Part:BBa_K3133004

mTagRFP-mWasabi-LC3

mWasabi-LC3 was used because it was more acid sensitive than EGFP-LC3 in lysosomes and brighter when monitoring autolysosomes in autophagy flux. Also, the exposure time needed by mWasabi-LC3 is shorter than the one of EGFP-LC3, which yielded photos of higher quality. Because LC3 could also be present in non-autophagy cells, mWasabi-LC3∆G is more effective at discriminating them during autophagy flux (Zhou 2012).

Usage and Biology

BBa_K3133004 mTagRFP-mWasabi-LC3

Method:

Activation on autophagy was investigated by using mTagRFP-mWassbi-LC3 translocation assay. Briefly, SH-SY5Y cells were transfected with mRFP-GFP-LC3 plasmid via Lipofectamine 2000 according to the manufacturer’s protocol. The cells were treated with different testing compounds and then fixed with 4% paraformaldehyde. The images were acquired using a Leica DMI8 confocal microscope and analyzed by the Image J software. Autophagy activator Nitazoxanide (NTZ) and inhibitor Chloroquine (CQ) were applied to an assay of SH-SY5Y as positive and negative control, respectively.

Result:

At early stage of autophagosomes biogenesis, puncta of mTagRFP-mWasabi-LC3 accumulated and displayed both green and red fluorescent signals (mRFP+GFP+ yellow). Notably, when autophagosomes were fused with lysosomes, they formed acidic autolysosomes (mRFP-GFP+ red), and the co-located mTagRFP-mWassbi-LC3 emitted only a red signal because the green signal quenched immediately under acidic conditions. Thus an increase in numbers of red and yellow puncta would be an indicator of promoted autophagy. Compound C4, C5, C7, C8, C9, C10 show an increase in numbers of both yellow and red puncta compared with the control, indicated they are the activators of autophagy.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 622
  Illegal BsaI.rc site found at 760
  Illegal BsaI.rc site found at 1171