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Interviews with Imaging Experts - Takanari Inoue, Ph.D.

Johns Hopkins University School of Medicine logo

Please tell us about your research.

We are interested in dissecting the spatiotemporal characteristics of intricate signaling network in cells. We are currently focusing on cell migration, a dynamic cellular process that occurs on a timescale of minutes.

To investigate the signaling network that drives cell migration, we have recently developed molecular tools for the inducible, quick-onset, and specific perturbation of various types of signaling molecules in the subcellular compartments of single, living cells. (See below for figures, images, movies.) Our goal is to enhance our understanding of the molecular mechanisms that are responsible for cell migration by using these molecular tools together with the fluorescence imaging technique.

Application

Rapidly-inducible, specifically-targeted recruitment of cytoplasmic proteins to various organelles

a) A diagram illustrating the inducible recruitment of signaling molecules (green dots). c: cytoplasm, o: organelle
b) Confocal fluorescence images of HeLa cells showing translocation of cytoplasmic proteins to specific organelles upon addition of a chemical dimerizer (iRap). Scale bar: 20um. The treatment leads to translocation of cytoplasmic proteins to specific organelles such as Golgi, endoplasmic reticulum, lysosome, mitochondria and plasma membrane.

golgi

Movie 1 - golgi
play

endoplasmic reticulum

Movie 2 - endoplasmic reticulum
play

lysosome

Movie 3 - lysosome
play

mitochondria

Movie 4 - mitochondria
play

What role does live imaging with the CSU play in your research?

The time-lapse, live-cell confocal fluorescence imaging capabilities of the CSU have been indispensable in quantifying a variety of molecular players with high spatio-temporal resolution at various locations inside living cells. Every day our CSU generates about 5 GB of video data.

CSU System

When did you start using the CSU?

It was 2003, when I was starting my postdoctoral research in the Tobias Meyer Lab at Stanford University. The lab had two CSU10 units. That was the first time I came across the CSU.

How would you sum up your feelings about the CSU?

After using the CSU almost every day for the last six years, I feel as if it has become an extension of my body.

What are the good points/bad points about the CSU?

Pros: Unrivaled capability of taking fast time-lapse confocal images of living cells. It is unbelievably reliable and robust. We have never experienced a malfunction with it. And most importantly, we have been getting great technical support from Yokogawa.

Cons: Cannot think of any bad points. It might be aesthetically appealing to see some variation in its colors, like with the Sony VAIO laptops (half joking). We sometimes wish it were possible to do triple-color confocal imaging with relative ease. (Perhaps a factor there is the limited availability of dichroic mirrors?)

What are your future plans?

I hope to be able to contribute to science as well as the scientific community by providing knowledge and tools of fine quality. As soon as funds become available, we would like to get another confocal microscope with the latest version of the CSU.

Tell us something about yourself.

My hobbies are playing with my son, traveling to the far corners of this country and the world, and procrastinating on paperwork.


Famous for his discoveries in signal transduction including the regulatory mechanism governing potassium ion channels and the membrane targeting mechanisms of small GTPase, Dr. Inoue opened his own lab at Johns Hopkins University in April 2008. It is a great pleasure to know that the CSU10 system has been a key technology in his research and that part of his enormous collection of live cell imaging data has appeared in the latest edition of Nature Methods.


Takanari Inoue, Ph.D.
http://www.hopkinsmedicine.org/pharmacology_molecular_sciences/faculty/bios/inoue.html
Department of Cell Biology (Primary)
Johns Hopkins University School of Medicine
Center for Cell Dynamics, Institute of Basic Biomedical Sciences
Department of Pharmacology (Secondary)
Johns Hopkins University School of Medicine


Interview date: December 2009

The Johns Hopkins University School of Medicine
Takanari Inoue 12/03/2009

Biography

Current Appointments
Department of Cell Biology (Primary)
Johns Hopkins University School of Medicine
Center for Cell Dynamics, Institute of Basic Biomedical Sciences

Department of Pharmacology (Secondary) Johns Hopkins University School of Medicine

Personal Data
855 N. Wolfe St. 453 Rangos, Baltimore, MD, 21205 tel.#: (443)287-7668; fax# (410)614-8375; e-mail jctinoue@jhmi.edu

Education and Training

Undergraduate 1994-98 B.S. University of Tokyo Pharmaceutical Sciences
Doctoral/graduate 1998-03 PhD. University of Tokyo Pharmaceutical Sciences
Postdoctoral 2003-08 Stanford University Chemical and Systems Biology

