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Loss of ER Ca 2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca 2+ . Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca 2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca 2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca 2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In the last two decades, fluorescent proteins became an indispensable tool to noninvasively label a protein in living cells. The discovery of photoswitchable fluorescent proteins expanded the applications of the fluorescent proteins to techniques such as molecular tracking and highlighting on a microscope. Recently, a new microscopic modality to achieve a superresolution circumventing the diffraction limit of light (photoactivated localization microscopy, PALM) has been developed based on the photoswitchable fluorescent proteins. Here we introduce a basic protocol of PALM through the visualization of actin bundles with superresolution.