Abstract
In our recently published paper, we highlight that during normal aging of C. elegans age-dependent aggregates of proteins form and lead to functional decline of tissues. The protocol described here details the isolation of two proteins from C. elegans in their aggregated amyloid-like form, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1). We used nickel beads to isolate His-tagged KIN-19 and RHO-1, and thus permitting the isolation of both small and large aggregated or fibrillary forms of the proteins. We characterized their morphology by transmission electron microscopy. We further expressed RFP-tagged proteins and stained them with a fluorescent molecule, thioflavin T, which identifies β-sheet structures, and which is a defining feature of amyloid fibrils. We further applied structured illumination microscopy to determine the level of colocalization between RFP and thioflavin T.
Keywords: His-tag, RFP-tag, Amyloid, Aggregates, Structured illumination microscopy, Transmission electron microscopy, RHO-1, KIN-19
Background
During aging, there is often a dysregulation in protein homeostasis and therefore an accumulation of damaged or non-functional proteins. Protein damage can occur by reactive oxygen species or post-translational modifications, along with mutations and transcriptional changes that naturally occur with age. One of the most studied forms of protein malfunction during aging are protein aggregates referred to as amyloids. These amyloids are found in many age-associated diseases such as Alzheimer’s disease and Parkinson’s disease. Soluble monomeric protein converts into an insoluble state where structure alters and proteins aggregate to form β-sheet rich fibrils. It is not clear whether the body is sequestering damaged or misfolded proteins into these amyloid fibrils when protein homeostasis is dysregulated or whether the formation of amyloid fibrils further dysregulates protein homeostasis, or both. Screening for proteins most likely to become insoluble upon aging Caenorhabditis elegans (C. elegans) yielded two target proteins, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1) (David et al., 2010). These two proteins were further studied to investigate their aggregation propensity during the ageing of C. elegans, whether they formed amyloid-like structures and whether they correlated with a functional decline (Huang et al., 2019). In this protocol we first express the amyloid-like proteins KIN-19 and RHO-1 with a 6xHis-tag, this permits us to isolate the proteins with nickel beads. There can often be fibrous structures within small organisms such as C. elegans, but isolating His-tagged proteins ascertains that we isolate the protein of interest. Use of nickel beads rather than a nickel chromatography column allowed us to isolate large structures such as fibrils and aggregates which can get stuck in nickel columns. Subsequent imaging of eluted His-tagged proteins on a transmission electron microscope (TEM) permitted us to investigate the morphology of potential amyloid fibrils due to the sub-nanometer resolution provided by the TEM. However, a defining feature of amyloid fibrils is the presence of highly structured β-sheets which cannot be determined using TEM. Thus, in order to determine the presence of β-sheet structures in our proteins of interest we used structure illumination microscopy (SIM) which has ~200 nm resolution. We expressed red fluorescent protein (RFP)-tagged proteins in C. elegans and concomitantly stained them with thioflavin-T (ThT), a molecule which fluoresces when it intercalates into β-sheet structures. We then consequently investigated the colocalization of RFP and ThT fluorescence.
Materials and Reagents
Equipment
Software
1. Fiji open source image processing program (https://fiji.sc)
Procedure
This protocol describes the preparation of worms for imaging techniques. For protocols on the number of worms to grow, growth conditions, collection and freezing of worms please refer to William B. W. (1988) and Groh et al. (2017).
Data analysis
Acknowledgments
G.S.K.S. acknowledges funding from the Wellcome Trust, the UK Medical Research Council (MRC), Alzheimer Research UK (ARUK), and Infinitus China Ltd. This protocol was based on the research paper (Huang et al., 2019) in collaboration with Dr Della David.
Competing interests
None.
References
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