Marker Gene Monthly Newsletter - Volume 11, Number 12 - December, 2011

Fluorophore Ligases for Specific Cell Labeling.

Proteomic methods to identify proteins from a specific cell, intracellular organelle, or cellular structure are often complicated by complex and disruptive purification methods. Such methods can often perturb the levels of individual proteins in a sample, or can even reduce their stability and labeling. In addition, co-expression with GFP, SNAP-Tag or Halo-Tag systems introduce large proteins that can perturb the protein intracellular function or trafficking. Recently several elegant techniques have been developed by Dr. Alice Ting and co-workers at Massacheusetts Institute of Technology that utilize engineered enzymes inside living cells to tag proteins with either a biotin or fluorophore. In one technique, termed "PRobe Incorporation Mediated by Enzymes" (PRIME), a mutant E.coli lipoic acid ligase (LplA),whose natural function is to ligate lipoic acid onto acceptor proteins involved in oxidative metabolism was developed. This mutant enzyme has been altered to instead ligate fluorophore analogs containing a short-chain fatty acid tail, onto a specific 13-16 AA receptor sequence on the protein of interest. Using this method, they were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin and other specific recombinant proteins inside living cells, on the cell surface or inside acidic endosomes. By expressing the mutant ligase on the surface of one cell, and the recognition sequence on cell surface proteins of a second cell, they were able to label only those cells which exhibited cell-cell contact. In addition, in a side-by-side comparison with FlAsH (fluorescein arsenical hairpin), the PRIME labeling was found to be faster, more specific, and less toxic. Marker Gene is currently working with MIT to bring these new fluorophores to the market for use by researchers worldwide. For more information about these techniques and systems, please see the references below or visit our website.

• Uttamapinant C , White KA, Baruah H, Thompson S, Fernández-Suárez M, Puthenveetil S, Ting AY(2010) "A fluorophore ligase for site-specific protein labeling inside living cells." PNAS 107(24): 10914–10919.
• Cohen JD, Thompson S, Ting AY (2011) "Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells." Biochemistry 50: 8221-8225.
• Slavoff SA, Liu DS, Cohen JD, Ting AY (2011) "Imaging protein-protein interactions inside living cells via interaction-dependent fluorophore ligation." J. Amer. Chem. Soc. 133: 19769-19776.
• Jin X,Uttamapinant C, Ting,AY (2011) "Synthesis of 7-Aminocoumarin by Buchwald-Hartwig Cross Coupling for Specific
  Protein Labeling in Living Cells." ChemBioChem 12:65-70.
• Fernández-Suárez M, Baruah H, Martínez-Hernández L, Xie KT, Baskin JM, Bertozzi CR, Ting AY, (2007) "Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes." Nature Biotechnol. 25: 1483-1487.
• Chen I, Howarth M, Lin W, Ting AY, (2005) "Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase." Nature Meth. 2: 99-104.

iPALM Improved Cellular Imaging.

The Abbe Diffraction Limit restricts optical resolution of fluorescently labeled proteins or dye stains in aqueous systems during cellular microscopy to an amount roughly λ/2, where λ is the wavelength of light, with water having a refractive index of about one. Single molecules emitting green light (about 500 nm) will therefore exhibit a cone of light approximately 250 nm in diameter at the position of the fluorophore. Since many intracellular structures are in the size range of 1 um, this inherent diffraction causes poor resolution and limits analytical discrimination of intracellular staining patterns. Recent work from the laboratories of Dr. Jennifer Lippincott-Schwartz and co-workers at the Cell Biology and Metabolism Program, NICHD, NIH have developed several ingenious methods of reducing this diffraction limit utilizing labeling with photoactivatable fluorescent proteins. These photoactivatable fluorescent proteins (PA-FPs) can switch to a new fluorescent state in response to excitation and generate a high level of contrast. There are a variety of PA-FPs now available that fluoresce green or red upon activation, or convert from green to red in response to the activating light. Others can reversibly switch between ‘off’ and ‘on’ in response to light. These properties can be used to enhance optical "highlighting" capability of PA-FPs by tracking the "on-off" or changes upon repeated short, low-light excitation sequences. In one such method, their group used combined eGFP and mCherry PA-FP labeled proteins to identify individual molecules inside living cells in three dimensions and with a resolution of about 25 nm. By coexpressing PAmCherry1 and PA-GFP, each protein can be switched on simultaneously from a dark state after a brief pulse of 405 nm or 488 nm laser light with imaging at 564 nm. In addition, by using two optical objectives, 3D-PALM data can be generated in a technique termed interferometric PALM or iPALM. These improved imaging techniques promise to allow the ability to dissect protein trafficking patterns and inter-organelle exchange of molecules within cellular systems. For more information about these techniques and systems, please see the references below or visit our website.

• Lippincott-Schwartz J, Patterson GH. (2009) "Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging." Trends Cell Biol. 19(11): 555-565.
• Lippincott-Schwartz J, Manley S. (2009) "Putting super-resolution fluorescence microscopy to work." Nature Methods 6(1): 21-3.
• Subach FV, Patterson GH, Manley S, Gillette JM, Lippincott-Schwartz J, Verkhusha VV. (2009) "Photoactivatable mCherry for high-resolution two-color fluorescence microscopy." Nature Methods 6(2):153-9.

Improved Delivery of Sulfonated Fluorophores.

Many fluorophores, including the CY3/CY5 carbocyanines and modified AlexaFluor-type dyes contain added sulfonate groups to impart greater water solubility, improve fluorescence quantum yields or decrease photobleaching. But these same sulfonate groups often act to restrict the passage of the dyes or conjugates containing them through the cell membrane. Recent work from the laboratory of Dr. Steven Miller and co-workers at the Department of Biochemistry and Molecular Biology at the University of Massacheusetts Medical School has developed a method of loading these dyes into living cells using a self-immolative sulfonate protecting group which, upon ubiquitous intracellular esterase activity, decomposes to release the free sulfonate dye compound. Using this method they were able to demonstrate loading of several highly photostable, near-IR oxazine fluorophores into live cells, with excellent staining levels and intracellular retention. The use of this new loading method for potential labeling of live cells and tissues is significant. For more information about these techniques and systems, please see the references below or visit our website.

• Pauff SM, Miller SC (2011) " Synthesis of Near-IR Fluorescent Oxazine Dyes with Esterase-Labile Sulfonate Esters." Org. Lett. 13(23): 6196-6199.
• Miller SC, (2010) "Profiling Sulfonate Ester Stability: Identification of Complementary Protecting Groups for Sulfonates." J. Org. Chem. 75(13): 4632-4635.
• Pauff S, Miller SC (2011) "A chemical strategy for the delivery of sulfonated fluorophores into live cells." Mol. Biol. Cell 22(24): 4705.

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