<i>In vivo</i> measurement of autophagic flux by fluorescence correlation spectroscopy

Song, Hang; Dong, Chaoqing; Ren, Jicun

doi:10.1039/d3an00651d

Cited by 2 publications

(2 citation statements)

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“…Recently, our group developed a method for assessing autophagic flux at the single-molecule level by fluorescence autocorrelation spectroscopy. 25 This method realizes the monitoring of autophagosomes by the difference in the diffusion time of fluorescently labeled autophagosomes and fluorescent monomers in the cytoplasm. However, monitoring multiple autophagic processes is constrained by the labeling of single fluorescent proteins.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Therefore, it is difficult for the accurate differentiation, localization, and quantification of autophagic structures in living cells by this method. Recently, our group developed a method for assessing autophagic flux at the single-molecule level by fluorescence autocorrelation spectroscopy . This method realizes the monitoring of autophagosomes by the difference in the diffusion time of fluorescently labeled autophagosomes and fluorescent monomers in the cytoplasm.…”

Section: Introductionmentioning

confidence: 99%

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Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy

Song,

Dong,

Ren

2024

Anal. Chem.

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Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter “delivery efficiency of autophagosome (DE AP)” to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

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Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy

Song,

Dong,

Ren

2024

Anal. Chem.

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Autophagy: A Silent Protagonist in Kidney Transplantation

Ponticelli,

Reggiani,

Moroni

2023

Transplantation

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Autophagy is a lysosome-dependent regulated mechanism that recycles unnecessary cytoplasmic components. It is now known that autophagy dysfunction may have a pathogenic role in several human diseases and conditions, including kidney transplantation. Both defective and excessive autophagy may induce or aggravate several complications of kidney transplantation, such as ischemia–reperfusion injury, alloimmune response, and immunosuppressive treatment and side effects. Although it is still complicated to measure autophagy levels in clinical practice, more attention should be paid to the factors that may influence autophagy. In kidney transplantation, the association of low doses of a mammalian target of rapamycin inhibitor with low doses of a calcineurin inhibitor may be of benefit for autophagy modulation. However, further studies are needed to explore the role of other autophagy regulators.

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In vivo measurement of autophagic flux by fluorescence correlation spectroscopy

Abstract: Autophagy is a fundamental and phylogenetically conserved self-degradation process and plays very important role in the selective degradation of deleterious proteins, organelles, and other macromolecules. Although flow cytometry and fluorescence...

Cited by 2 publications

References 46 publications

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy

Autophagy: A Silent Protagonist in Kidney Transplantation

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