Wild-type huntingtin protein (25Q) interacts with Huntingtin aggregate (94Q) in a living cell.
Building tools to watch individual molecules at work in living cells
Many questions about gene regulation come down to the behavior of individual molecules: How does a single transcription factor find its target among millions of decoy sites? How fast does it bind, and how long does it stay? What happens to molecular transport in neurons across millimeter-scale axons?
Standard fluorescence microscopy cannot resolve these dynamics. We develop single-molecule tracking, labeling, and imaging methods that can.
Core technologies developed in our lab:
Lattice light-sheet single-molecule tracking. By combining lattice light-sheet microscopy with single-molecule detection, we image transcription factor dynamics in 3D across the entire nucleus with minimal phototoxicity, enabling residence-time measurements that distinguish stable binding from transient interactions (Chen et al., Cell 2014; Liu et al., eLife 2014).
Stochastic protein labeling (SPL). We devised a method for sparse, stochastic labeling of endogenous proteins by engineering highly-tunable stop-codon read-though, enabling long-term single-molecule tracking over hours in living zebrafish and cultured neurons without overexpression artifacts (Liu et al., PNAS 2018).
Biological discoveries enabled by these tools:
Applying these methods, we uncovered the principles of enhancer-cluster assembly in embryonic stem cells (Chen et al., Cell 2014), showed that huntingtin aggregates divert transcription factor search and disrupt gene regulation (Li et al., eLife 2016), identified enhanced anterograde trafficking of synaptic vesicle precursors at the axon initial segment (Liu et al., PNAS 2018), discovered neuron-to-astrocyte lipid transfer as a neuroprotective mechanism (Ioannou et al., Cell 2019), and resolved compartment-specific sorting of voltage-gated sodium channels Nav1.2 and Nav1.6 in neurons (Liu et al., J. Neurosci. 2022).
Broad adoption and collaborative applications. Our single-molecule tools have been adopted by collaborators to study CRISPR/Cas9 genome interrogation dynamics (Knight et al., Science 2015), transcription factor hubs in B-cell activation and cancer (Kieffer-Kwon et al., Mol. Cell 2017; Chong et al., Science 2018), MeCP2 chromatin dynamics and Rett syndrome mutations (Piccolo et al., eLife 2019), extrachromosomal DNA hubs driving oncogene expression (Hung et al., Nature 2021), and CRISPR/Cas12-based multiplex genomic locus imaging (Yang et al., Nat. Methods 2024).
Related Publications
1. Chen, J., Zhang, Z., Li, L., Chen, B., Revyakin, A., Hajj, B., Legant, W., Dahan, M., Lionnet, T., Betzig, E., Tjian, R., and Liu, Z.@ (2014). Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell, 156(6):1274-1285.
2. Li, L., Liu, H., Dong, P., Li, D., Legant, W.R., Grimm, J.B., Lavis, L.D., Betzig, E., Tjian, R. @, Liu, Z.@ (2016). Real-time imaging of Huntingtin aggregates diverting target search and gene transcription. eLife, 5:e17056
3. Liu, H., Dong, P., Ioannou, M.S., Shea, J., Pasolli, A., Grimm, J., Rivlin, P., Lavis, L.D., Koyama, M., Liu, Z.@ (2018). Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling. Proc Natl Acad Sci U S A, 115(2):343-348.
4. Ioannou, M.S.@, Jackson, J., Sheu, S.H., Chang, C.L., Weigel, A.V., Liu, H., Pasolli, H.A., Xu, S., Pang, S., Hess, H.F., Lippincott-Schwartz, J.@, Liu, Z.@ (2019). Neuron-astrocyte metabolic coupling protests against activity-induced fatty acid toxicity. Cell, 177(6):1522-1535.
5. Liu, H., Wang, P.G., Pitt, G., Liu, Z.@ (2022) Direct observation of compartment-specific localization and dynamics of voltage-gated sodium channels. Journal of Neuroscience, 42(28):5482-5498. (Cover story)
Collaborations:
1. Chen, B., Legant, R.W., Wang, K., Shao, L., Milkie, D.E., Davidson, M.W., Janetopoulos, C., Wu, X.S., Hammer, J.A. 3rd, Liu, Z., English, B.P., Mimori-Kiyosue, Y., Romero, D.P., Ritter, A.T., Lippincott-Schwartz, J., Fritz-Laylin, L., Mullins, R.D., Mitchell, D.M., Bembenek, J.N., Reymann, A.C., Böhme, R., Grill, S.W., Wang, J.T., Seydoux, G., Tulu, U.S., Kiehart, D.P., Betzig, E. (2014). Lattice light sheet microscopy: imaging molecules, cells, and embryos at high spatiotemporal resolution. Science, 346(6208):1257998.
2. Knight, SC., Xie, L., Deng, W., Guglielmi, B., Bosanac, L., Witkowsky, LB., Zhang, ET., El-Beheiry, M., Dahan, M., Liu, Z.@, Doudna, JA.@, and Tjian, R.@ (2015). Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science, 350(6262):823-6.
