National Institute for Basic Biology

Cell Biology

DIVISION OF QUANTITATIVE BIOLOGY

Professor

AOKI, Kazuhiro

Assistant Professor:

KONDO, Yohei
GOTO, Yuhei
Technical Staff:
BINO, Takahiro
Postdoctoral Fellow:
NAKAMURA, Akinobu
JSPS Postdoctoral Fellow: SUGIYAMA, Hironori
Research Staff:
EBINE, Emi
TANII, Ryosuke
GOTO, Yoko
SOKENDAI Graduate Student: MUKAI, Masaya
YAMAMOTO, Kei
SAKAI, Keiichiro
TSURUOKA, Tatsuki
ITOH, Thoma
Visiting Scientist:
ITO, Reina
Admin Support Staff:
ONODA, Kaorigene mutation is widely known to result in various pathologies, such as malignant tumors.
The intracellular signal transduction system has been extensively studied over the past few decades through approaches
utilizing both biochemistry and cell biology approaches. As
a result, many proteins and regulations have been identified,
which has resulted in an increase in the pathway’s complexity. The complicated signaling network makes it difficult tounderstand how cells process information and quantitatively
make decisions.
To address these issues, we are currently focusing on thedevelopment of two types of research tools that enable us
to (1) visualize and (2) manipulate intracellular signaling
pathways (Figure 1).

Figure 1. Information processing by intracellular signaling devices andnetworks.

I. Visualization of cell signaling

Visual overview of this lab’s work, showing fluorescence images ofmammalian cells, fission yeast, and C.elegans

Living cells act as input-output (I/O) units, in which theirenvironment and/or internal states are recognized on thecell surface and processed within the cell, thus leading toan adaptive response (Figure 1). This cellular information processing is mainly implemented by an intracellularsignal transduction system, which is comprised of a series ofchemical reactions such as protein-protein interactions andprotein phosphorylation. Dysregulation of cell signaling by14

With the advent of fluorescent proteins, it has become
possible to visualize kinetic reactions at the single-cell level.
Förster (or fluorescence) resonance energy transfer (FRET)
is a non-radiative process in which the excitation energy of adonor fluorophore is transferred to a nearby acceptor fluorophore. FRET-based biosensors allow us to detect PKA, ERK,Akt, JNK, PKC, and S6K’s kinase activity in living cells at ahigh temporal and spatial resolution (Komatsu N, Mol. Biol.
Cell, 2011; Aoki K, Mol Cell, 2013; Aoki K, Dev Cell, 2017).
Although FRET biosensors enable us to monitor cell signaling events, FRET imaging is not suitable for multiplexed
imaging because the biosensors are composed of two different fluorophores in many cases. To circumvent this limitation, we are currently developing single-fluorophorebased biosensors. We recently developed a red-fluorescence
dopamine (DA) reporter. DA, a neuromodulator, is involvedin many neuronal functions, and to monitor it in living
cells and animals, we have designed a red-fluorescent DA
reporter. This reporter features a circular-permutated mApple(cpmApple), a red fluorescent protein, and a cpmApple wasinserted into the third intracellular loop of a DA receptor,
DRD1 (Figure 2A). The linker sequences between DRD
and cpmApple were optimized by random mutagenesis andsubsequent live-cell screening. Finally, we picked up the best
performance reporter and named the red genetically encoded
GPCR activation reporter for DA, R-GenGAR-DA. Thered fluorescence intensity decreased in response to DA andreturned to the basal level upon a DRD1 antagonist treatment (Figures 2B and 2C). In addition, by using a previously
reported green fluorescence norepinephrine (NE) reporter,
we succeeded in simultaneously visualizing DA and NE inliving neurons (Nakamoto C, Goto Y, Mol Brain, 2021).

A DRD1A

iRFP

Biliverdin

B
HO1

R-GenGAR-DA
SPFLAGICL1 ICL2B HeLa cells
BasalFdHeme

ICL3

GAFPAS

FnrcpmApple

R-GenGAR-DA1.2
DACSCH⊿F/F00.2

PcyAFdFdox

DMSO

Biliverdin
(BV)

Fdox
Fnr

Biliverdin

Phycocyanobilin(PCB)
Phycocyanobilin

DIC+iRFP–0.6

C Primary neurons
Basal

R-GenGAR-DA1.2
DAiRFPSCH⊿F/F00.3–0.8

Figure 2. Development of a genetically encoded red fluorescent dopamine(DA) reporter. (A) Schematic representation of a dopamine receptor, DRD1(left), and Red DA reporter, R-GenGAR-DA. (B and C) HeLa cells (B) ormouse primary hippocampal neurons (C) expressing R-GenGAR-DA were
stimulated with DA, followed by a DRD1 antagonist, SCH.

