A Fluorescence‐Based Sensor Assay that Monitors General Protein Aggregation in Human Cells

Abstract

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ-peptide. Exposure to proteome destabilizers lead to re-localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide-genetic screens to identify genes and pathways involved in protein misfolding and aggregation.

Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior

“…Quantifying low probability nucleation events under cellular conditions is crucial to answering this question but doing so necessitates probing millions of reaction vessels of microscopic volumes. Existing assays for protein self-assembly rely on the formation of visible puncta or the inactivation of fusion partners (Alberti et al, 2009; Morell et al, 2011; Narayanaswamy et al, 2009; Newby et al, 2017; Noree et al, 2010; Pereira et al, 2018; Ramdzan et al, 2012; Waldo et al, 1999; Zhao et al, 2016). They require restrictive subcellular localization (Sivanathan and Hochschild, 2013), and/or necessitate expression from dual constructs (Arslan et al, 2015; Blakeley et al, 2012; Cabantous et al, 2013; Holmes et al, 2014; Shyu and Hu, 2008).…”

Quantifying nucleationin vivoreveals the physical basis of prion-like phase behavior

“…It has no restrictions on subcellular localization, solubility, or size of the assemblies, thereby mitigating false-negatives and false-positives. In contrast, existing high-throughput assays for protein aggregation report on large puncta (Narayanaswamy et al, 2009;Noree et al, 2010;Pereira et al, 2018;Ramdzan et al, 2012) , inactivation of fusion partners (Alberti et al, 2009;Morell et al, 2011;Newby et al, 2017;Waldo et al, 1999;Zhao et al, 2016) , require restrictive subcellular localization (Sivanathan and Hochschild, 2013) , and/or necessitate expression from dual constructs (Arslan et al, 2015;Blakeley et al, 2012;Cabantous et al, 2013;Shyu and Hu, 2008) . Most importantly, DAmFRET simultaneously reports total protein concentration, and thereby the concentration-dependence of self-assembly, in every single sample.…”

Quantification of Cellular Proteostasis in Live Cells by Fluorogenic Assay Using the AgHalo Sensor

“…To this end, fusion of fluorescent protein tags to these sensors or labeling with fluorescent probes has been utilized to visualize the sensors’ aggregation by observing fluorescent granules in live cells. These sensors are exemplified by destabilized client proteins that are fused to green fluorescent protein (GFP) (Gupta et al., ; Winkler et al., ), thermally labile proteins incorporating fluorescent unnatural amino acids (Hsieh et al., ), thermally labile proteins that are fused to a tetracysteine motif and labeled with FlAsH dye (Ignatova & Gierasch, ), destabilized retroaldolase enzyme that is labeled with a fluorophore (Liu, Zhang, Chen, Tan, & Kelly, ), destabilized barnase enzyme (Wood et al., ), GFP‐fused heat‐shock proteins (Pereira et al., ), and destabilized GFP (Waldo, Standish, Berendzen, & Terwilliger, ; Wigley, Stidham, Smith, Hunt, & Thomas, ). The limitation of these sensors is that they exhibit fluorescence before and after aggregation (i.e., they are non‐fluorogenic), so their aggregation can only be judged based on formation of fluorescent granules.…”