3. Grimm, J.B., English, B.P., Choi, H., Muthusamy, A.K., Mehl, B.P., Dong, P., Brown, T.A., Lippincott-Schwartz, J., Liu, Z., Lionnet, T., Lavis, L.D. (2016). Bright photoactivatable fluorophores for single-molecule imaging. Nature Methods, 13(12):985-988
4. Kieffer-Kwon, K.R., Nimura, K., Rao, S.S.P., Xu, J., Jung, S., Pekowska, A., Dose, M., Stevens, E., Mathe, E., Dong, P., Huang, S.C., Ricci, M.A., Baranello, L., Zheng, Y., Tomassoni Ardori, F., Resch, W., Stavreva, D., Nelson, S., McAndrew, M., Casellas, A., Finn, E., Gregory, C., St Hilaire, B.G., Johnson, S.M., Dubois, W., Cosma, M.P., Batchelor, E., Levens, D., Phair, R.D., Misteli, T., Tessarollo, L., Hager, G., Lakadamyali, M., Liu, Z., Floer, M., Shroff, H., Aiden, E.L., Casellas, R. (2017). Myc regulates chromatin decompaction and nuclear architecture during B cell activation. Molecular Cell, 67(4):566-78.
5. Xie, L., Torigoe, S., H., Xiao, J., Mai, D.H., Li, L., Davis, F.P., Maire-Nelly, H., Grimm, J.B., Lavis, L.D., Darzacq, X., Cattoglio, C., Liu, Z.@ and Tjian, R.@ (2017). A dynamic interplay of Klf4 enhancer elments drives naïve-pluripotency. Genes & Development, 31(17):1795-1808.
6. Chong, S., Dugast-Darzacq, C., Liu, Z., Dong, P., Dailey, G.M., Cattoglio, C., Heckert, A., Sambashiva, B., Lavis, L.D., Darzacq, X., Tjian, R. (2018). Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science, 361(6400).
7. Abdelfattah, A.S., Kawashima, T., Singh, A., Novak, O., Liu, H., Shuai, Y., Huang, Y.C., Campagnola, L., Seeman, S.C., Yu, J., Zheng, J., Grimm, J.B., Patel, R., Friedrich, J., Mensh, B.D., Paninski, L., Macklin, J.J., Murphy, G.J., Podgorski, K., Lin, B.J., Chen, T.W., Turner, G.C., Liu, Z., Koyama, M., Svoboda, K., Ahrens, M.B., Lavis, L.D., Schreiter, E.R. (2019). Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Science, 365(6454):699-704.
8. Piccolo, F.M., Liu, Z., Deng, D., Hsu, C., Stoyanova, E.I., Rao, A., Tjian, R., Neintz, N. (2019). MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions. eLife, 8:e51449.
9. Grimm, J.B., Tkachuk, A.N., Xie, L., Choi, H., Mohar, B., Falco, N., Schaefer, K., Patel, R., Zheng, Q., Liu, Z., Lippincott-Schwartz, J., Brown, T.A., Lavis, L.D. (2020). A general method to optimize and functionalize red-shifted rhodamine dyes. Nature Methods, 17(8):815-821
10. Lerner, J., Gomez-Garcia, P.A., McCharhy, R.L., Liu, Z., Lakadamyali, M., Zaret, K.S. (2020). Two-parameter mobility assessments discriminate diverse regulatory factor behaviors in chromatin. Molecular Cell, 79(4):677-688.
11. Hung, K.L.*, Yost, K.E.*, Xie, L.*, Shi, Q., Helmsauer, K., Luebeck, J., Schöpflin, R., Lange, J.T., Chamorro González, R., Weiser, N.E., Chen, C., Valieva, M.E., Wong, I.T., Wu, S., Dehkordi, S.R., Duffy, C.V., Kraft, K., Tang, J., Belk, J.A., Rose, J.C., Corces, M.R., Granja, J.M., Li, R., Rajkumar, U., Friedlein, J., Bagchi, A., Satpathy, A.T., Tjian, R., Mundlos, S., Bafna, V., Henssen, A.G., Mischel, P.S., Liu, Z., Chang, H.Y. (2021) ecDNA hubs drive cooperative intermolecular oncogene expression. Nature, 600(7890):731-736.
12. Lange, J.T., Rose, J.C., Chen, C.Y., Pichugin, Y., Xie, L., Tang, J., Hung, K.L., Yost, K.E., Shi, Q., Erb, M.L., Rajkumar, U., Wu, S., Taschner-Mandl, S., Bernkopf, M., Swanton, C., Liu, Z., Huang, W., Chang, H.Y., Bafna, V., Henssen, A.G., Werner, B., Mischel, P.S. (2022) The evolutionary dynamics of extrachromosomal DNA in human cancers. Nature Genetics, 54(10):1527-1533.
13. Hao, S., Ye J.L., Goldfajn, N.B., Flores, E., Liang, J., Fuehrer, H., Demmerte, J., Lippincott-Schwartz, J., Liu, Z., Sukenik, S., Cai, D. (2024) YAP condensates are highly organized hubs. iScience, 27(6):109927.
14. Wong, V.C., Houlihan, P.R., Liu, H., Walpita, D., DeSantis, M.C., Liu, Z., O'Shea, E.K. (2024) Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking. Elife, 15;13:e80622.
15. Yang, L., Min, Y., Liu, Y., Gao, B., Liu, X., Huang, Y., Wang, H., Yang, L., Liu, Z., Chen, L. CRISPR-array-mediated imaging of non-repetitive and multiplex genomic loci in living cells. (2024) Nature Methods, 21(9):1646-1657.