The list below shows the target protein followed by different methods we have applied. Click the method or go to the get tools page to see caveats and advantages for each method. References are being added for each molecule. Check back for multiple new biosensors and optogenetic tools currently submitted, or write Dr. Hahn to ask what has been targeted.
Visualizing protein activity - biosensors
AR = affinity reagent, a protein or protein fragment that binds selectively to a given state of the target molecule.
- Rac1 (dual chain AR)
- RhoA (dual chain AR, single chain AR)
- RhoC (single chain AR)
- Cdc42 (dual chain AR, dye-labeled AR)
- RhoGDi-Rac1 interaction (antenna)
- RhoGDI-RhoA interaction (antenna)
- RhoGDI-Cdc42 interaction (antenna)
- Src family proteins sensed as a group (dye-labeled AR)
- FAK (biosensor in collaboration with Schaller group)
- Calmodulin (dye-labeled protein)
- S100 protein (dye-labeled protein)
- G9a transcription factor (small molecule sensor)
Activating proteins with light
- Rac1 (LOV fusion, LOVTRAP)
- Cdc42 (LOVTRAP)
- RhoA (LOVTRAP)
- ROCK (LOVTRAP)
- Formins (embedded peptides, in collboration with Zaidel-Bar group)
Inhibiting proteins with light
- Rac1 (loopology)
- RhoA (loopology)
- Cdc42 (loopology)
- Vav2 (loopology)
- ITSN (loopology)
- GEF-H1 (loopology)
- MLCK (embedded peptide)
- PKA (embedded peptide)
Engineered activation by membrane-permeable small molecules
Notes:- Currently responds to rapamycin and rapmycin analogs that do not interact with mTor.
- We can use a light-activated rapamycin to control with light.
- Activation is irreversible. Karginov lab has developed a reversible version.
- Src when activated interacts only with p130Cas (RapR TAP)
- Src when activated interacts only with FAK (RapR TAP)
- Src is driven to the membrane (RapR TAP)
- ITSN (SPELL)
- RhoGDI1 (SPELL)
- TEV (SPELL)
Other light-controlled techniques
- Light induced translocation into or out of the nucleus (embedded peptide and/or Zdk, in collaboration with Kuhlman lab)
- Light-controlled microtubule tip labeling (Zdk and Eb3, with Wittman lab)
Some references, soon to be associated with above tools
- Hahn, K.M., A.S. Waggoner and D.L. Taylor. A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin- binding fluorophore. J. Biol. Chem., 265: 20335-20345, 1990. Online article | PDF
- Hahn, K.M., R. DeBiasio and D.L. Taylor. Patterns of elevated free calcium and calmodulin activation in living cells. Nature, 359:736-738, 1992. Online article | PDF
- Hahn, K.M., P.A. Conrad, J. Chao, D.L. Taylor and A.S. Waggoner. A photocrosslinking, fluorescent indicator of mitochondrial membrane potential. J. Histochem. Cytochem., 41: 631-634, 1993. Online article | PDF
- Bark, S. J., and K.M. Hahn. Fluorescent indicators of peptide cleavage inside living cells. Methods, 20: 429-435, 2000. Online article | PDF
- Chamberlain, C.E., Kraynov, V. and Hahn, K.M. Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. Methods Enzymol., 325: 389-400, 2000. Online article | PDF
- Kraynov, V. S., Chamberlain, C. E., Bokoch, G. M., Schwartz, M. A., Slabaugh, S., and K. M. Hahn. Localized Rac activation dynamics visualized in living cells. Science, 290:333-337, 2000. Online article | PDF
- Nalbant, P., L. Hodgson, V. Kraynov, A. Toutchkine, K.M. Hahn. Activation of Endogenous Cdc42 Visualized in Living Cells. Science,305:1615-1619, 2004. Online article | PDF
