Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates

Sin, Olga; Mata‐Cabana, Alejandro; Seinstra, Renée I.; Nollen, Ellen A. A.

doi:10.21769/bioprotoc.3042

Cited by 9 publications

(9 citation statements)

References 8 publications

(12 reference statements)

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“…The filter trap assay was performed to analyze SDS-insoluble aggregates according to the published literature [ 45 , 46 ]. After 48 h of transient transfection with pHM6-HA-Q23 and pHM6-HA-Q74, cells were rinsed with 1X PBS and lysed with lysis buffer (40 mM HEPES, pH 7.5, 50 mM KCl, 1% ( v / v ) Triton X-100, 2 mM DTT, 5 mM EDTA) supplemented with 1X cOmplete™, Mini, EDTA-free protease inhibitor cocktail.…”

Section: Methodsmentioning

confidence: 99%

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin

et al. 2020

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The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington’s disease (HD) research.

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Section: Methodsmentioning

confidence: 99%

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin

et al. 2020

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“…For experiments assaying worms at different ages, NGM plates containing 50 μM 5-fluorodeoxyuridine (FUDR) were used instead of standard NGM plates to inhibit egg-laying, as described previously (Sutphin & Kaeberlein, 2009). In these experiments, worms were transferred to fresh FUDR-containing plates whenever OP50 bacteria were close to being depleted (approximately every 3 days).…”

Section: Worm Synchronisation and Sample Preparationmentioning

confidence: 99%

“…In addition, these worms have a short reproductive cycle and are easy to manipulate genetically. Consequently, they have been investigated extensively in forward and reverse genetic studies to identify genes that modulate biochemical processes including those involved in protein aggregation and neurodegeneration (Kraemer et al, 2006;Nollen et al, 2004;van Ham et al, 2008). They are also highly amenable to screens of pharmacological compounds and are often used as an initial model system for potential therapeutics before conducting rodent-based studies (Chen et al, 2015;Ikenaka et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…A fundamental component of studying the aggregation of proteins in cells and organisms, and identifying modulators of this process, is the capacity to quantify the number and type of inclusions that are formed. The quantification of aggregation in C. elegans often requires laborious manual counting of inclusions from images generated through fluorescence microscopy, or other biochemical methods, such as fractionation of detergent soluble and insoluble fractions or filter trap assays, both of which rely on quantifying the amount of insoluble protein in the lysate (Sin et al, 2018; Walther et al, 2017). Recently, there have been several advances in automated imaging methods in an attempt to increase the throughput of microscopy‐based experiments (Cornaglia et al, 2016; Gosai et al, 2010; Mondal et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

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Exploiting flow cytometry for the unbiased quantification of protein inclusions in Caenorhabditis elegans

Claesson

Chew

Ecroyd

2022

Journal of Neurochemistry

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The aggregation of proteins into inclusions or plaques is a prominent hallmark of a diverse range of pathologies including neurodegenerative diseases. The quantification of such inclusions in Caenorhabditis elegans models of aggregation is usually achieved by fluorescence microscopy or other techniques involving biochemical fractionation of worm lysates. Here, we describe a simple and rapid flow cytometry‐based approach that allows fluorescently tagged inclusions to be enumerated in whole worm lysate in a quantitative and unbiased fashion. We demonstrate that this technique is applicable to multiple C. elegans models of aggregation and importantly, can be used to monitor the dynamics of inclusion formation in response to heat shock and during ageing. This includes the characterisation of physicochemical properties of inclusions, such as their apparent size, which may reveal how aggregate formation is distinct in different tissues or at different stages of pathology or ageing. This new method can be used as a powerful technique for the medium‐ to high‐throughput quantification of inclusions in future studies of genetic or chemical modulators of aggregation in C. elegans.

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“…A fundamental component of studying the aggregation of proteins in cells and organisms, and identifying modulators of this process, is the capacity to quantify the number and type of inclusions that are formed. The quantification of aggregation in C. elegans often requires laborious manual counting of inclusions from images generated through fluorescence microscopy, or other biochemical methods, such as immunoblotting or filter trap assays, which rely on quantifying the amount of insoluble protein in the lysate (Sin et al 2018; Walther et al 2017). Recently, there have been several advances in automated imaging methods in an attempt to increase the throughput of microscopy-based experiments (Cornaglia et al 2016; Gosai et al 2010; Mondal et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Exploiting flow cytometry for the unbiased quantification of protein inclusions inCaenorhabditis elegans

Claesson

Chew

Ecroyd

2021

Preprint

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The aggregation of proteins into inclusions or plaques is a prominent hallmark of a diverse range of pathologies including neurodegenerative diseases. The quantification of such inclusions in Caenorhabditis elegans models of aggregation is usually achieved by fluorescence microscopy or other techniques involving biochemical fractionation of worm lysates. Here, we describe a simple and rapid flow cytometry-based approach that allows fluorescently-tagged inclusions to be enumerated in whole worm lysate in a quantitative and unbiased fashion. We demonstrate that this technique is applicable to multiple C. elegans models of aggregation and importantly, can be used to monitor the dynamics of inclusion formation in response to heat shock and during aging. This includes the characterisation of physicochemical properties of inclusions, such as their size, which may reveal how aggregate formation is distinct in different tissues or at different stages of pathology or aging. This new method can be used as a powerful technique for the medium- to high-throughput quantification of inclusions in future studies of genetic or chemical modulators of aggregation in C. elegans.

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Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates

Cited by 9 publications

References 8 publications

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin

Exploiting flow cytometry for the unbiased quantification of protein inclusions in Caenorhabditis elegans

Exploiting flow cytometry for the unbiased quantification of protein inclusions inCaenorhabditis elegans

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