One of the major challenges in cell biology is to understand how biochemical reactions at the molecular scale self-organize to generate physiological forms and functions at the cellular and tissue scale. Although genetic and biochemical approaches have been invaluable in identifying key regulatory molecules, these classical approaches rarely have the temporal and spatial resolution required to reveal the molecular mechanisms underlying such self-organization in vivo. Our research aims to address this knowledge gap by developing innovative molecular actuators that can be used to perturb and assay molecular actions in live cells with high temporal and spatial precision. In the past decade, we have applied these tools to studies of innate immune functions such as chemotaxis, phagocytosis and degranulation, as well as subcellular organizations including primary cilia, microtubules and stress granules. Please see below for our recent achievements under this theme:
- Non-catalytic allostery in α-TAT1 by a phospho-switch drives dynamic microtubule acetylation
Journal of Cell Biology, 2022:221:11:e1-20
“Location, Location, Location”
We report an unconventional regulatory mechanism of α-TAT1, a posttranslational modification enzyme that acetylates microtubules. More specifically, we identified a unique signal motif in the intrinsically disordered region of α-TAT1. Multidisciplinary characterization subsequently revealed that this previously uncharacterized motif functions as an intricate molecular switch that integrates upstream kinase signaling to actuate the intracellular localization of α-TAT1, hence achieving dynamic microtubule acetylation. Activity of cellular enzymes is often regulated allosterically, where the interaction between a given enzyme and its substrate is initiated by a third molecule binding to the enzyme to induce conformational change. In contrast, it appears to be the spatial regulation that mainly controls the functional output of α-TAT1. While there are enzymes that translocate between nucleus and cytosol, α-TAT1 is the only enzyme to our knowledge that uses the nucleus as a sequestration hideout.
Technological advance:
In parallel to characterizing α-TAT1 regulatory mechanisms, we developed a co-recruitment assay based on chemically inducible dimerization tools to assess protein-protein interactions. This assay is like performing Co-IP assays in living cells, and is quantitative, robust and reliable. Most existing protein-protein interaction assays require purification of proteins and/or lysing cells where procedural concerns need to be taken into consideration for data interpretation. Our co-recruitment assays circumvent this problem. And thanks to the modular design principles, the assay is generalizable to virtually any soluble proteins of your choice. Therefore, we expect its wide application to diverse systems.
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2. A molecular trap inside microtubules probes luminal access by soluble proteins
Nature Chemical Biology, 2021:17:888-895
“Illuminating the path to cellular bower”
We present the first method to chemically and inducibly trap proteins inside microtubules that enables direct assessment of biomolecular accessibility in living cells. This work overcame previous technical limitations and provides a novel mechanistic insight of how protein regulators and effectors gain access to the site of their action inside the cellular tunnel.
Microtubules are the fundamental basis for many cellular functions including cell division, cell migration, neuronal plasticity and ciliogenesis. The multi-tasking schemes of microtubule functions is believed to be encoded in a spatio-temporal pattern as well as a combinatorial set of their posttranslational modification status, a concept put forth as the tubulin code. While these modifications mostly occur on the outer surface, microtubules can also be regulated via the inner part, such as via acetylation. As a cellular tunnel, the microtubules uniquely present a lumen that is physically insulated by the cylindrical wall of densely-packed tubulins, with limited connections to the cytoplasm at their open ends. It is natural to wonder how regulatory and effector proteins of acetylated tubulins reach this secluded site.
The bottleneck of microtubule studies thus far is the limited number of experimental systems. To overcome this challenge, we rationally established a chemically-inducible technique for conditional trapping of a cytosolic protein in the microtubule lumen, with which we subsequently discovered that soluble proteins can enter the lumen via diffusion through openings at the MT ends and sides. Additionally, proteins forming a complex with tubulins can be incorporated to the lumen through the plus ends. We believe that our molecular tool is unique and powerful in probing biomolecular accessibility, with a strong potential in decoding tubulin codes in the near future, thus appealing ubiquitously to a large audience in cell biology.
