LiMETER

Definition

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

Design

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]

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. 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.

References

  1. 1 2 3 Jing, Ji; He, Lian; Sun, Aomin; Quintana, Ariel; Ding, Yuehe; Ma, Guolin; Tan, Peng; Liang, Xiaowen; Zheng, Xiaolu (2015-01-01). "Proteomic mapping of ER–PM junctions identifies STIMATE as a regulator of Ca2+ influx". Nature Cell Biology. 17: 1339–47. doi:10.1038/ncb3234. PMID 26322679.
  2. ↑ Soboloff, Jonathan; Rothberg, Brad S.; Madesh, Muniswamy; Gill, Donald L. (2012-01-01). "STIM proteins: dynamic calcium signal transducers". Nature Reviews Molecular Cell Biology. 13 (9): 549–565. doi:10.1038/nrm3414. PMC 3458427Freely accessible. PMID 22914293.
  3. ↑ Gudlur, Aparna; Zhou, Yubin; Hogan, Patrick G. (2013-01-01). Prakriya, Murali, ed. Chapter Two - STIM–ORAI Interactions That Control the CRAC Channel. Store-Operated Calcium Channels. 71. Academic Press. pp. 33–58. doi:10.1016/b978-0-12-407870-3.00002-0.
  4. ↑ Hogan, Patrick G.; Lewis, Richard S.; Rao, Anjana (2010-01-01). "Molecular Basis of Calcium Signaling in Lymphocytes: STIM and ORAI". Annual Review of Immunology. 28 (1): 491–533. doi:10.1146/annurev.immunol.021908.132550. PMC 2861828Freely accessible. PMID 20307213.
  5. ↑ Ma, Guolin; Wei, Ming; He, Lian; Liu, Chongxu; Wu, Bo; Zhang, Shenyuan L.; Jing, Ji; Liang, Xiaowen; Senes, Alessandro (2015-07-17). "Inside-out Ca2+ signalling prompted by STIM1 conformational switch". Nature Communications. 6: 7826. doi:10.1038/ncomms8826. PMC 4509486Freely accessible. PMID 26184105.
  6. ↑ Gudlur, Aparna; Quintana, Ariel; Zhou, Yubin; Hirve, Nupura; Mahapatra, Sahasransu; Hogan, Patrick G. (2014-01-01). "STIM1 triggers a gating rearrangement at the extracellular mouth of the ORAI1 channel". Nature Communications. 5: 5164. doi:10.1038/ncomms6164. PMC 4376667Freely accessible. PMID 25296861.
  7. ↑ Zhou, Yubin; Srinivasan, Prasanna; Razavi, Shiva; Seymour, Sam; Meraner, Paul; Gudlur, Aparna; Stathopulos, Peter B; Ikura, Mitsuhiko; Rao, Anjana (2013-01-01). "Initial activation of STIM1, the regulator of store-operated calcium entry". Nature Structural & Molecular Biology. 20 (8): 973–981. doi:10.1038/nsmb.2625. PMC 3784406Freely accessible. PMID 23851458.
  8. ↑ Zhou, Yubin; Meraner, Paul; Kwon, Hyoung T; Machnes, Danya; Oh-hora, Masatsugu; Zimmer, Jochen; Huang, Yun; Stura, Antonio; Rao, Anjana (2009-01-01). "STIM1 gates the store-operated calcium channel ORAI1 in vitro". Nature Structural & Molecular Biology. 17 (1): 112–116. doi:10.1038/nsmb.1724. PMC 2902271Freely accessible. PMID 20037597.
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