- Pertz, O., L. Hodgson, R. R. Klemke, and K.M. Hahn. Spatio-temporal dynamics of RhoA activity in migrating cells. Nature, 440(7087):1069-1072, 2006. Online article | PDF
- Hodgson, L. Nalbant, P. Shen, F., and Hahn, K. Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol., 406:140-156, 2006. Online article | PDF`
- Cai, X., Lietha, D. Ceccarelli, D.F., Karginov, A.V., Rajfur, Z., Jacobson, K., Hahn, K.M., Eck, M.J., and Schaller, M.D. Spatial and temporal regulation of focal adhesion kinase activity in living cells. Mol. Cell Biol. 28:201-14, 2008. PMC2223290. Online article | Free PMC article
- Garrett, S.C., Hodgson, L., Rybin, A., Toutchkine, A., Hahn, K.M., Lawrence, D.S., and Bresnick, A.R. A Biosensor of S100A4 Metastasis Factor Activation: Inhibitor screening and Cellular Activation Dynamics. Biochemistry, 47:986-996, 2008. PMC3227476. Online article | Free PMC article
- Machacek, M., Hodson, L., Welch, C., Elliot, H., Pertz, O., Nalbant, P., Abell, A., Johnson, G., *Hahn, K.M. and *Danuser, G. Coordination of Rho GTPase activities during cell protrusion. Nature, 461: 99-103, 2009. PMC2885353. Online article | Free PMC article
- Wu, Y, Frey, D., Lungu, O. I., Jaehrig, A., Schlichting, I., Kuhlman, B. and Hahn, K.M. A genetically-encoded photoactivatable Rac reveals spatiotemporal coordination of Rac and Rho during cell motility. Nature, 461: 104-110, 2009. PMC2766670. Online article | Free PMC article
- Yoo, S.K., Deng, Q., Cavnar, P. J., Wu,Y.I., Hahn, K.M., Huttenlocher, A. Differential Regulation of Protrusion and Polarity by PI(3)K during Neutrophil Motility in Live Zebrafish. Developmental Cell, 18(2):226-236, 2010. PMC2824622. Online article | Free PMC article
- Karginov, A.V., Ding, F., Kota, P., Dokholyan, N.V., and Hahn, K.M. Engineered allosteric activation of kinases in living cells. Nature Biotech, July; 28(7):743-7, 2010. PMC2902629. Online article | Free PMC article
- Wang, X., He L, Wu, Y.I., Hahn, K.M., and Montell, D.J. Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nature Cell Biol., 12(6):591-7, 2011. PMC2929827. Online article | Free PMC article
- Karginov, A.V., Zou, Y., Shirvanyants, D., Kota, P., Dokholyan, N.V., Young, D.D., *Hahn, K.M. and *Deiters, A. Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP. J. Am. Chem. Soc., 133: 420-423, 2011. PMC3133816. Online article | Free PMC article
- Gulyani, A., Vitriol, E., Allen, R., Wu, J., Gremyachinskiy, D., Lewis, S., Dewar, B., Graves, L.M., Kay, B.K., Kuhlman, B., Elston T., and Hahn, K.M. A biosensor generated via high-throughput screening quantifies cell edge Src dynamics. Nature Chem. Biol., 7: 437-444, 2011. PMC3135387. Online article. | Free PMC article
- Wu, Y.I., Wang, X., He, L., Montell, D. and Hahn, K.M. Spatiotemporal control of small GTPases with light using the LOV domain. Methods Enzymol., 497: 393-407, 2011. PMC3407667. Online article | Free PMC article
- Karginov, A.V. and Hahn, K.M. Allosteric activation of kinases: design and application of RapR kinases. Curr. Protoc. Cell Biol., 53: 14.13.1 – 14.13.16, 2011. PMC3269071. Online article | Free PMC article
- Hinde, E., Digman, M.A., Welch, C., Hahn, K.M. and Gratton, E. Biosensor FRET detection by the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM). Microsc. Res. Tech., 75(3): 271-281, 2012. PMC3523109 . Online article | Free PMC article
- Lungu, O.I., Hallett R.A., Choi E.J., Aiken M.J., *Hahn K.M., *Kuhlman B. Designing Photoswitchable Peptides Using the AsLOV2 Domain. Chemistry and Biology, 19(4):507-17, 2012. PMC334866. Online article | Free PMC article