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3. Rational design and implementation of a chemically inducible hetero-trimerization system
Nature Methods, 2020:17:928–936
“The Three Musketeers”
We report the first chemically inducible “trimerization” system (CIT), where a small molecule rapidly brings three unique proteins together in living cells, offering unprecedented intracellular operation in addressing new biological questions, harnessing cell functions and behaviors, as well as performing synthetic operations.
Since the development of a chemically inducible dimerization system (CID) in the late 90’s, a number of CID tools were generated and used to advance life sciences in answering biological questions. In addition, the CID-mediated operation became a foundation for designing optogenetic dimerization tools in recent years. Despite the prevalent trimerization events in nature (death receptors inducing apoptosis, G proteins initiating signal transduction, etc.), and despite the self-evident utility of CIT, we do not have CIT tools yet in our hands; this is simply because developing CIT is extremely challenging.
In this study, our multidisciplinary team constructed a trimerization system by co-opting unique properties of the well-established FKBP/FRB/rapamycin dimerization system, thus rationally avoiding the challenging task of engineering multiple cooperative protein-small molecule interactions de novo. Through computational analysis, we identified split sites for the FKBP or FRB proteins such that the two split protein halves and the cognate protein partner form the three stable protein components of the trimerization system. Live-cell imaging experiments and structural validation with protein crystallography showed that the split-based CIT system undergoes rapid trimerization upon rapamycin addition. Increased capability in spatiotemporal control conferred by CIT allowed us to demonstrate its potential, for example, in the emerging field of organelle-organelle membrane contact sites. Since CIT molecular tools are modular in design, these first CIT tools pave the way for future generalizable CIT-based approaches that alter function at organelle junctions and offer higher spatiotemporal control in other biological contexts. The work was highlighted in Nature Chemical Biology, “Three’s company”.![]()
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Publications
Kim AK, Wu HD, Inoue T “Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch” Scientific Reports 2021;11:16421
Nakamura H, Rho E, Deng D, Razavi S, Matsubayashi HT, Inoue T “ActuAtor, a molecular tool for generating force in living cells” bioRxiv03.30.016360
Deb Roy A, Gross EG, Pillai GS, Seetharaman S, Etienne-Manneville S, Inoue T “Non-catalytic allostery in α-TAT1 by a phospho-switch drives dynamic microtubule acetylation” Journal of Cell Biology 2022;221:11:e1-20
Matsubayashi HT, Mountain J, Yao T, Peterson AF, Deb Roy A, Inoue T “Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP-2-mediated endocytosis” bioRxiv, 2022.12.31.522383
Imoto, Y., Raychaudhuri, S.*, Ma, Y.*, Fenske, P., Sandoval, E., Itoh, K., Blumrich, E., Matsubayashi, H.T., Mamer, L., Zarebidaki, F., Söhl-Kielczynski, B., Trimbuch, T., Nayak, Shraddha, Iwasa, J., Liu, J., Wu, B., Ha, T., Inoue, T., Jorgensen, E.M., Cousin, M., Rosenmund, C., Watanabe, S., (2022), Dynamin is primed at endocytic sites for ultrafast endocytosis, Neuron, S0896-6273(22)00548-7.
