LiMETER

LiMETER stands for light-inducible membrane-tethered peripheral endoplasmic reticulum (ER).

LiMETER is an optogenetics tool designed to reversibly label cortical ER[1] or the apposition between plasma membrane (PM) and endoplasmic reticulum (ER) membranes (termed as ER-PM junctions).

The ER luminal domain of LiMETER contains a signal peptide and the transmembrane domain derived from STIM1,[2] with GFP placed in between as a reporter. STIM1 is an ER-resident calcium sensor protein responsible for sensing calcium changes in internal calcium stores and communicate with ORAI calcium channels in the plasma membrane.[3][4][5][6][7][8] The cytoplasmic region of LiMETER contains a flexible linker and a genetically encoded lightswitch LOV2 domain (light oxygen voltage-sensing domain, residues 404–546) derived from Avena sativa phototropin 1, followed by a C-terminal PM-targeting polybasic tail that associates with negative charged phosphoinositides in the inner half of the leaflet of plasma membrane.[1]

In the dark, the Jα helix docks to the LOV2 domain and cages the polybasic tail to prevent its interaction with negatively charged PM-resident phosphoinositides. Following blue light illumination, photoexcitation generates a covalent adduct between a cysteine residue and the flavin cofactor in LOV2, and subsequently promotes the undocking and unwinding of the Jα helix, thereby exposing the polybasic C-tail to enable translocation of the protein towards PM to form puncta-like structures.

As a result, LiMETER undergoes photo-inducible translocation toward ER–PM junctions to specifically label cER. A video showing this process can be found here. This process can be reversibly repeated with multiple light–dark cycles without significant loss in the magnitude of response.[1]

This optical tool enables cell biologists to quantitatively examine the effect of regulators that modulate the dynamics of cER accumulation at defined spatiotemporal resolution in living cells.