- Dietz, D.M., Sun, H., Lobo, M.K., Cahill, M.E., Chadwick, B., Gao, V., Koo, J.W., Mazei-Robison, M.S., Dias, C., Maze, I., Damez-Werno, D., Dietz, K.C., Scobie, K.N., Ferguson, D., Christoffel, D., Ohnishi, Y., Hodes, G.E., Zheng, Y., Neve, R.L., Hahn, K.M., Russo, S.J. and Nestler, E.J. Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons. Nature Neurosci., 15(6): 891-896, 2012. PMC3565539. Online article | Free PMC article
- MacNevin, C.J., Gremyachinskiy, D., Hsu, C.W., Li L., Rougie, M., Davis, T.T., Hahn, K.M. Environment-sensing merocyanine dyes for live cell imaging applications. Bioconjug. Chem. 24(2):215-23, 2013. PMC3667669. Online article | Free PMC article
- Dagliyan, O., Shirvanyants, D., Karginov, A.V., Ding, F., Fee, L., Chandrasekaran, S.N., Freisinger, C.M., Smolen, G.A., Huttenlocher, A., *Hahn, K.M., *Dokholyan, N.V. Rational design of a ligand-controlled protein conformational switch. Proc. Natl. Acad. Sci. U.S.A.,110(17):6800-4, 2013. PMC3637791. Online article | Free PMC article
- Konze, K.D., Ma, A., Li, F., Barsyte-Lovejoy, D., Parton, T., Macnevin, C.J., Liu, F., Gao, C., Huang, X.P., Kuznetsova, E., Rougie, M., Jiang, A., Pattenden, S.G., Norris, J.L., James, L.I., Roth, B.L., Brown, P.J., Frye, S.V., Arrowsmith, C.H., Hahn, K.M., Wang, G.G., Vedadi, M. and Jin, J. An orally bioavailable chemical probe of the lysine methyltransferases EZH2 and EZH1. ACS Chem. Biol., 8(6): 1324–1334,2013. PMC3773059. Online article | Free PMC article
- Rao, M.V., Chu, P.H., Hahn, K.M. and Zaidel-Bar, R. An optogenetic tool for the activation of endogenous diaphanous-related formins induces thickening of stress fibers without an increase in contractility. Cytoskeleton, 70(7): 394-407, 2013. PMC4064713.Online article | Free PMC article
- Zawistowski, J.S., Sabouri-Ghomi, M., *Danuser, G., *Hahn, K.M. and *Hodgson, L. A. RhoC biosensor reveals differences in the activation kinetics of RhoA and RhoC in migrating cells. PLoS One, 8(11): e79877, 2013. PMC3818223. Online article | Free PMC article
- Karginov, A., Tsygankov, D., Berginski, M., Chu, P-H., Trudeau, E., Yi, J.J., Gomez, Shawn, Elston, T.C. and Hahn, K.M. Dissecting motility signaling through activation of specific Src-effector complexes. Nature Chem. Bio., 10(4):286-90, 2014. PMC4064790. Online article | Free PMC article
- Yi, J.J., Wang, H., Vilela, M., Danuser, G., and Hahn, K.M. Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides. ACS Synth. Biol., 3(11):788-95, 2014. PMC4277778. Online article | Free PMC article
- Chu, P-H., Tsygankov, D., Berginski, M.E., Dagliyan, O., Gomez, S.M., Elston, T.C., Karginov, A.V. and Hahn, K.M. Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms. Proc. Natl. Acad. Sci. U.S.A.,111(34): 12420–12425, 2014. PMC4151743. Online article | Free PMC article
- Yumerefendi, H., Dickinson, D.J., Wang, H., Zimmerman, S., Bear, J.E., Goldstein, B., *Hahn, K. and *Kuhlman, B. Control of protein activity and cell fate specification via light-mediated nuclear translocation. PLoS One, 10(6): e0128443, 2015. PMC4471001. Online article | Free PMC article
- Hayashi-Takagi, A., Yagishita, S., Nakamura, M., Shirai, F., Wu, Y.I., Loshbaugh, A.L., Kuhlman, B., Hahn, K.M., and Kasai, H. Labelling and optical erasure of synaptic memory traces in the motor cortex. Nature, 525(7569): 333-8, 2015. PMC4634641. Online article |Free PMC article
