Chang Chi-Lun, Lee Wan-Ru, Li Wei-Ting, Liou Jen
Department of Cell and Molecular Biology, St. Jude Children's Hospital, Memphis, TN, USA.
Department of Physiology, UT Southwestern Medical Center, Dallas, TX, USA.
Methods Mol Biol. 2025;2888:23-34. doi: 10.1007/978-1-0716-4318-1_3.
Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P) in the plasma membrane (PM) important for Ca signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM contact sites, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P replenishment following receptor stimulation as a readout of PI transfer using a PI(4,5)P biosensor and total internal reflection microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM contact sites and mediate PI(4,5)P replenishment with PI in the ER in stimulated cells. These methods provide spatial and temporal analysis of ER-to-PM PI transfer during receptor stimulation and can be adapted to other research questions related to this topic.
磷脂酰肌醇（PI）是一种在内质网（ER）中合成的含肌醇磷脂。PI是质膜（PM）中PI 4,5-二磷酸（PI(4,5)P）的前体脂质，对响应细胞外刺激的钙信号传导很重要。因此，内质网到质膜的PI转运对于细胞在受体刺激期间维持PI(4,5)P稳态至关重要。在本章中，我们讨论两种活细胞成像方案，以分析内质网与质膜接触位点处的内质网到质膜的PI转运，在这些位点，两个膜区室紧密并列以适应PI转运。首先，我们描述如何使用PI(4,5)P生物传感器和全内反射显微镜监测受体刺激后PI(4,5)P的补充，作为PI转运的读数。第二个方案直接可视化在受刺激细胞中在内质网与质膜接触位点积累并介导内质网中的PI补充PI(4,5)P的PI转运蛋白。这些方法提供了受体刺激期间内质网到质膜PI转运的时空分析，并且可以适用于与该主题相关的其他研究问题。
