The coincidence biosensor tubbyCT reveals local phosphatidylinositol-4,5-bisphosphate synthesis at endoplasmic reticulum-plasma membrane junctions

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays a prominent role in plasma membrane (PM) physiology. It is implicated in the regulation of a variety of cellular functions including exo- and endocytosis, cytoskeleton anchorage, and ion channel activity. Activation of Gq-coupled receptors indu...

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Bibliographic Details
Main Author: Thallmair, Veronika
Contributors: Oliver, Dominik (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2020
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Summary:Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays a prominent role in plasma membrane (PM) physiology. It is implicated in the regulation of a variety of cellular functions including exo- and endocytosis, cytoskeleton anchorage, and ion channel activity. Activation of Gq-coupled receptors induces rapid break-down of PI(4,5)P2 by PLCβ. The thereby generated second messengers I(1,4,5)P3 and diacylglycerol (DAG), in turn also stimulate the resynthesis of PI(4,5)P2. Because its precursor phosphytidylinositol (PI) is synthesized in the endoplasmic reticulum (ER), PI transport to the PM is an essential step in replenishment of PI(4,5)P2. This transport has recently been shown to occur by a non-vesicular mechanism at highly specialized contact sites between both membranes, the ER-PM junctions. These membrane contact sites are mediated by membrane tethering proteins, including the Extended Synaptotagmins (E-Syts) and tightened upon intracellular Ca2+ rise, allowing PI transfer to occur. In my work, I discovered the preferential localization of tubbyCT, a known PI(4,5)P2 recognition domain, to E-Syt3-rich ER-PM junctions. Junctional recruitment is mediated by coincidence detection of E-Syt3 and PI(4,5)P2, as shown by co-localization experiments, co-immunoprecipitations and manipulations of PM PI(4,5)P2 content. These dual binding properties allowed, for the first time, the selective investigation of local PI(4,5)P2 dynamics at ER-PM junctions. Using Total Internal Reflection Fluorescence (TIRF) microscopy, TubbyCT revealed the unexpected increase of a local PI(4,5)P2 pool at ER-PM junctions, that was dependent on local synthesis, despite concurrent global PI(4,5)P2 consumption by PLCβ. Pharmacological inhibition of PI(4,5)P2 resynthesis revealed that these local PI(4,5)P2 pool dynamics are required for maintenance and tightening of ER-PM contact sites during PLCβ signaling. Together, my data suggest a model of local metabolic turnover of locally supplied PI, i.e. ‘metabolic channeling’ of PI(4,5)P2 production in the PM. Enrichment at ER-PM contact sites was not restricted to the isolated tubby domain, but was likewise observed with the full-length tubby protein and its close relative TULP3. So far, tubby-like proteins (TULPs) have been implicated in delivery of G protein-coupled receptors to primary cilia. My findings suggest an additional role of TULP proteins at ER-PM junctions not previously recognized.
Physical Description:126 Pages
DOI:https://doi.org/10.17192/z2021.0200