Nihongaki Y, Matsubayashi HT, Inoue T. “A molecular trap inside microtubules probes luminal access by soluble proteins” Nature Chemical Biology 2021;17:888-895
Wolfe K., Kamata R., Coutinho K., Inoue T. and Sasaki A.T. “Metabolic Compartmentalization at the Leading Edge of Metastatic Cancer Cells” Frontiers in Oncology (in press)
Kim AK, Wu HD, Inoue T, “Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter” Scientific Reports 2020:10:9365
Wu HD, Kikuchi M, Dagliyan O, Aragaki AK, Nakamura H, Dokholyan NV, Umehara T, Inoue T, “Rational design and implementation of a chemically inducible hetero-trimerization system” Nature Methods 17, 928–936 (2020). Note: Research Highlight in Nature Chemical Biology, “Three’s company”
Kim AK, Wu HD, Inoue T “Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter” Scientific Reports 2020;10:9365
Wu HD, Kikuchi M, Dagliyan O, Aragaki AK, Nakamura H, Dokholyan NV, Umehara T, Inoue T “Rational design and implementation of a chemically inducible hetero-trimerization system” Nature Methods 2020;17:928–936
Georgess D, Padmanaban V, Sirka OK, Coutinho K, Choi A, Frid G, Neumann NM, Inoue T, Ewald AJ. Twist1-induced epithelial dissemination requires Prkd1signaling. Cancer Res, 2020 Jan 15, 80 (2), 204-218.
Nakamura H, DeRose R, Inoue T. “Harnessing biomolecular condensates in living cells” Journal of Biochemistry (in press, 2019)
Miao Y, Battacharya S, Banerjee T, Abudaker-Sharif B, Long Y, Inoue T, Iglesias P and Devreotes P. (2019). Wave patterns organize cellular protrusions and control cortical dynamics. Molecular Systems Biology 15(3). DOI 10.15252/msb.20188585| Published online 11.03.2019.
Nakamura H, Lee AA, Afshar AS, Watanabe S, Rho E, Razavi S, Suarez A, Lin YC, Tanigawa M, Huang B, DeRose R, Bobb D, Hong W, Gabelli SB, Goutsias J, Inoue T “Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions” Nature Materials 2018;17:79-89
Cuei-Ling Wang, Yu-Chen Chang, Ya-Chu Chang, Shi-Rong Hong, Chun-Yu Lin, Ning Hsu, Hsiao-Chi Cheng, Yueh-Chen Chiang, Wei-En Huang, Nathan C. Shaner, Rajat Rohatgi, Inoue T, Yu-Chun Lin. (2018).“Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling.”Nature Communications 2018:9:1732:1-13
Naoto Takada, Tomoki Naito, Inoue T, Kazuhisa Nakayama, Hiroyuki Takatsu, Hye-Won Shin. “Enhanced phospholipid flipping by P4-ATPase promotes membrane deformation.” 2018. EMBO Reports (in press).
Nakamura H, Lee AA, Afshar AS, Lin YC, Tanigawa M, Suarez A, Razavi S, DeRose R, Bobb D, Hong W, Gabelli SB, Goutsias J, Inoue T. 2017. Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions. Nature Materials (In press, doi:10.1038/nmat5006)
Phua SC, Chiba S, Suzuki M, Su E, Roberson EC, Pusapati GV, Schurmans S, Setou M, Rohatgi R, Reiter JF, Ikegami K, Inoue T “Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.” Cell 2017;168:264-279
Miao Y, Bhattacharya S, Edwards M, Cai H, Inoue T, Iglesias P, Devreotes PN. 2017. Altering the threshold of an excitable signal transduction network changes cell migratory modes. Nat Cell Biol 19:329-340.
Niu J., Johny M.B., Dick I.E., Inoue T. 2016. Following Optogenetic Dimerizers and Quantitative Prospects. Biophysical Journal 111:1132–1140
Phua SC, Chiba S, Suzuki M, Su E, Roberson EC, Pusapati GV, Setou M, Rohatgi R, Reiter JF, Ikegami K, Inoue T. 2017. Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision. Cell 2017;168:264-279
Gaus K., Inoue T. 2016. New Biological Frontiers Illuminated by Molecular Sensors and Actuators. Biophysical journal. 2016; 111(6):E01-2.
Meier EL, Razavi S, Inoue T, Goley ED. A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus. Mol Microbiol. 2016 Jul;101(2):265-80.