- Gentry, L.R., Karginov, A.V., Hahn, K.M. and Der, C.J. Characterization of an engineered Src kinase to study Src signaling and biology. Methods Mol. Biol., 1360: 157-167, 2016. PMC4621786. Online article | Free PMC article
- MacNevin, C.J., Toutchkine, A., Marston, D.J., Hsu, C.W., Tsygankov, D., Li, L., Liu, B., Qi, T., Nguyen, D.V. and Hahn, K.M. Ratiometric imaging using a single dye enables simultaneous visualization of Rac1 and Cdc42 activation. J. Am. Chem. Soc., 138(8): 2571-2575, 2016. PMC4825053. Online article | Free PMC article
- Yumerefendi, H., Lerner, A.M., Zimmerman, S.P., Hahn, K.M., Bear, J.E., Strahl, B.D. and Kuhlman, B. Light-induced nuclear export reveals rapid dynamics of epigenetic modifications. Nature Chem. Biol., 12(6): 399-401, 2016. PMC488806. Online article | Free PMC article
- *Hodgson, L., *Spiering, D., Sabouri-Ghomi, M., DerMardirossian, C., *Danuser, G., and *Hahn, K.M. FRET binding antenna reports spatioteGDI-Cdc42 GTPase interactions. Nature Chem. Biol., 12(10): 802-9, 2016. PMC5030135. Online article | Free PMC article
- Wang, W., Vilela, M., Winkler, A., Tarnawski, T., Schlichting, I., Yumerefendi, H., Kuhlman, B., Liu, R., Danuser, G., and Hahn, K.M. LOVTRAP, An Optogenetic System for Photo-induced Protein Dissociation. Nature Methods, 13(9): 755-8, 2016. PMC5137947. Online article | Free PMC article
- Wang, H. and Hahn, K.M. LOVTRAP: a versatile method to control protein function with light. Curr. Protoc. Cell Biol., 73: 21.10.1 - 21.10.14, 2016. PMC5137945. Online article | Free PMC arqticle
- Dagliyan, O., Tarnawski, M., Chu, P-H., Shirvanyants, D., Schlichting, I., *Dokholyan, N.V., and *Hahn, K.M. Engineering extrinsic disorder to control protein activity in living cells. Science, 354(6318): 1441-1444, 2016. PMC5362825. View free in Science online: Full text & PDF | Free PMC article
- Dagliyan, O., Karginov, A.V., Yagishita, S., Gale, M.E., Wang, H., DerMardirossian, C., Wells, C.M., Dokholyan, N.V., Kasai, H. and Hahn, K.M. Engineering Pak1 Allosteric Switches. ACS Synth. Biol., 6 (7): 1257–1262, 2017. PMC5562282. Online article | Free PMC article
- Van Haren, J., Charafeddine, R.A., Ettinger, A., Wang, H., Hahn, K.M., and Wittman, T. Local Control of Intracellular Microtubule Dynamics by EB1 Photo-Dissociation. Nature Cell Biol., 20(3):252–261, 2018. PMC5826794. Online article | Free PMC article
- Yumerefendi, H., Wang, H., Dickinson, D.J., Lerner, A., Malkus, P., Goldstein, B., Hahn, K., and Kuhlman, B. Light-dependent cytoplasmic recruitment enhances the dynamic range of a nuclear import photoswitch.Chembiochem., 9(12):1319-1325, 2018. PMC6013380. Online article | Free PMC article
- Dagliyan, O., Krokhotin, A., Ozkan-Dagliyan, I., Deiters, A., Der, C.J., Hahn, K.M. and Dokholyan, N.V. Computational design of chemogenetic and optogenetic split proteins. Nature Communications, 9: 1-8, 2018. Doi: 10.1038/s41467-018-06531-4. online article | Free PMC article
- MacNevin, C.J., Watanabe, T., Weitzman, M., Gulyani, A., Fuehrer, S., Pinkin, N.K., Tian, X., Liu, F., Jin, J., and Hahn, K.M. Membrane-Permeant, Environment-Sensitive Dyes Generate Biosensors within Living Cells. J. Am. Chem. Soc. 141(18):7275-7282., 2019. Online article
- Dagliyan, O., Dokholyan, N.V., and Hahn, K.M. Engineering proteinsfor allosteric control by light or ligands. Nature Protocols, 2019 Jun;14(6): 1863-1883. doi: 10.1038/s41596-019-0165-3. [Epub before print] Online article