Osborne, Shona Louise; (2001) Phosphatidylinositol (4,5) bisphosphate: An essential lipid involved in membrane dynamics and nuclear physiology. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) regulates many cellular processes, as an intact molecule and as a precursor for InsP3, diacylglycerol and PtdIns(3,4,5)P3. One example where PtdIns(4,5)P2 itself is required is the calcium- dependent exocytosis of secretory granules, although the site of PtdIns(4,5)P2 synthesis and the identity of PtdIns(4,5)P2 effector(s) have yet to be determined. The synaptic vesicle protein synaptotagmin I (Syt I) is a strong candidate for calcium sensor in neurotransmitter release. Syt I contains two tandem C2 domains. In the presence of calcium, the first C2 domain (C2A) binds acidic phospholipids, while the second (C2B) binds PtdIns(4,5)P2. The C2B also mediates the calcium-dependent homooligomerisation of Syt I. Syt II is highly homologous to Syt I and is also found on synaptic vesicles. We have shown that Syt I and II can be present in the same synaptic vesicle. In addition, both native and recombinant cytoplasmic Syt I and II hetero- oligomerise in the presence of calcium, suggesting different Syt isoforms can combine to create synaptic calcium sensors with novel calcium-dependent binding properties.To investigate PtdIns(4,5)P2 distribution at the synapse, we produced monoclonal antibodies against PtdIns(4,5)P2. Immunofluorescence in the absence of detergent reveals a punctate staining in the neurite tips of NGF-differentiated PC 12 cells. In the presence of detergent, there is an intense nuclear staining in PC 12 cells and other cell-types from different species. The characterisation of this detergent-resistant nuclear PtdIns(4,5)P2 is described. Nuclear PtdIns(4,5)P2 co-localises with the splicing factor SC35 in interphase. Its distribution changes during mitosis. In late telophase, detergent- resistant PtdIns(4,5)P2 concentrates in discrete SC35-containing structures that are excluded from the reforming daughter nuclei. Nuclear PtdIns(4,5)P2 is associated with both snRNAs and proteins, including splicing factors and RNA Polymerase II. Immunodepletion and add-back experiments demonstrate that PtdIns(4,5)P2 and interacting factors are necessary, but not sufficient, for pre-mRNA splicing in vitro, suggesting PtdIns(4,5)P2 may be involved in pre-mRNA processing in vivo.

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