SUNtag = making FP's brighter by localization ... and multiplexing

[George McNamara]

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making FP's brighter by localization ... see my text and PDF download
at   http://works.bepress.com/gmcnamara/63 
<http://works.bepress.com/gmcnamara/63>   for multiplexing ideas.

http://www.ncbi.nlm.nih.gov/pubmed/25307933


Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD 2014 A Protein-Tagging System for Signal Amplification in Gene Expression and
Fluorescence Imaging. Cell. 2014 Oct 8. pii: S0092-8674(14)01227-6. doi: 10.1016/j.cell.2014.09.039. [Epub ahead of print]

Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we
have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the
SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a
potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and
demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform
for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.


//


for those of you who do not use PubMed Commons, I posted the following
comments:

George McNamara
<http://www.ncbi.nlm.nih.gov/myncbi/george.mcnamara.1/comments/>

This is a nice paper. The abstract refers to using 24 epitope tags
(24mer), much of the paper uses a 10mer. Just doing GFP is boring. When
I came up with the "Tattletales" (TALE-FPn ... I came up with the idea
before sgRNA:Cas9 became popular), I immediately realized that
multimerizing FP biosensors. The current paper is the same as my what I
refer to as "Binary Tattletales", as in: 1. TALE-(linker-epitope tag)n
2. "binder"-(linker-FP)m with Tattletales being T-cells -- TALE
FPs/Biosensors. Since I moved to MD Anderson Cancer Center, the first T
now refers to "T-cells and Tumor cells". Likewise T-bow refers to
rainbow T-cells and Tumor cells for promoter bashing and otherwise
multicolor dots labeling cells (rainbow in homage of course to Brainbow
mice etc, and especially to real rainbows). For more on Tattletales,
Binary Tattletales, and T-Bow, see
http://works.bepress.com/gmcnamara/63 http://works.bepress.com/gmcnamara/42

http://works.bepress.com/gmcnamara/63 
http://works.bepress.com/gmcnamara/63 
<http://works.bepress.com/gmcnamara/42>

The PDF download at http://works.bepress.com/gmcnamara/63 has a table of
130 FP biosensors (if you are Laconic about ATeam and Fire, too bad) and
an extensive reference list with ZF-FP, TALE-FP, Cas9-FP (the latter
from the Weissman group), and more (PUF's and PPR's are RNA binding
protein families with structural similarities to TALEs). My favorite
name -- besides Tattletales and T-Bow, of course -- is "TALE-Lights"
from Yuan, Shermoen, O'Farrell 2014,
http://www.ncbi.nlm.nih.gov/pubmed/24556431

Giving credit where credit is due: The authors really should have cited
the first mammalian cell paper localizing a lot of FPs in one spot (they
came 'close' with a Gordon 1997 Cell paper on GFP:LacO in E.coli, but
the Tanenbaum paper is all mammalian cells): Robinett et al 1996 JCB
http://www.ncbi.nlm.nih.gov/pubmed/8991083 
http://jcb.rupress.org/content/135/6/1685.long See their figure 4A.
Straight, Robinett et al also published a yeast paper in 1996,
http://www.ncbi.nlm.nih.gov/pubmed/8994824 and it would have been useful
to cite that.


<http://www.ncbi.nlm.nih.gov/pubmed/24556431>


//

I hope some of you also find of interest my October 24, 2012 post,
http://lists.umn.edu/cgi-bin/wa?A2=ind1210&L=CONFOCALMICROSCOPY&P=15301
and maybe even look at

http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg  <http://home.earthlink.net/%7Epubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg>  

//

Finally, if reviewer #1 of our (L.J.N. Cooper, principal investigator)
2014 R21 Single Cell Analysis Program grant reads this post, please read
VERY CAREFULLY the section on photobleaching in the Tanenbaum et al
paper. Then please re-read your review of our proposal, please [text
edited out since this is a public forum], and especially, please recuse
yourself from reviewing any more NIH grant proposals.


enjoy,

George




--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Vitaly Boyko]

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Dear All,
we gave a couple of users who is using clearing agents for large tissue samples (CUBIC, ScaleA2 etc.). However, clearing causing tissue expansion and therefore difficulties in alignment when the samples scanned 12+ hrs apart. Does anyone have any experience or tested expansion rate over time, let say every 6 hrs, or at what time during clearing tissue expansion would reach the plateau?
Many thanks in advance,
Vitaly 

[George McNamara]

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*****

Hi Vitaly and listserv,

those are so last week. Latest is iDISCO

DOI: 10.1016/j.cell.2014.10.010


    Highlights

•

    iDISCO is a method for immunolabeling and volume imaging of large
    biological samples

•

    We optimize iDISCO for use in large mouse embryos and adult organs

•

    iDISCO is easy to implement, inexpensive, fast, and reliable

•

    We use iDISCO to study neurodevelopment and degeneration in embryos
    and adults mice


The visualization of molecularly labeled structures within large intact
tissues in three dimensions is an area of intense focus. We describe a
simple, rapid, and inexpensive method, iDISCO, that permits whole-mount
immunolabeling with volume imaging of large cleared samples ranging from
perinatal mouse embryos to adult organs, such as brains or kidneys.
iDISCO is modeled on classical histology techniques, facilitating
translation of section staining assays to intact tissues, as evidenced
by compatibility with 28 antibodies to both endogenous antigens and
transgenic reporters like GFP. When applied to degenerating neurons,
iDISCO revealed unexpected variability in number of apoptotic neurons
within individual sensory ganglia despite tight control of total number
in all ganglia. It also permitted imaging of single degenerating axons
in adult brain and the first visualization of cleaved Caspase-3 in
degenerating embryonic sensory axons in vivo, even single axons. iDISCO
enables facile volume imaging of immunolabeled structures in complex
tissues.

at Cell Press
http://www.cell.com/cell/abstract/S0092-8674(14)01297-5
at ScienceDirect (many Universities have full text access through SD,
not Cell)
http://www.sciencedirect.com/science/article/pii/S0092867414012975


enjoy,

George

On 10/30/2014 2:32 PM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear All,
> we gave a couple of users who is using clearing agents for large tissue samples (CUBIC, ScaleA2 etc.). However, clearing causing tissue expansion and therefore difficulties in alignment when the samples scanned 12+ hrs apart. Does anyone have any experience or tested expansion rate over time, let say every 6 hrs, or at what time during clearing tissue expansion would reach the plateau?
> Many thanks in advance,
> Vitaly
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42