Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

Whiten, Daniel R.; Gil, Rebecca San; McAlary, Luke; Yerbury, Justin J.; Ecroyd, Heath; Wilson, Mark R.

doi:10.1038/srep31138

Cited by 31 publications

(82 citation statements)

References 20 publications

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“…We also describe the use of the rapid flow cytometry technique FloIT 13 to quantify seeding and co-aggregation of proteins expressed in 2 separate populations of cells. In previous coculture experiments using SH-SY5Y (human neuroblastoma) cells, it was shown that TDP-43 can transfer between cells and that phosphorylated TDP-43 aggregates can be released from donor cells and taken up into recipient cells.…”

Section: Discussionmentioning

confidence: 99%

“…FloIT can quantify the proportion of inclusions containing 2 different proteins fused to distinct fluorescent protein tags. 13 Here we took advantage of this and quantified the proportion of inclusions that contained both TDP-43…”

Section: Tdp-43mentioning

confidence: 99%

“…TDP-43 inclusions have been quantified by FloIT previously. 13 Here, we quantified inclusion formation of either TDP-43…”

Section: Floit Quantifies Tdp-43 Aggregation and Inclusion Formation mentioning

confidence: 99%

“…1B & E). Inclusions were then distinguished from cellular debris by exploiting the fluorescent protein tag fused to TDP-43 13 ( Fig. 1C & F).…”

Section: Floit Quantifies Tdp-43 Aggregation and Inclusion Formation mentioning

confidence: 99%

“…12 Recently a novel method for quantifying protein inclusions was described. 13 We reasoned that this method might be used to provide a quick and robust measure of prion-like propagation.…”

Section: Introductionmentioning

confidence: 99%

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Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

Zeineddine

Whiten

Farrawell

et al. 2017

Prion

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ABSTRACT. Amyotrophic lateral sclerosis is a devastating neuromuscular degenerative disease characterized by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology including TAR DNA-binding protein of 43 kDa (TDP-43) aggregates. Previous work suggests that TDP-43 can move between cells. Here we used a novel flow cytometry technique (FloIT) to analyze TDP-43 inclusions and propagation. When cells were transfected to express either mutant G294A TDP-43 fused to GFP or wild type TDP-43fused to tomato red and then co-cultured, flow cytometry detected intact cells containing both fusion proteins and using FloIT detected an increase in the numbers of inclusions in lysates from cells expressing wild type TDP-43-tomato. Furthermore, in this same model, FloIT analyses detected inclusions containing both fusion proteins. These results imply the transfer of TDP-43 fusion proteins between cells and that this process can increase aggregation of wild-type TDP-43 by a mechanism involving co-aggregation with G294A TDP-43.

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Section: Discussionmentioning

confidence: 99%

Section: Tdp-43mentioning

confidence: 99%

“…TDP-43 inclusions have been quantified by FloIT previously. 13 Here, we quantified inclusion formation of either TDP-43…”

Section: Floit Quantifies Tdp-43 Aggregation and Inclusion Formation mentioning

confidence: 99%

“…1B & E). Inclusions were then distinguished from cellular debris by exploiting the fluorescent protein tag fused to TDP-43 13 ( Fig. 1C & F).…”

Section: Floit Quantifies Tdp-43 Aggregation and Inclusion Formation mentioning

confidence: 99%

“…12 Recently a novel method for quantifying protein inclusions was described. 13 We reasoned that this method might be used to provide a quick and robust measure of prion-like propagation.…”

Section: Introductionmentioning

confidence: 99%

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Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

Zeineddine

Whiten

Farrawell

et al. 2017

Prion

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Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation

Zeineddine

Farrawell

Lambert-Smith

et al. 2017

Cell Stress and Chaperones

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ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent propagation, accumulation and deposition in motor neurons may explain such a pattern of pathology. Previous work has suggested that the internalization of aggregates triggers stress granule formation. Given the close association of stress granules and TDP-43, we wondered whether internalisation of SOD1 aggregates stimulated TDP-43 cytosolic aggregate structures. Addition of recombinant mutant G93A SOD1 aggregates to NSC-34 cells was found to trigger a rapid shift of TDP-43 to the cytoplasm where it was still accumulated after 48 h. In addition, SOD1 aggregates also triggered cleavage of TDP-43 into fragments including a 25 kDa fragment. Collectively, this study suggests a role for protein aggregate uptake in TDP-43 pathology.

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Small heat shock proteins: multifaceted proteins with important implications for life

Carra

Alberti

Benesch

et al. 2019

Cell Stress and Chaperones

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Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated

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Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

Cited by 31 publications

References 20 publications

Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation

Small heat shock proteins: multifaceted proteins with important implications for life

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