EB1 binding restricts STIM1 translocation to ER-PM junctions and regulates store-operated Ca²⁺ entry

The endoplasmic reticulum (ER) Ca²⁺ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca²⁺ entry (SOCE) after ER Ca²⁺ depletion. STIM1 also interacts with EB1 and dynamically tracks microtubule (MT) plus ends. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with a synthetic construct approach, we found that EB1 binding constitutes a trapping mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca²⁺-depleted cells. By trapping STIM1 molecules at dynamic contacts between the ER and MT plus ends, EB1 binding delayed STIM1 translocation to ER-PM junctions during ER Ca²⁺ depletion and prevented excess SOCE and ER Ca²⁺ overload. Our study suggests that STIM1-EB1 interaction shapes the kinetics and amplitude of local SOCE in cellular regions with growing MTs and contributes to spatiotemporal regulation of Ca²⁺ signaling crucial for cellular functions and homeostasis.