lines
interfered with epithelial differentiation, reducing cell number and altering
morphology, to produce large flat cells. We attribute this phenotype, stronger
than any other so far described experimentally, to a defect in tight junction
maturation
