Super-Resolution Microscopy Reveals That Stromal Interaction Molecule 1 Trafficking Depends on Microtubule Dynamics.

Yi-Ting Huang,Ya-Ting Hsu,Meng-Ru Shen,Yih-Fung Chen

doi:10.3389/fphys.2021.762387

Abstract

Store-operated Ca2+ entry (SOCE) is an essential pathway for Ca2+ signaling, and regulates various vital cellular functions. It is triggered by the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1). Illustration of STIM1 spatiotemporal structure at the nanometer scale during SOCE activation provides structural and functional insights into the fundamental Ca2+ homeostasis. In this study, we used direct stochastic optical reconstruction microscopy (dSTORM) to revisit the dynamic process of the interaction between STIM1, end-binding protein (EB), and microtubules to the ER-plasma membrane. Using dSTORM, we found that“powder-like”STIM1 aggregates into “trabecular-like” architectures toward the cell periphery during SOCE, and that an intact microtubule network and EB1 are essential for STIM1 trafficking. After thapsigargin treatment, STIM1 can interact with EB1 regardless of undergoing aggregation. We generated STIM1 variants adapted from a real-world database and introduced them into SiHa cells to clarify the impact of STIM1 mutations on cancer cell behavior. The p.D76G and p.D84Y variants locating on the Ca2+ binding domain of STIM1 result in inhibition of focal adhesion turnover, Ca2+ influx during SOCE and subsequent cell migration. Inversely, the p.R643C variant on the microtubule interacting domain of STIM1 leads to dissimilar consequence and aggravates cell migration. These findings imply that STIM1 mutational patterns have an impact on cancer metastasis, and therefore could be either a prognostic marker or a novel therapeutic target to inhibit the malignant behavior of STIM1-mediated cancer cells. Altogether, we generated novel insight into the role of STIM1 during SOCE activation, and uncovered the impact of real-world STIM1 variants on cancer cells.

Highlights

Store-operated Ca2+ entry (SOCE), a major Ca2+ influx mechanism in most non-excitable cells, is an essential pathway for Ca2+ signaling (Cahalan, 2009)
The morphology of stromal interaction molecule 1 (STIM1) overexpressed in SiHa cells after TG treatment is identical to the endogenous expression of STIM1 (Supplementary Figure 1), but the former is conductive for characterizing the architecture of STIM1 (Chen et al, 2011, 2013a)
The full width at half maximum (FWHM) of STIM1 estimated by direct stochastic optical reconstruction microscopy (dSTORM) imaging was ∼250 nm, much smaller than the ∼650 nm measured by WF and confocal microscopy (Figure 1B)

Summary

Introduction

Store-operated Ca2+ entry (SOCE), a major Ca2+ influx mechanism in most non-excitable cells, is an essential pathway for Ca2+ signaling (Cahalan, 2009). STIM1 is an ER-resident transmembrane protein with several functional domains and protein-protein interaction motifs, which has a critical role in SOCE activation (Yeung et al, 2020). Several studies have reported the importance of the microtubule plusend trafficking mechanism in the redistribution of STIM1 toward the ER-plasma membrane junctions, and the subsequent activation of Orai proteins (Chen et al, 2013a, 2019a). The ultrastructural organization of STIM1 during activation, aggregation, and translocation, as well as the interaction between STIM1, microtubules, and end-binding proteins (EBs) during SOCE remain unclear

Objectives

Methods

Results

Conclusion