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

optogenetics Optogenetics is a biological technique to control the activity of neurons or other cell types with light. This is achieved by Gene expression, expression of Channelrhodopsin, light-sensitive ion channels, Halorhodopsin, pumps or Photoactivated ade ...

tool designed to reversibly label cortical ER or the apposition between plasma membrane (PM) and

endoplasmic reticulum The endoplasmic reticulum (ER) is a part of a transportation system of the eukaryote, eukaryotic cell, and has many other important functions such as protein folding. The word endoplasmic means "within the cytoplasm", and reticulum is Latin for ...

(ER) membranes (termed as ER-PM junctions).

Design

The ER luminal domain of LiMETER contains a

signal peptide A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16–30 amino acids long) present at the ...

and the

transmembrane domain A transmembrane domain (TMD, TM domain) is a membrane-spanning protein domain. TMDs may consist of one or several alpha-helices or a transmembrane beta barrel. Because the interior of the lipid bilayer is hydrophobic, the amino acid residues in ...

derived from

STIM1 Stromal interaction molecule 1 is a protein that in humans is encoded by the ''STIM1'' gene. STIM1 has a single transmembrane domain, and is localized to the endoplasmic reticulum, and to a lesser extent to the plasma membrane. Even though the pr ...

, 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 Orai is a city in Uttar Pradesh, India India, officially the Republic of India, is a country in South Asia. It is the List of countries and dependencies by area, seventh-largest country by area; the List of countries by population ( ...

calcium channels in the plasma membrane. 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 Phototropins are blue light photoreceptor proteins (more specifically, flavoproteins) that mediate phototropism responses across many species of algae, fungi and higher plants. Phototropins can be found throughout the leaves of a plant. Along with ...

1, followed by a C-terminal PM-targeting polybasic tail that associates with negative charged

phosphoinositides Phosphatidylinositol or inositol phospholipid is a biomolecule. It was initially called "inosite" when it was discovered by Léon Maquenne and Johann Joseph von Scherer in the late 19th century. It was discovered in bacteria but later also found ...

in the inner half of the leaflet of plasma membrane.

Function

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 Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

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. This process can be reversibly repeated with multiple light–dark cycles without significant loss in the magnitude of response. 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.

References

{{Reflist Biological techniques and tools Membrane biology