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Hi all-
I had read with interest last year a modified way of measuring autophagy
flux in live cells using TagRFP and mWasabi tandem-tagged to LC3. The
TagRFP signal survives the acidic environment of the lysosome while the
mWasabi signal dies due to its higher pKa. This allows one to track how
much of the autophagy component LC3 that exists in the cell (green signal)
actually makes its way to a functional lysosome (red signal). This
construct was supposed to represent an improvement over the old GFP-RFP-LC3
construct because the pKa of mWasabi is higher than GFP (so the green
signal would more fully die in the presence of an acidic lysosome) and the
pKa of TagRFP is lower than mRFP (Tag-RFP-T doesn't work because of a
higher pKa):
Zhou C, Zhong W, Zhou J, Sheng F, Fang Z, Wei Y, Chen Y, Deng X, Xia B, Lin
J. Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3
(mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic
flux in cancer cells. Autophagy 2012; 8:1215 - 1226; PMID: 22647982
Unfortunately, I have been unable to get in touch with the authors to
request this construct. Does anyone on the listserve have this construct
and permission to distribute? If not, does anyone have experience with the
original GFP-RFP-LC3 construct - does it perform as advertised or are the
improvements in pKa seen with the newer mWasabi-TagRFP construct necessary?
Sincere thanks in advance.
-Jason Miller
>
> --
>
> Jason Miller, MD, PhD
>
> University of Michigan Kellogg Eye Center
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> The first thing which I can record concerning myself is, that I was born.
> These are wonderful words. This life, to which neither time nor eternity
> can bring diminution - this everlasting living soul, began. My mind loses
> itself in these depths. -- Groucho Marx
>
--
Jason Miller, MD, PhD
University of Michigan Kellogg Eye Center
E-mail: *
[hidden email] <
[hidden email]> *
The first thing which I can record concerning myself is, that I was born.
These are wonderful words. This life, to which neither time nor eternity
can bring diminution - this everlasting living soul, began. My mind loses
itself in these depths. -- Groucho Marx
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