Professional Experience

April 2008-present Assistant Professor Department of Cell Biology, Johns Hopkins University School of Medicine

Publications

  1. Inoue T., Kikuchi K., Hirose K., Iino M., and Nagano T. (1999) Bioorg. Med. Chem. Lett. 9, 1697-1702.  “Synthesis and evaluation of 1-position-modified inositol 1,4,5-trisphosphate analogs”
  2. Inoue T., Kikuchi K., Hirose K., Iino M., and Nagano T. (2001) Chem. Biol. 8, 9-15.  “Small molecule-based laser inactivation of inositol 1,4,5-trisphosphate receptor”
  3. Nakanishi W., Kikuchi K., Inoue T., Hirose K., Iino M., and Nagano T. (2002) Bioorg. Med. Chem. Lett. 12, 911-913.  “Hydrophobic modifications at 1-phosphate of inositol 1,4,5-trisphosphate analogues enhance receptor binding”
  4. Inoue T., Kikuchi K., Hirose K., Iino M., and Nagano T. (2003) Chem. Biol.*10, 503-509. “Spatiotemporal Laser Inactivation of Inositol 1,4,5-Trisphosphate Receptors Using Synthetic Small-molecule Probes” (*Cover Article)
  5. Yogo T., Kikuchi K., Inoue T., Hirose K., Iino M. and Nagano T. (2004) Chem. Biol. 11, 1053-1058.  “Modification of intracellular Ca2+ dynamics by laser inactivation of inositol 1,4,5-trisphosphate receptor using membrane-permeant probes.”
  6. Inoue T., Heo W.D., Grimley J.S., Wandless T.J., and Meyer T. (2005) Nature Methods 2, 415-418.  “Inducible translocation strategies to rapidly activate and inhibit small GTPase signaling pathways”
  7. Heo W.D., Inoue T., Park W.S., Kim M.L., Park B.O., Wandless T.J. and Meyer T. Science 314, 1458-1461 (2006) “PI(3,4,5)P3 and PI(4,5)P2 lipids target Ras, Rho, Arf and Rab GTPases to the plasma membrane”
  8. Suh B.C. *, Inoue T. *, Meyer T. and Hille B. Science 314, 1454-1457 (2006) “Rapid chemically-induced changes of PtdIns(4,5)P2 gate KCNQ ion channels” (*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)) and “Review” (invited by Nature Chem. Biol.).
  9. Liou J., Fivaz M.,Inoue T. and Meyer T. Proc. Natl. Acad. Sci 104, 9301-9306 (2007) “Live-cell imaging reveals sequential oligomerization and local PM targeting of STIM1 following Ca2+store depletion”
    Note: “Research Roundup” (J. Cell Biol. 177 (6), (2007)).
  10. Aoki. K, Nakamura T., Inoue T., Meyer T., and Matsuda M.  J. Cell Biol. 177, 817-827 (2007) “An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor–stimulated PC12 cells”
  11. Yogo T., Urano Y., Mizushima A., Sunahara H., Inoue T., Hirose K., Iino M., Kikuchi K. and Nagano T., Proc. Natl. Acad. Sci., 105, 28-32 (2008)  "Selective photoinactivation of protein function through environment-sensitive switching of singlet oxygen generation by photosensitizer"
  12. Fivaz M., Bandara S., Inoue T. and Meyer T., Current Biology 18, 44-50 (2008), "Robust neuronal symmetry breaking by Ras-triggered local positive feedback"
  13. Abe N., Klein L., Inoue T., Galvez T. and Meyer T. J. Cell Sci. 121, 1488 (2008) “Dissecting the role of PI(4,5)P2 in transferrin receptor endocytosis and recycling”
  14. Inoue T.* and Meyer T.* PLoS ONE 3(8), e3068 (2008)  “Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration” (*Corresponding author)
  15. Rahdar M., Inoue T., Meyer T., Zhang J., Vazquez F., Devreotes PN. Proc. Natl. Acad. Sci.U S A. (2009) “A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN” 106(2):480-5.

Related Products & Solutions

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Your microscope can be easily upgraded to confocal microscope with Confocal Scanner Unit. The multi-beam scanning method offers not only high-speed imaging but also significantly reduced photo-toxicity and photo bleaching because of very reduced laser power of each beamlet. The CSU series have been already delivered more than 2,000 units and supported leading-edge research around the world.

 

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