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Method for measuring tension in clawed frog embryos

Researchers succeed in measuring tension in living tissue without damaging cells

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Graduate School of Arts and Sciences / College of Arts and Sciences
2016/08/09

© 2016 Satoshi Yamashita, Tatsuo Michiue. Transferred from the paper (experiments carried out by SY).(Top) The overview of FRET analysis. When the tension is low on the cell membrane, the distance between two fluorescent proteins becomes smaller, resulting in high FRET. On the other hand, higher tension on the membrane gives low FRET. (Bottom) Measurement of focal tension. Tension was high in presumptive neural ectoderm, and low in presumptive epidermal ectoderm.

Measurement of tension in xenopus (African clawed frog) embryo with FRET probe
(Top) The overview of FRET analysis. When the tension is low on the cell membrane, the distance between two fluorescent proteins becomes smaller, resulting in high FRET. On the other hand, higher tension on the membrane gives low FRET. (Bottom) Measurement of focal tension. Tension was high in presumptive neural ectoderm, and low in presumptive epidermal ectoderm.
© 2016 Satoshi Yamashita, Tatsuo Michiue. Transferred from the paper (experiments carried out by SY).

A University of Tokyo research group and their collaborators succeeded in non-invasive measurement of tension in living tissue by introducing into African clawed frog embryos a tension detection probe—comprising two fluorescent proteins connected by an elastic linker protein, and actin-binding protein actinin that captured tension over the entire ectoderm, the outer germ layer of the embryo.

In recent years, mechanobiology research demonstrating the importance of cellular tension in cell differentiation and morphogenesis, which gives organisms their shape, has gained traction as an emerging trend in biology. Various methods to measure cellular tension have been developed so far, but challenges remain, including not being able to capture the tensile forces in many cells simultaneously or measure the physical forces non-invasively without damaging the cells. Given this situation, measuring tension using fluorescence resonance energy transfer, or FRET, in which energy transferred from a donor fluorescent protein creates fluorescence in an acceptor fluorescent protein, has been drawing attention since it was first reported in 2011. However, few studies using FRET to measure the tension of cells in the whole embryo have been reported to date.

In this study, the joint research group of Professor Tatsuo Michiue, researcher Satoshi Yamashita, graduate student Nanako Ishinabe, and Associate Professor Takashi Tsuboi, Department of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, constructed a novel tension probe consisting of two fluorescent proteins, GFP and mCherry, spider silk protein as an elastic linker between them, and actin-binding protein actinin. The group first tested the tension probe on cultured cells, which worked as expected. The researchers then injected a trace amount of the tension probe into the African clawed frog embryo and found that the tensile forces varied according to the type of ectoderm being measured: For instance, stronger forces were detected in neural ectoderm, which develop into nerve tissue, than epidermal ectoderm, which become skin.

“This study measuring the tensile forces of cells in whole African clawed frog embryos without invasion and fixation is one of the only successful cases in the world, the only other example being that confirmed in a nematode,” says Michiue. He continues, “Changing the combination of both fluorescent proteins and an elastic linker used in the current research was the key in giving us the outcome we got. Even though clawed frog embryos are not necessarily suitable for fluorescent imaging, that we succeeded in FRET measurement shows that the probe may be applicable for measuring tension in many other types of embryos, tissues, and organs. I expect many researchers will use this probe in the future.”

The research was conducted jointly with Associate Professor Tetsuya Kitaguchi of Waseda University.

Paper

Satoshi Yamashita, Takashi Tsuboi, Nanako Ishinabe, Tetsuya Kitaguchi, Tatsuo Michiue, "Wide and high resolution tension measurement using FRET in embryo", Scientific Reports Online Edition: 2016/06/23 (Japan time), doi: 10.1038/srep28535.
Article link (Publication)

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Graduate School of Arts and Sciences

Department of Life Sciences, Graduate School of Arts and Sciences

Michiue Laboratory, Department of Life Sciences, Graduate School of Arts and Sciences (Japanese)

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