Tattletales for Multiplex Fluorescent Reporters in Single Cells for Metabolomics

George McNamara

As of April 2013: L.J.N. Cooper & D.A. Lee Cellular Immunotherapy Lab, University of Texas M.D. Anderson Cancer Center, Houston, TX

Email: gtmcnamara@mdanderson.org, geomcnamara@earthlink.net

Tattletales is my concept for spatial multiplexing many fluorescent protein (FP) biosensors in the same live cell. For example, there are excellent FP biosensors to Ca++ ions, pH, glucose, ribose, glutamine, glutamate, ATP, redox, ROS, pyruvate, cAMP, cGMP, IP3, PI(3,4,5)P3, cell cycle indicators (Fucci2), PKA, PKC, photsphatases, caspase(s) [1, 2]. However, these are typically used one biosensor per experiment, due in part to flooding the cell with soluble biosensor. That is, conventionally, either a metabolite (glucose) reporter or a signal transduction (Ca++) reporter can be imaged. By flooding the cell with the reporter, signal to noise ratio is compromized by autofluorescence.

Tattletales takes advantage of spatial multiplexing to both increase the number of different reporters, and improve signal to noise ratio by localizing each biosensor to a small volume. I started with the observation by Robinett et al [3] who localized 512 GFP-nls-LacI to a 256 LacO array as a 200 nm diameter diffraction limited spot (nuclear background due to overexpression). Many thousands of DNA binding proteins, of known sequence specifities, exist (LacI, TetR, GalR, etc for cell line studies; ZF-FPs, TALE-FPs to STRs, telomere repeat binding factors-FPs, etc for primary cells) and can be fused (as cDNAs) to different fluorescent proteins and FP biosensors.

Many biosensors are available as affinity series [1, 4], now enabling extended dynamic range. I realized that spatial multiplexing of many DNA binding protein-reporters by localizing to different spots in the cell nucleus and distinguished by combinatorial addressing, where N address colors provide 2^N addresses (example, 3 colors is 2^3 = 8 combinations). Multiplexing enables many critical nodes to be quantified simultaneously in live cells.

[1] Frommer WF 2013 http://biosensor.dpb.carnegiescience.edu/ [2] Newman RH, et al. Chem Rev 111: 3614-3666, 2011. [3] Robinett CC, et al. J Cell Biol 135: 1685-1700, 1996. [4] Okumoto S et al. Proc Natl Acad Sci USA 102: 8740-8745, 2005.

My thanks to Dr. Brian Rabinovich, University of Texas M.D. Anderson Cancer Center, for telling me about the L.J.N. Cooper & D.A. Lee high throughput TALEN assembly robot that inspired my Tattletales concept. In April 2013 I plan to move to the Cooper & Lee lab in Houston to be the lab's single cells image analyst. I anticipate working in the lab to "reduce to practice" Tattletales in T-cells and NK cells later in 2013.

A PDF with figures and table is included in the ZIP file.

Additional web posters are online at

http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg

and

http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_TAM_and_Tattletales_TIK_Public_Domain.jpg

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November 4, 2013 update:

I have been making further progress on Tattletales, inspired in part by FingR (Gross et al 2013 Neuron), more information about human variable number tandem repeats (VNTRs), and more. It turns out that some people have ~200 repeats (20aa, 60 base pairs) of the MUC1 VNTR; some have ~400 repeats (16 aa, 48 base pairs) of the MUC4 VNTR. I propose using synthetic versions of parts of these, as well as entirely synthetic VNTRs (synVNTR or sVNTR) as landing sites.

The 201311 pdf now includes a description of how to use "rainbow" T-cell and tumor cells, that is, T-Bow, to mark each cell, or clone, with a molecular and visual "dot code" that goes beyond current "molecular barcodes".

I also include more details on the 90+ fluorescent protein biosensors that have been published - many available through addgene.org.

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Feb 22, 2013 update. I revised the zip file and added a README file with the following information:

George McNamara, Ph.D. University of Miami (for one more month) Cooper and Lee Labs, M.D. anderson Cancer Center, Houston, TX (starting April 1, 2013, if all goes to schedule). gmcnamara2med.miami.edu geomcnamara@earthlink.net

February 22, 2013,

Tattletales multiplex fluorescent (and bioluminescent) reporters ZIP file archive.

Note: I had encouraged some researchers to check out my Feb 5, 2013, pdf and ppt (pptx) files here. These have been replaced by the two newer files below.

McNamara 20130220 Tattletales 2013 update - 43x47inches metabolomics.pdf * my current "Tattletales metabolomics" poster. I am presenting the physical poster at several events in Feb and March 2013.

McNamara 20130221 - Tattletales Concept for Multiplex Fluorescent Reporters in Single Cells - update.pdf * two page abstract for the above "Tattletales metabolomics".

**** older files ****

McNamara 201210 HHMI Poster GFP 20121023Tue draft - Tattletales - 43x47inches - Public Domain date.pptx * my original poster.

McNamara 201210 HHMI Poster Turning Images into Knowledge 20121023Tue draft - TAM and Tattletales - 43x47inches - Tattletales Public Domain date.pptx * another poster from my "public domain release date", this one mostly on quantification of cell motility and chemotaxis, with some information on Tattletales.

McNamara 201210 HHMI Text from poster GFP 20121027Sat draft - Tattletales multiplex biosensors and expression activity reporters.pdf * text related to my original 20121023Tue poster.