Applications of flow cytometry for measurement of autophagy

Demishtein, Alik; Porat, Ziv; Elazar, Zvulun; Shvets, Elena

doi:10.1016/j.ymeth.2014.12.020

Cited by 27 publications

(25 citation statements)

References 47 publications

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“…This approach unites flow cytometry with high-resolu-tion, fluorescence imaging (38), thus combining high sampling with spatial information obtained by high-resolution imaging, respectively. Therefore, the sensitivity and information content on autophagic flux are very much increased compared to standard flow cytometry (39)(40)(41). From each cell, an image was created, and segmentation masks for the cell and for AVs were generated and used to calculate the total cellular and vesicular GFP-LC3 fluorescence (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Metz

Chiramel

Chatel‐Chaix

et al. 2015

J Virol

103

104

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Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. IMPORTANCEAutophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus. Dengue virus (DENV) is a member of the family Flaviviridae and is responsible for one of the most common infections transmitted to humans by mosquitoes. DENV is a positive-st...

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Section: Resultsmentioning

confidence: 99%

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Metz

Chiramel

Chatel‐Chaix

et al. 2015

J Virol

103

104

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“…To quantify the cells with autophagic vacuoles, the fluorochrome MDC was used to stain the vacuoles. Staining was detected by flow cytometry, which measured the absorbance at 488 nm as previously described . To further determine the autophagic vacuoles, LysoTracker staining method was used and detected by confocal microscopy as previously described …”

Section: Methodsmentioning

confidence: 99%

Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin‐related protein 14‐mediated autophagy

Zhen¹,

Yang

et al. 2017

Cancer Science

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Paclitaxel is not as effective for neuroblastoma as most of the front‐line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel‐associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy‐associated proteins were assessed by western blot. Autophagy was induced and the autophagy‐associated proteins LC3‐I, LC3‐II, Beclin 1, and thioredoxin‐related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1‐mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel‐induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel‐induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14.

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“…The uptake of AO by lysosomes of live cells is a consequence of protonation and entrapment that leads to the accumulation and aggregation of this basic fluorochrome within the acidic organelles. It should also be noted that AO is the key marker of lysosomes used in wideranging studies of autophagy (Demishtein, Porat, Elazar, & Shvets, 2015;SenthilKumar, Skiba, & Kimple, 2019;Thomé et al, 2016;Warnes, 2015). The uptake of AO by lysosomes has therefore been explored as a means of photodynamic-induced cytotoxicity (Zdolsek, Olsson, & Brunk, 1990) for selectively targeting cancer cells (Kusuzaki et al, 2012;Lin et al, 2017).…”

Section: Of 14mentioning

confidence: 99%

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

et al. 2019

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The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G 0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain singleversus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. C 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single-versus double-stranded DNA with acridine orange Keywords: acridine orange r apoptosis r autophagy r cell cycle r lysosomes r mitosis r sperm chromatin fertility assay How to cite this article: Darzynkiewicz, Z., Halicka, D. H., Zhao, H., & Li, J. (2019). Assessment of DNA susceptibility to denaturation as a marker of chromatin structure. BASIC PROTOCOL DNA DenaturationThe process of splitting double-stranded DNA (dsDNA) into single strands, which involves breaking hydrogen bonds between the bases in the DNA double-helical structure, is defined as DNA denaturation. DNA in solution undergoes denaturation when heated, and the temperature at which half remains in double-stranded conformation while the other half is single stranded is called the melting temperature (T m ). Because the G-C base pair, maintained by three hydrogen bonds, is stronger that the two-base pairing of AO has unique metachromatic properties: under the correct conditions (AO concentration, ionic strength, temperature), it can differentially stain ssDNA and dsDNA. Specifically, when AO intercalates between the base pairs of dsDNA, its fluorescence is maximally excited at 480 nm, its emission is at 530 nm, and its excitation lifetime is ß2 ns (Kapuscinski & Darzynkiewicz, 1984Kapuscinski, Darzynkiewicz, & Melamed, 1982. These values reflect singlet excitation of AO. The interaction of AO with ssDNA is more complex: it involves the insertion of the AO cation between adj...

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Applications of flow cytometry for measurement of autophagy

Cited by 27 publications

References 47 publications

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin‐related protein 14‐mediated autophagy

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

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