Gaus K., Inoue T. 2016. New Biological Frontiers Illuminated by Molecular Sensors and Actuators. Biophysical Journal. 2016;111(6):E01–E02
Coutinho K, Inoue T. 2016. Deconstructing and constructing innate immune functions using molecular sensors and actuators. Proc. SPIE 9871, Sensing and Analysis Technologies for Biomedical and Cognitive Applications 2016, 987102 (May 19, 2016); doi:10.1117/12.2225185
Antonczak A.K., Mullee L., Wang Y., Comartin D., Inoue T., Pelletier L. and Morrison C.G. 2016. Opposing effects of pericentrin and microcephalin on the pericentriolar material regulate CHK1 activation in the DNA damage response. Oncogene. 30:2003-10.
Kim AK, DeRose R, Ueno T, Lin B, Komatsu T, Nakamura H, Inoue T. Toward total synthesis of cell function: Reconstituting cell dynamics with synthetic biology. Sci Signal. 2016 Feb 9;9(414). Review.
Phua SC, Lin YC, Inoue T. An intelligent nano-antenna: Primary cilium harnesses TRP channels to decode polymodal stimuli. Cell Calcium. 2015 Oct;58(4):415-22. Review.
Miyamoto T, Rho E, Inoue T. Deconvoluting AMPK dynamics. Oncotarget. 2015 Oct 13;6(31):30431-2.
Garcia-Gonzalo FR, Phua SC, Roberson EC, Garcia G 3rd, Abedin M, Schurmans S, Inoue T, Reiter JF. Phosphoinositides regulate ciliary protein trafficking to modulate hedgedog signaling. Dev Cell. 2015 Aug 24;34(4):400-9.
Nguyen H-N, Yang J-M, Miyamoto T, Itoh K, Rho E, Zhang Q, Inoue T, Devreotes PN, Sesaki H, and Iijima M. 2015. Opening the conformation is a master switch for the dual localization and phosphatase activity of PTEN. Sci Rep Jul 28;5:12600. doi: 10.1038/srep12600. PMCID: PMC4517176.
Miyamoto T., Rho E, Sample V., Akano H., Magari M., Ueno T., Chen M., Tokumitsu H., Zhang J., and Inoue T.* “Compartmentalized AMPK Signaling Illuminated by Genetically Encoded Molecular Sensors and Actuators” (Accepted at Cell Reports)
Lin B., Yin T., Wu Y.I., Inoue T.* and Levchenko A.* “Interplay between chemotaxis and contact inhibition of locomotion determines exploratory cell migration” (Accepted at Nature Communications)
Onuma H, Komatsu T, Arita M, Hanaoka K, Ueno T, Terai T, Nagano T, Inoue T. 2014. Rapidly rendering cells phagocytic through a cell surface display technique and concurrent Rac activation. Sci Signal. Jul 15;7(334):rs4. doi: 10.1126/scisignal.2005123.
Suarez A., Ueno T., Huebner R., McCaffery J.M., and Inoue T.* “Bin/Amphiphysin/Rvs (BAR) family members bend membranes in cells” Scientific Reports 4, 4693 (2014)
Kobayashi T., Kim S., Lin Y.C., Inoue T., and Dynlacht B.D. “CP110-interacting proteins, Talpid3 and Cep290, play overlapping and distinct roles in cilia assembly.” Journal of Cell Biology 204, 215 (2014)
Su S., Phua S.C., DeRose R., Chiba S., Narita K., Kalugin P.N., Katada T., Kontani K., Takeda S. and Inoue T. “Genetically encoded calcium indicator illuminates calcium dynamics in cilia.” Nature Methods 10, 1105 (2013)
Thevathasan, J.V., Tan E., Hui Z., Lin Y.C., Li Y., Inoue T. and Fivaz M. “The small GTPase HRas shapes local PI3K signals through positive feedback and regulates persistent membrane extension in migrating fibroblasts.” Molecular Biology of the Cell 24, 2228 (2013)
