ER-PM Junction Proteins and Their Roles in Regulating Cell Homeostasis and Signaling

Date

2021-05-01T05:00:00.000Z

Authors

Quintanilla, Carlo Giovanni

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Abstract

Homeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2) in receptor-stimulated cells is a critical step in the phosphoinositide cycle (PI-Cycle). This regulatory feedback mechanism is mediated by the lipid transfer protein (LTP) Nir2. Nir2 is dynamically recruited to endoplasmic reticulum-plasma membrane (ER-PM) junctions to facilitate replenishment of PM PIP2 hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP2 is limited. The work presented in this dissertation, describes the functions of Nir1, a previously unidentified ER-PM junction tether and regulator of Nir2 and PM PIP2 replenishment. Manipulation of Nir1 levels in live cells via overexpression or transient knockdown drives remodeling of ER-PM junction properties. Additionally, Nir1 potentiates Nir2 targeting to ER-PM junctions during receptor-mediated signaling and is required for efficient PM PIP2 replenishment. Importantly, I found that Nir1 localization at ER-PM junctions is a requirement for Nir2 potentiation, highlighting the importance of this subcellular site in regulating the PI-Cycle. The Live-cell and biochemical analysis revealed that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Lastly, I describe a novel localization of Nir proteins near the nucleus and demonstrate the requirement of the minimally characterized domain, DDHD. In summary, the results from these studies identify Nir1 as a novel ER-PM junction tether as well as a positive regulator of the PI-Cycle and of the LTP, Nir2. My observations of Nir proteins near the nucleus implicate a novel subcellular site for phosphoinositide metabolic regulation beyond ER-PM junctions.

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Pages x-xiii are misnumbered as pages xi-xiv, page xiv is misnumbered as page 1, and pages 1-96 are misnumbered as pages 2-97.

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