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 expression of light-sensitive ion channels, pumps or enzymes specifically in the target cells. On the level of individ ...
tool designed to reversibly label cortical ER
or the apposition between plasma membrane (PM) and
endoplasmic reticulum (ER) membranes (termed as
ER-PM junctions).
Design
The ER luminal domain of LiMETER contains a
signal peptide and the
transmembrane domain
A transmembrane domain (TMD) is a membrane-spanning protein domain. TMDs generally adopt an alpha helix topological conformation, although some TMDs such as those in porins can adopt a different conformation. Because the interior of the lipid bi ...
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 pro ...
, 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. It is the administrative headquarters of Jalaun District. All administrative offices of Jalaun District, including the District Collectorate, police, telecom and various other government organizations are ...
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 photoreceptor proteins (more specifically, flavoproteins) that mediate phototropism responses in higher plants. Phototropins can be found throughout the leaves of a plant. Along with cryptochromes and phytochromes they allow plants ...
1, followed by a C-terminal PM-targeting polybasic tail that associates with negative charged
phosphoinositides
Phosphatidylinositol (or Inositol Phospholipid) consists of a family of lipids as illustrated on the right, where red is x, blue is y, and black is z, in the context of independent variation, a class of the phosphatidylglycerides. In such molecul ...
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 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