Rapid manipulation of mitochondrial morphology in a living cell with iCMM

Miyamoto, Takafumi; Uosaki, Hideki; Mizunoe, Yuhei; Han, Song‐iee; Goto, Shozo; Yamanaka, Daisuke; Masuda, Masato; Yoneyama, Yoshimasa; Nakamura, Hideki; Hattori, Naoko; Takeuchi, Yoshinori; Ohno, Hiroshi; Sekiya, Motohiro; Matsuzaka, Takashi; Hakuno, Fumihiko; Takahashi, Shinichiro; Yahagi, Naoya; Ito, Koichi; Shimano, Hitoshi

doi:10.1016/j.crmeth.2021.100052

Cited by 11 publications

(11 citation statements)

References 77 publications

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“…The plasmid pAG1142 allowing the expression of FRB- FIRE mate-IRES-mTurquoise2 was constructed by replacing the sequence coding for FAST 1-114 by FIRE mate sequence, in the previously described vector pAG64 11 . The plasmid pAG1159 allowing the expression of TOM20-ECFP- FIRE mate was constructed by replacing the sequence coding for FRB by FIRE mate sequence in the Addgene vector #171461 Tom20-CR coding for TOM20-ECFP-FRB 25 . Tom20-CR was a gift from Takafumi Miyamoto (Addgene plasmid # 171461 ; http://n2t.net/addgene:171461 ; RRID:Addgene_171461).…”

Section: Methodsmentioning

confidence: 99%

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A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

Bottone

Cakil

Joliot

et al. 2023

Preprint

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Proximity between proteins plays an essential and ubiquitous role in many biological processes. Molecular tools enabling to control and observe the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced REcognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible, and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allowed furthermore the design of innovative biosensors for the study of various processes, such as signal transduction and apoptosis.

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Section: Methodsmentioning

confidence: 99%

“…HBR-2,5DM), match600 (a.k.a HBR-3,5DOM), matchdark (a.k.a. HBIR-3M) were previously reported 13,14,25 .…”

Section: Data Availabilitymentioning

confidence: 99%

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

Bottone

Cakil

Joliot

et al. 2023

Preprint

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“…Imaging was performed using an Eclipse Ti2-E microscope equipped with NIS-Elements AR imaging software. The representative images were processed by Clarify.ai and Denoise.ai using NIS-Elements AR 5.30, as previously described (42).…”

Section: Methodsmentioning

confidence: 99%

Activity-dependent glassy cell mechanics I : Mechanical properties measured with active microrheology

Katsuhiro

Nishizawa

Ngao

et al. 2022

Preprint

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The active microrheology was conducted in living cells by applying optical-trapping force to vigorously-fluctuating tracer beads with the feedback-tracking technology. Complex shear viscoelastic modulus G(ω) was measured in HeLa cells in an epithelial-like confluent monolayer. We found that G(ω) \vprop (-i ω)^{0.5} in large range of frequencies (0.1 Hz <ω/(2 \π) <10 kHz). Actin disruption and cell-cycle progression from G1 to S, G2 phases only limitedly altered G(ω) in living cells. On the other hand, G(ω) was dependent on cell metabolism; the ATP-depleted cells increased elastic modulus G'(ω) at low frequencies by giving rise to a constant plateau as G(ω)=G_0+A(-iω)^0.5. Both the plateau and the additional frequency dependency proportional to (-i ω)^{0.5} of ATP-depleted cells are consistent to the typical rheological response of colloidal jamming. On the other hand, the plateau G_0 disappeared in the ordinary metabolically active cells, implying that the living cells fluidize their internal states towards critical jamming point.

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“…We next measured mitochondrial ATP using mitAT1.03, an organelle-targeted Förster resonance energy transfer (FRET) indicator of ATP 33 . Methods for measurement and analysis of CFP-to-YFP FRET were described previously 8,9,34 . Cells expressing mitAT1.03 were subjected to mitochondrial recruitment of 18TM for 2 h. As a result, the measured FRET values normalized by CFP intensity were significantly smaller than that with no recruitment of 18TM (0.44 ± 0.02 and 0.37 ± 0.01, without and with 18TM recruitment, respectively, p = 0.0027) (Fig.…”

Section: Defunctionalization Of Mitochondria Following Chemically Ind...mentioning

confidence: 99%

“…With this in mind, we recently engineered actin nucleation promoting factors to generate constrictive force against target organelles for their deformation 8 . Interestingly, when the technique was applied to mitochondria, their major functions such as ATP synthesis were not significantly altered 8,9 . This suggested that induction of deformation is not necessarily sufficient for functional perturbation, and thus radical approaches such as striking remodeling of the membrane composition should be explored.…”

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confidence: 99%

Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases

et al. 2022

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Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.

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Rapid manipulation of mitochondrial morphology in a living cell with iCMM

Cited by 11 publications

References 77 publications

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

Activity-dependent glassy cell mechanics I : Mechanical properties measured with active microrheology

Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases

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