Lin Y.C., Niewiadomski P., Lin B., Nakamura H., Phua S.C., Jiao J., Levchenko A., Inoue T., Rohatgi T., and Inoue T. “Chemically-inducible diffusion trap reveals molecular sieve-like barrier at primary cilia“ Nature Chemical Biology 9, 437 (2013)
Lin YC, Liu T-Y, Razavi S. and Inoue T. “Rapidly Reversible Manipulation of Molecular Activities Using Dual Chemical Dimerizers” Angewandte Chemie 52, 6450 (2013)
Thevathasan, J.V., Tan E., Hui Z., Lin Y.C., Li Y., Inoue T. and Fivaz M. “Local positive feedback from PI3K to Ras drives cell polarization and migration” Molecular Biology of the Cell (doi:10.1091/mbc.E12-12-0905)
Miyamoto T., DeRose R., Suarez A., Ueno T., Chen M., Sun T.-p., Wolfgang M.J., Mukherjee C., Meyers D. and Inoue T. “Generation of Intracellular Logic Gates with Two Orthogonal Chemically Inducible Systems” Nature Chemical Biology (Accepted 2012)
Umeda N., Ueno T., Pohlmeyer C., Nagano T. and Inoue T. “A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity” Journal of American Chemical Society 133(1), 12-14 (2011) Note: “Scientist use light to move molecules within living cells” Science News article in ScienceDaily
Ueno T., Falkenburger B.H., Pohlmeyer C., and Inoue T. “Triggering Actin Comets Versus Membrane Ruffles: Distinctive Effects of Phosphoinositides on Actin Reorganization” ScienceSignaling 4(203), ra87 (2011) (Cover Article)
Komatsu T., Kukelyansky I., McCaffery J.M., Ueno T., Varela L.C. and Inoue T. “Organelle-Specific, Rapid Induction of Molecular Activities and Membrane Tethering” Nature Methods 7, 206-208 (2010) Note: “Hopkins researchers put proteins right where they want them” Breaking News article in Genetic Engineering and Biotechnology News
Rahdar M., Inoue T., Meyer T., Zhang J., Vazquez F., and Devreotes P.N. “A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN” Proc. Natl. Acad. Sci. U S A. 106(2):480-5 (2009)
Inoue T. and Meyer T. “Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration” PLoS ONE 3(8), e3068 (2008)
Fivaz M., Bandara S., Inoue T. and Meyer T. "Robust neuronal symmetry breaking by Ras-triggered local positive feedback" Current Biology 18, 44-50 (2008)
Suh BC*, Inoue T*, Meyer T, and Hille B. “Rapid chemically-induced changes of PtdIns(4,5)P2 gate KCNQ ion channels” Science 314, 1454-1457 (2006) (*Contributed Equally) Note: “Perspectives” (Science 314, 1402-1403 (2006)), “Editor’s Choice” (Science STKE 364, tw410 (2006)), “Spotlight” (ACS Chem. Biol. 1, 608 (2006)), “Research Highlights” (Nature Methods 4, 7 (2007))
Heo WD, Inoue T, Park WS, Kim ML, Park BO, Wandless TJ, and Meyer T. “PI(3,4,5)P3 and PI(4,5)P2 lipids target Ras, Rho, Arf and Rab GTPases to the plasma membrane” Science 314, 1458-1461 (2006)
Inoue T, Heo WD, Grimley JS, Wandless TJ, and Meyer T. “Inducible translocation strategies to rapidly activate and inhibit small GTPase signaling pathways” Nature Methods 2, 415-418 (2005)
Inoue T, Kikuchi K, Hirose K, Iino M, and Nagano T. “Spatiotemporal Laser Inactivation of Inositol 1,4,5-Trisphosphate Receptors Using Synthetic Small-molecule Probes” Chem. Biol. 10, 503-509 (2003) Note: “Cover art”
Inoue T, Kikuchi K, Hirose K, Iino M, and Nagano T. “Small molecule-based laser inactivation of inositol 1,4,5-trisphosphate receptor” Chem. Biol. 8, 9-15 (2001)