Near-infrared fluorescent protein (iRFP) is a fluorescentprotein with near-infrared excitation and emission maxima.
Unlike the other conventional fluorescent proteins, iRFP
requires biliverdin (BV) as a chromophore (Figure 3A). We
accidentally found that phycocyanobilin (PCB) functions asa brighter chromophore for iRFP than BV and that biosynthesis of PCB allows live-cell imaging with iRFP in the fission
yeast Schizosaccharomyces pombe (Figure 3B). We initiallyfound that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence (Figure 3C). The
brightness of iRFP–PCB was higher than that of iRFP–BV
both in vitro and in fission yeast. We introduced SynPCB2.1,a PCB biosynthesis system (Uda, Miura, ACS Chem Biol,2020), into fission yeast, resulting in the brightest iRFP
fluorescence. These tools not only enable the easy use ofmultiplexed live-cell imaging in fission yeast with a broader
color palette, but also open the door to new opportunities
for near-infrared fluorescence imaging in a broader range ofliving organisms (Sakai, JCS, 2021).

II. Manipulation of cell signaling

Artificial manipulation of biochemical networks is wellestablished in the quantitative understanding of biologicalsystems. Multiple methods have been suggested for control-050000

Figure 3. Phycocyanobilin (PCB) as a brighter chromophore for iRFP.
(A) Schematic representation of iRFP with its chromphore Bliverdin. (B)The metabolic pathway of PCB. (C) DIC and iRFP fluorescence images
are shown in fission yeats treated with DMSO, Biliverdin, or PCB.

ling signal transduction, including light-induced dimerization(LID) and chemically induced dimerization (CID) systems.
Of these two, the LID system is more beneficial in termsof temporal and spatial manipulation. The photo-responsive proteins derived from fungi, cyanobacteria, plants, andmodified fluorescent proteins are used in this system.
We have reported a novel optogenetic tool to induce relaxationof actomyosin contractility with a blue light-responsive protein,such as cryptochrome 2 (CRY2) and iLID. Actomyosin contractility, which is generated cooperatively by nonmuscle myosin II(NMII) and actin filaments, plays essential roles in a wide rangeof biological processes such as cell motility and cytokinesis. Yet,it remains elusive as to how actomyosin contractility generates
force and maintains cellular morphology. To elucidate this, wehave developed a novel optogenetic tool, OptoMYPT, which
combines a catalytic subunit of type I phosphatase-binding
domain of MYPT1 with an optogenetic dimerizer so that itallows light-dependent recruitment of endogenous PP1c to theplasma membrane (Figure 4A). We confirmed that blue-lightillumination was sufficient to induce dephosphorylation ofMLC, resulting in lamellipodial membrane protrusion (Figure4B). The OptoMYPT was further employed to understand themechanics of actomyosin-based cortical tension and contractile
ring tension during cytokinesis (Figure 4C). We found that therelaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, thus revealing that the corticaltension substantially antagonizes constriction of the cleavage
furrow. These results provide new opportunities to freely design
cell and tissue morphology through light (Yamamoto, NatComm, 2021).

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National Institute for Basic Biology

Cell Biology

Figure 5. Optogenetic control of cell cycle in fission yeast. (A) Lightinduced recruitment of PIF3-mNeonGreen (mNG) to the plasmamembrane (left) and nucleus (right). (B) Opto-G2/M: Optical control ofG2/M transition. (left) Opto-G2/M OFF cells showed normal morphology, while Opto-G2/M ON cells exhibited longer cell length, a typicalphenotype of G2/M arrest fission yeast. (C) Opto-SAC: Optical controlof spindle assembly checkpoint. Representative cells undergoing metaphase arrest (right) by Opto-SAC. (D) Long-term metaphase arrest by
Opto-SAC leads to a mitotic failure and cut phenotype.
Figure 4. PhyB-PIF light-inducible dimerization (LID) system. (A) The
metabolic pathway of phytochrome chromophores, PCB. (B and C)
Light-induced translocation of PIF3-mEGFP to the plasma membrane,
where PhyB is localized (B). Membrane translocation of PIF3-mEGFP isquantified (C).

The Blue light-responsive optical dimerizers are useful, but its
activation light often overlaps with the excitation light of fluorescent proteins, which in turn hampers the application of GFPor FRET biosensors together with their optogenetic tools. To
circumvent this issue, we focus on the phytochrome B (PhyB)PIF LID system. Upon red-light illumination, PhyB binds toPIF, and the PhyB-PIF complex dissociate from each other by
far-red light exposure. One drawback of PhyB-PIF LID is thata chromophore, e.g., PCB, is required for the light sensing. To
overcome this issue, we have developed a method for PCB
biosynthesis, called SynPCB, in mammalian cells by introducing the gene products of HO1, PcyA, Fd, and Fnr into the mitochondria (Uda Y, PNAS, 2017; Uda Y, ACS Chem Biol, 2020).
To take full advantage of this system, we applied theSynPCB system to S. pombe and C. elegans, both of which
are incapable of having purified PCB delivered to them.
As we expected, the expression of HO1, PcyA, Fd, and Fnr
genes induced PCB biosynthesis in S. pombe and C. elegans.
Using this system, we developed optogenetic control of thecell cycle in two ways: the Opto-G2/M checkpoint triggered
G2/M cell cycle arrest, and Opto-SAC induced a spindle
assembly checkpoint (SAC) in response to red light (Figure5) (Goto Y, and Aoki K., bioRxiv, 2020).

