Tunable Chemical and Optical Modulation of ER-Plasma Membrane Contact-Site Geometry, Dynamics, and Protein Recruitment with High-Fidelity Visualization.

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites are dynamic membrane interfaces that regulate essential cellular processes, including calcium signaling and lipid homeostasis. Emerging evidence suggests that contact-site geometry and organization critically influence these functions, yet existing tools cannot systematically manipulate individual geometric parameters while providing high-fidelity visualization in living cells. Here, we develop complementary chemical and optical platforms for inducible ER-PM contact-site reconstitution. A nontoxic, reversible abscisic acid-inducible system based on the plant-derived ABIcs/PYLcs pair and a rapidly reversible optogenetic iLID/SspB system enable robust visualization and dose-dependent control of contact-site assembly and disassembly. Increasing inducer dose selectively increases contact-site density and total contact area per cell without substantially changing the average size of individual contact sites. In contrast, systematic variation of tether length or tether abundance selectively increases average contact-site size and total contact area without altering contact-site density, providing orthogonal strategies to modulate contact-site density and size. Importantly, engineered contact sites recruit ER-PM contact-site-associated proteins, including MAPPER, Kv2.1, and Stromal Interaction Molecule 1 (STIM1), demonstrating key organizational features of native ER-PM contacts. MAPPER recruitment is maintained across tether lengths tested, whereas Kv2.1/STIM1 recruitment depends strongly on tether length, with longer tethers promoting recruitment by increasing intermembrane spacing. In contrast, varying tether abundance has little effect on protein recruitment, revealing distinct roles for intermembrane spacing and tether density in organizing ER-PM contact sites. Together, this work establishes a quantitative platform for engineering ER-PM contact sites with defined geometric properties and provides a framework for investigating how membrane contact-site architecture regulates cellular function.