Crosstalk Between Calcium Signaling and Lipid Metabolism at Endoplasmic Reticulum-Plasma Membrane Junctions




Chang, Chi-Lun

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Receptor-induced Ca2+ signaling is the key to many cellular functions, such as secretion, migration, differentiation, and proliferation. The increase in cytosolic Ca2+ signals is dependent on the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane (PM). To enable subsequent signaling activation and maintain cellular homeostasis, it is necessary to replenish the consumed PIP2. However, the molecular mechanisms underlying PM PIP2 replenishment after hydrolysis remain elusive. PIP2 is generated at the PM by sequential phosphorylation of phosphatidylinositol (PI) originating from the endoplasmic reticulum (ER). Delivering PI from the ER to the PM by PI transfer proteins (PITPs) is therefore postulated to support PM PIP2 replenishment. Such transfer is more likely to take place at ER-PM junctions, since the close apposition of the ER and the PM enables PITPs to efficiently interact with two heterologous membranes. To study ER-PM junctions and their roles in PM PIP2 replenishment, I generated a genetically-encoded fluorescent marker to selectively label ER-PM junctions. With this marker, minute ER-PM junctions were easily observed in live cells using multiple imaging techniques. At the resting state, approximately two hundred stable ER-PM junctions were detected at the adhesion surface of a single HeLa cell. Photo-activated localization microscopy (PALM) super-resolution imaging further demonstrate that ER-PM junctions labeled by this marker were remarkably uniform in size and slightly elongated in shape with a long axis of 255.5 nm and a short axis of 157.7 nm. Furthermore, analysis of the distance to nearest neighbor of individual ER-PM junctions show that these junctions are distributed uniformly in the cells. Following the activation of Ca2+ signaling, I observed an enhanced ER-to-PM connection resulting from new junction formation and a decrease in the gap distance of ER-PM junctions. The enhanced ER-to-PM connection depends on cytosolic Ca2+ levels and extended synaptotagmin-like protein 1 (E-Syt1), a C2 domain-containing ER membrane protein. E-Syt1 detects the increase in cytosolic Ca2+ via its C2C domain and translocates from the bulk of ER to ER-PM junctions to enhance ER-to-PM connection. This in turn facilitates the recruitment of Nir2, an ER-associated PITP, to ER-PM junctions to promote PM PIP2 replenishment. In summary, these results indicate a feedback loop for PM PIP2 replenishment via E-Syt1 and Nir2 at ER-PM junctions. Disruption of this feedback mechanism by knockdown of E-Syt1 or Nir2 abolished PM PIP2 replenishment and therefore, impaired receptor-induced Ca2+ signaling. This work reveals the long-sought mechanism of PM PIP2 replenishment following hydrolysis and sheds light on the functional roles of poorly characterized ER-PM junctions. Furthermore, given the fact that PIP2 and Ca2+ are pivotal signaling molecules for many cellular functions, these findings are of significance for providing new mechanistic insights into the signaling crosstalk and may have a broader impact on fields beyond cell signaling, organelle dynamics, and lipid trafficking.

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