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Organelle-specific, rapid induction of molecular activities and membrane tethering

Abstract

Using new chemically inducible dimerization probes, we generated a system to rapidly target proteins to individual intracellular organelles. Using this system, we activated Ras GTPase at distinct intracellular locations and induced tethering of membranes from two organelles, endoplasmic reticulum and mitochondria. Innovative techniques to rapidly perturb molecular activities and organelle-organelle communications at precise locations and timing will provide powerful strategies to dissect spatiotemporally complex biological processes.

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Figure 1: Chemically inducible recruitment of cytoplasmic proteins to various organelles.
Figure 2: Implementation of organelle recruiting system to probe the role of signaling molecules and interorganelle interactions.

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Acknowledgements

We thank B. Wattenberg (University of Louisville) for Cb5/MoA plasmids; N. Ishihara (Toyko Medical and Dental University) for Tom20 plasmid; C. Machamer (Johns Hopkins University) for Giantin plasmid; W.D. Heo (Korea Advanced Institute of Science and Technology) for RasGRF plasmid; F. Tsuruta (University of Tsukuba) for LAMP plasmid; M. Fivaz (Duke-National University of Singapore Graduate Medical School Singapore) YFP-RBD plasmid; and T. Wandless (Stanford University) for providing rapamycin analogs (iRap, synthetic ligand of FKBP, FK506M); F. Fernandez, M. Fivaz, M. Meffert, J. Zhang, V. Sample, D. Montell, M. Caterina, P. Devreotes, A. Ewald and D. Robinson for critical review of the manuscript; R. Pagano for helpful comments on PS assays; and Z. Wei, W. Wong, K. Venkatachalam and J. Cordon for a technical assistance with western blot analyses. Supported in part by US National Institutes of Health (MH084691 to T.I., NCRR1S10RR023454-01 to J.M.M.). T.K. and T.U. are recipients of a fellowship from Japanese Society for the Promotion of Science.

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Authors

Contributions

T.K., I.K., T.U., L.C.V. and T.I. designed and conducted molecular/cellular biology experiments with following data analysis. J.M.M. took and analyzed TEM images. T.I. conceived and supervised the project, and wrote the paper.

Corresponding author

Correspondence to Takanari Inoue.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Table 1 (PDF 2476 kb)

Supplementary Video 1

Time-lapse confocal fluorescence images of HeLa cells showing translocation of cytoplasmic proteins to the Golgi upon iRap addition. (AVI 9732 kb)

Supplementary Video 2

Time-lapse confocal fluorescence images of HeLa cells showing translocation of cytoplasmic proteins to the ER upon iRap addition. (AVI 5997 kb)

Supplementary Video 3

Time-lapse confocal fluorescence images of HeLa cells showing translocation of cytoplasmic proteins to the lysosomes upon iRap addition. (AVI 8229 kb)

Supplementary Video 4

Time-lapse confocal fluorescence images of HeLa cells showing translocation of cytoplasmic proteins to the mitochondria upon iRap addition. (AVI 25185 kb)

Supplementary Video 5

Time-lapse confocal fluorescence images of HeLa cells showing ER-mitochondria tethering upon iRap addition. This movie corresponds to ER. (AVI 5480 kb)

Supplementary Video 6

Time-lapse confocal fluorescence images of HeLa cells showing ER-mitochondria tethering upon iRap addition. This movie corresponds to mitochondria. (AVI 5480 kb)

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Komatsu, T., Kukelyansky, I., McCaffery, J. et al. Organelle-specific, rapid induction of molecular activities and membrane tethering. Nat Methods 7, 206–208 (2010). https://doi.org/10.1038/nmeth.1428

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