Molecular Prosthetics for Long-Term Functional Imaging with Fluorescent Reporters

Grenier, Vincent; Martinez, Kayli; Benlian, Brittany; García-Almedina, Derek M.; Raliski, Benjamin K.; Boggess, Steven; Maza, Johnathan C.; Yang, Samantha; Gest, Anneliese; Miller, Evan W.

doi:10.1021/acscentsci.1c01153

Cited by 18 publications

(16 citation statements)

References 16 publications

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“…As nanoscopy techniques generally require higher light doses than diffraction-limited approaches, photodamage and photobleaching can become a key, yet often under-evaluated technical hurdle, for analyzing four-dimensional dynamics ( 19 – 21 ). We previously demonstrated that cyanine-COT conjugates ( 22 – 26 ) are gentle mitochondrial markers that allow prolonged SIM recordings of cristae ( 25 ) ( SI Appendix , Fig. S1 ).…”

mentioning

confidence: 99%

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Liu

Stephan

Chen

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Capturing mitochondria’s intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.

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mentioning

confidence: 99%

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Liu

Stephan

Chen

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Not surprisingly, therefore, it is possible to modify a potent bilayer-modifying molecule (e.g., resveratrol; Ingólfsson et al, 2014 ), to minimize its bilayer-perturbing effects while maintaining its desired biological effects ( Bosquesi et al, 2020 ). To our knowledge, this is the first example of a drug candidate that was developed with explicit consideration of its bilayer-modifying properties; a similar strategy has been used to minimize the off-target effects of photostabilizers used to protect fluorescent reporter groups ( Grenier et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Screening for bilayer-active and likely cytotoxic molecules reveals bilayer-mediated regulation of cell function

Peyear

Andersen

2023

Journal of General Physiology

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A perennial problem encountered when using small molecules (drugs) to manipulate cell or protein function is to assess whether observed changes in function result from specific interactions with a desired target or from less specific off-target mechanisms. This is important in laboratory research as well as in drug development, where the goal is to identify molecules that are unlikely to be successful therapeutics early in the process, thereby avoiding costly mistakes. We pursued this challenge from the perspective that many bioactive molecules (drugs) are amphiphiles that alter lipid bilayer elastic properties, which may cause indiscriminate changes in membrane protein (and cell) function and, in turn, cytotoxicity. Such drug-induced changes in bilayer properties can be quantified as changes in the monomer↔dimer equilibrium for bilayer-spanning gramicidin channels. Using this approach, we tested whether molecules in the Pathogen Box (a library of 400 drugs and drug-like molecules with confirmed activity against tropical diseases released by Medicines for Malaria Venture to encourage the development of therapies for neglected tropical diseases) are bilayer modifiers. 32% of the molecules in the Pathogen Box were bilayer modifiers, defined as molecules that at 10 µM shifted the monomer↔dimer equilibrium toward the conducting dimers by at least 50%. Correlation analysis of the molecules’ reported HepG2 cell cytotoxicity to bilayer-modifying potency, quantified as the shift in the gramicidin monomer↔dimer equilibrium, revealed that molecules producing <25% change in the equilibrium had significantly lower probability of being cytotoxic than molecules producing >50% change. Neither cytotoxicity nor bilayer-modifying potency (quantified as the shift in the gramicidin monomer↔dimer equilibrium) was well predicted by conventional physico-chemical descriptors (hydrophobicity, polar surface area, etc.). We conclude that drug-induced changes in lipid bilayer properties are robust predictors of the likelihood of membrane-mediated off-target effects, including cytotoxicity.

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“…However, photobleaching of traditional reporters makes it challenging to perform prolonged imaging experiments, and phototoxicity results in alterations to neuronal physiology. To bypass these limitations, Miller and co-workers reported a creative approach wherein they appended a cyclooctatetraene (COT) scaffold to a VFdye . The COT group acts as a triplet state quencher (TSQ) to reduce both photobleaching of the dye and phototoxicity toward neurons and cardiomyocytes.…”

Section: Small-molecule Probe Platformsmentioning

confidence: 99%

Chemical Sensors and Imaging: Molecular, Materials, and Biological Platforms

Messina,

Chang

2023

ACS Cent. Sci.

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Molecular Prosthetics for Long-Term Functional Imaging with Fluorescent Reporters

Cited by 18 publications

References 16 publications

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Screening for bilayer-active and likely cytotoxic molecules reveals bilayer-mediated regulation of cell function

Chemical Sensors and Imaging: Molecular, Materials, and Biological Platforms

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