Novel Cell- and Tissue-Based Assays for Detecting Misfolded and Aggregated Protein Accumulation Within Aggresomes and Inclusion Bodies
Open Access
- 5 December 2010
- journal article
- research article
- Published by Springer Science and Business Media LLC in Cell Biochemistry and Biophysics
- Vol. 60 (3), 173-185
- https://doi.org/10.1007/s12013-010-9138-4
Abstract
Aggresomes and related inclusion bodies appear to serve as storage depots for misfolded and aggregated proteins within cells, which can potentially be degraded by the autophagy pathway. A homogenous fluorescence-based assay was devised to detect aggregated proteins inside aggresomes and inclusion bodies within an authentic cellular context. The assay employs a novel red fluorescent molecular rotor dye, which is essentially nonfluorescent until it binds to structural features associated with the aggregated protein cargo. Aggresomes and related structures were generated within cultured cells using various potent, cell permeable, proteasome inhibitors: MG-132, lactacystin, epoxomicin and bortezomib, and then selectively detected with the fluorescent probe. Employing the probe in combination with various fluorescein-labeled primary antibodies facilitated co-localization of key components of the autophagy system (ubiquitin, p62, and LC3) with aggregated protein cargo by fluorescence microscopy. Furthermore, cytoplasmic aggregates were highlighted in SK-N-SH human neuroblastoma cells incubated with exogenously supplied amyloid beta peptide 1–42. SMER28, a small molecule modulator of autophagy acting via an mTOR-independent mechanism, prevented the accumulation of amyloid beta peptide within these cells. The described assay allows assessment of the effects of protein aggregation directly in cells, without resorting to the use of non-physiological protein mutations or genetically engineered cell lines. With minor modification, the assay was also adapted to the analysis of frozen or formalin-fixed, paraffin-embedded tissue sections, with demonstration of co-localization of aggregated cargo with β-amyloid and tau proteins in brain tissue sections from Alzheimer’s disease patients.Keywords
This publication has 24 references indexed in Scilit:
- Molecular mechanism of Thioflavin-T binding to amyloid fibrilsBiochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2010
- p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell deathThe Journal of cell biology, 2005
- Global Impairment of the Ubiquitin-Proteasome System by Nuclear or Cytoplasmic Protein Aggregates Precedes Inclusion Body FormationMolecular Cell, 2005
- Protein aggregation and neurodegenerative diseaseNature Medicine, 2004
- The Deacetylase HDAC6 Regulates Aggresome Formation and Cell Viability in Response to Misfolded Protein StressCell, 2003
- A Novel Tool for Detecting Amyloid Deposits in Systemic Amyloidosis In Vitro and In VivoLaboratory Investigation, 2003
- Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing proteinHuman Molecular Genetics, 2003
- Aggresomes, inclusion bodies and protein aggregationTrends in Cell Biology, 2000
- [1] Staining methods for identification of amyloid in tissueMethods in Enzymology, 1999
- Calcein accumulation as a fluorometric functional assay of the multidrug transporterBiochimica et Biophysica Acta (BBA) - Biomembranes, 1994