*****
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.
*****

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