Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

Beam, Monica; Silva, M. Catarina; Morimoto, Richard I.

doi:10.1074/jbc.m112.362764

Cited by 27 publications

(31 citation statements)

References 85 publications

(54 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Evidence for in vivo intermediate aggregate states for transgenic worms expressing polyQ fusion constructs comes from microscopy experiments, applying FRAP and FCS, methods. FRAP analysis on the series of polyQ strains suggests such intermediates based on some fluorescence recovery, as shown in Supporting Information Figure S4B, and is consistent with the published work on these strains .…”

Section: Resultsmentioning

confidence: 99%

“…Worms were maintained according to standard methods, at 20° C on nematode growth media (NGM) with OP50 E. Coli . The polyglutamine strains, expressing different lengths of CAG‐repeats fused with YFP, have been described elsewhere: wild‐type (wt) Bristol strain N 2 ; Q 0 AM134 (rmls126[Punc‐54::Q 0 ::YFP]X; Q 24 AM138 (rmIs130[Punc‐54::Q 24 ::YFP]II); Q 35 AM140 (rmIs132[Punc‐54::Q 35 ::YFP]I; Q 40 AM141 (rmIs133[Punc‐54::Q 40 ::YFP]X) . The Q 82 ‐YFP strain, harboring an integrated extrachromosomal array, was created in our laboratory using a plasmid donated from the Morimoto laboratory .…”

Section: Methodsmentioning

confidence: 99%

“…Then, the original 10 μm 2 area was imaged with the exact same settings as during prebleach six times every 20 s at 10% transmission. Relative fluorescence intensity (RFI) was determined in the NIS‐Elements based on the formula RFI = T t / T 0 , where T 0 represents fluorescence intensity in the bleached area before bleaching and T t fluorescence intensity in the bleached area at time t after bleaching . Since our confocal microscope did not support an automated protocol, it was not possible to monitor a control unbleached area in tandem with the bleached area.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection

et al. 2015

View full text Add to dashboard Cite

This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for the analytical ultracentrifuge. Further, new sedimentation velocity methods, such as the multi-speed method for data capture and wide distribution analysis for data analysis, are applied to improve the resolution of the measures of heterogeneity over a wide range of sizes. The focus here is to test the ability to measure sedimentation of polyglutamine aggregates in complex mixtures as a prelude to future studies that will explore the effects of genetic manipulation and environment on aggregation and toxicity. Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging from robust analysis of the monomer species through an intermediate and quite heterogeneous population of oligomeric species, and all the way up to detecting species that likely represent intact inclusion bodies based on comparison to an analysis of fluorescent puncta in living worms by confocal microscopy. Our results support the hypothesis that misfolding of expanded polyglutamine tracts into insoluble aggregates involves transitions through a number of stable intermediate structures, a model that accounts for how an aggregation pathway can lead to intermediates that can have varying toxic or protective attributes. An understanding of the details of intermediate and large-scale aggregation for polyglutamine sequences, as found in neurodegenerative diseases such as Huntington's Dis-

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Both assays have been used for the identification of compounds with inhibitory properties against mHTT aggregation. More recently, in vitro assays have been used to define more clearly the toxic protein species [28,29]. For example, methylene blue (MB) reduced aggregation as measured by the filter trap method when added to monomers or preformed fibrils in vitro [30].…”

Section: Molecular Screening To Target Polyq Protein Levels and Aggrementioning

confidence: 99%

Experimental Models for Identifying Modifiers of Polyglutamine-Induced Aggregation and Neurodegeneration

Calamini

Kaltenbach

2013

Neurotherapeutics

View full text Add to dashboard Cite

Huntington's disease (HD) typifies a class of inherited neurodegenerative disorders in which a CAG expansion in a single gene leads to an extended polyglutamine tract and misfolding of the expressed protein, driving cumulative neural dysfunction and degeneration. HD is invariably fatal with symptoms that include progressive neuropsychiatric and cognitive impairments, and eventual motor disability. No curative therapies yet exist for HD and related polyglutamine diseases; therefore, substantial efforts have been made in the drug discovery field to identify potential drug and drug target candidates for disease-modifying treatment. In this context, we review here a range of early-stage screening approaches based in in vitro, cellular, and invertebrate models to identify pharmacological and genetic modifiers of polyglutamine aggregation and induced neurodegeneration. In addition, emerging technologies, including high-content analysis, threedimensional culture models, and induced pluripotent stem cells are increasingly being incorporated into drug discovery screening pipelines for protein misfolding disorders. Together, these diverse screening strategies are generating novel and exciting new probes for understanding the disease process and for furthering development of therapeutic candidates for eventual testing in the clinical setting.

show abstract

“…wrMTrck was developed to track the behavior of Caenorhabditis elegans and has also been applied successfully to track melanoblasts, mitochondria, Schistosoma mansoni, Xenopus tropicalis tadpoles, and yeast (12–17). Our method, which we named Mouse Behavior Tracker, measures distance traveled, average speed, and movement patterns/tracks in home cages using economical USB-webcams, free software, and a standard desktop computer.…”

Section: Introductionmentioning

confidence: 99%

Mouse Behavior Tracker: An Economical Method for Tracking Behavior in Home Cages

et al. 2017

View full text Add to dashboard Cite

Analysis of mouse behavior often requires expensive equipment and transfer of the mice to new test en- vironments, which could trigger confounding behavior alterations. Here, we describe a system for track- ing mouse behavior in home cages using a low-cost USB webcam and free software (Fiji and wrMTrck). We demonstrate the effectiveness of this method by tracking differences in distance traveled, speed, and movement tracks between wild-type mice and amyotrophic lateral sclerosis (ALS) model mice (SOD1G93A).

show abstract

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

Cited by 27 publications

References 85 publications

Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection

Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection

Experimental Models for Identifying Modifiers of Polyglutamine-Induced Aggregation and Neurodegeneration

Mouse Behavior Tracker: An Economical Method for Tracking Behavior in Home Cages

Contact Info

Product

Resources

About