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Publication List:〔Original papers〕

H., Ogoh, K., Kondo, Y., and Aoki, K. (2022). LIM Tracker:• Aragaki,a software package for cell tracking and analysis with advanced•
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interactivity. Sci. Rep. 12, 2702. DOI: 10.1038/s41598-022-06269-6
Ito, R.E., Oneyama, C., and Aoki, K. (2021). Oncogenic mutation oroverexpression of oncogenic KRAS or BRAF is not sufficient to confer
oncogene addiction. PLoS One 16, e0249388. DOI: 10.1371/journal.
pone.0249388
Kajiwara, K., Yamano, S., Aoki, K., Okuzaki, D., Matsumoto, K., andOkada, M. (2021). CDCP1 promotes compensatory renal growth byintegrating Src and Met signaling. Life Sci. Alliance 4, e202000832.
DOI: 10.26508/lsa.202000832
Lin, S., Hirayama, D., Maryu, G., Matsuda, K., Hino, N., Deguchi, E.,Aoki, K., Iwamoto, R., Terai, K., and Matsuda, M. (2022). Redundantroles of EGFR ligands in the ERK activation waves during collectivecell migration. Life Sci. Alliance 5, e202101206. DOI: 10.26508/lsa.202101206
Matsui, K., Okamoto, K., Hasegawa, T., Ohtsuka, H., Shimasaki, T.,Ihara, K., Goto, Y., Aoki, K., and Aiba, H. (2021). Identification of ksg1
mutation showing long-lived phenotype in fission yeast. Genes to Cells
26, 967–978. DOI: 10.1111/gtc.12897
Nakamoto, C., Goto, Y., Tomizawa, Y., Fukata, Y., Fukata, M., Harpsoe,K., Gloriam, D.E., Aoki, K., and Takeuchi, T. (2021). A novel red
fluorescence dopamine biosensor selectively detects dopamine in thepresence of norepinephrine in vitro. Mol. Brain 14, 173. DOI: 10.1186/s13041-021-00882-8
Nakayama, H., Rowland, S.D., Cheng, Z., Zumstein, K., Kang,J., Kondo, Y., and Sinha, N.R. (2021). Leaf form diversificationin an ornamental heirloom tomato results from alterations in two
different HOMEOBOX genes. Curr. Biol. 31, 4788+. DOI: 10.1016/j.
cub.2021.08.023
Sakai, K., Kondo, Y., Fujioka, H., Kamiya, M., Aoki, K., and Goto,•••Y. (2021). Near-infrared imaging in fission yeast using a geneticallyencoded phycocyanobilin biosynthesis system. J. Cell Sci. 134,
jcs259315. DOI: 10.1242/jcs.259315
Verma, A., Jena, S.G., Isakov, D.R., Aoki, K., Toettcher, J.E., andEngelhardt, B.E. (2021). A self-exciting point process to study
multicellular spatial signaling patterns. Proc. Natl. Acad. Sci. U.S.A.
118. e2026123118. DOI: 10.1073/pnas.2026123118
Yagi-Utsumi, M., Aoki, K., Watanabe, H., Song, C., Nishimura, S.,
Satoh, T., Yanaka, S., Ganser, C., Tanaka, S., Schnapka, V., Goh, E.W.,
Furutani, Y., Murata, K., Uchihashi, T., Arakawa, K., and Kato, K.
(2021). Desiccation-induced fibrous condensation of CAHS proteinfrom an anhydrobiotic tardigrade. Sci. Rep. 11. 21328. DOI: 10.1038/
s41598-021-00724-6
Yamamoto, K., Miura, H., Ishida, M., Mii, Y., Kinoshita, N., Takada,S., Ueno, N., Sawai, S., Kondo, Y., and Aoki, K. (2021). Optogenetic
relaxation of actomyosin contractility uncovers mechanistic roles ofcortical tension during cytokinesis. Nat. Commun. 12. 7145. DOI:
10.1038/s41467-021-27458-3〔Review Articles〕

P.E., Reed, E.H., Underhill, E.J., Aoki, K., and Toettcher, J.E.
• Farahani,(2021). Signaling, Deconstructed: Using Optogenetics to Dissect and••

Direct Information Flow in Biological Systems. Annu. Rev. Biomed.
Eng. 23, 61–87. DOI: 10.1146/annurev-bioeng-083120-111648
Hiiragi, T., Gladfelter, A.S., Miguel-Aliaga, I., Coller, H.A., Lau, O.S.,
McBride, H.M., Aoki, K., Linkermann, A., Santaguida, S., Wasteneys,G., Rivron, N.C., Shahbazi, M.N., Ebisuya, M., and Lancaster, M.A.
(2021). 20 years of Developmental Cell: Looking forward. Dev. Cell56, 3185–3191. DOI: 10.1016/j.devcel.2021.11.017
Nakamura, A., Goto, Y., Kondo, Y., and Aoki, K. (2021). Shedding lighton developmental ERK signaling with genetically encoded biosensors.
Development 148. dev199767. DOI: 10.1242/dev.199767

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