Browsing by Author "De Wet, Sholto"

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    Dissection of autophagy machinery and protein cargo flux
    (Stellenbosch : Stellenbosch University, 2020-04) De Wet, Sholto; Loos, Ben; Stellenbosch University. Faculty Science. Dept. of Physiology.
    ENGLISH ABSTRACT: Introduction: Neurodegenerative diseases, such as Alzheimer’s disease, are characterised by the increased histological expression of insoluble protein aggregates. Evidence has demonstrated that the soluble protein oligomers which constitute these insoluble protein aggregates, are a major source of neurotoxicity. Autophagy is known to be a major protein degradative pathway and has been shown to be active during neurodegenerative diseases. In the past, macroautophagy has been described as a non-specific means of degrading long-lived cytoplasmic proteins. However, recent evidence has demonstrated that various subtypes of macroautophagy (hereafter autophagy) exist, distinguished from one another by their preferred cargo. Of particular interest in the context of neurodegenerative diseases is regulating and controlling protein degradation through autophagy. p62 has been shown to be the preferred autophagy cargo receptor during protein degradation. In addition to p62, NBR1 has been shown to be another autophagy cargo receptor of proteins and has been shown to compensate for a loss of p62 expression levels. How these two receptors behave with respect to one another to support effective degradation over a 24 hour period of autophagy induction is unknown. Additionally, it is unknown to what extent these receptors interact with autophagosomes to contribute toward effective autophagy flux. Aims and methods: The aim of this study was to investigate the changes that occur to components of the autophagy system within an autophagy model system established in mouse embryonic fibroblasts (MEFs) not expressing any disease symptoms. MEFs were micropatterned as a means of standardising cell shape and size. Autophagy changes were induced using Rapamycin and Spermidine, respectively at relatively high and low concentrations. Studies were conducted over 24 hours to understand what impact time had on autophagy and its components. Western blotting was used to measure the abundance changes of LC3-II, p62, NBR1 and LAMP2a. Additionally, fluorescence microscopy was used to observe GFP-LC3, p62 and NBR1 puncta counts. Furthermore, studies were done in the presence and absence of Bafilomycin A1, an inhibitor of autophagosome/ lysosome fusion, to better understand the clearance activities of each protein. Results and discussion: Initial investigations using western blotting techniques demonstrated that Rapamycin caused an increase in LC3-II abundance levels but does not change receptor levels. Additionally, Spermidine treatment caused an increase in autophagosome clearance and receptor abundance but does not change receptor clearance levels. Fluorescence microscopy imaging revealed that autophagy induction with 1 μM Rapamycin caused an increase in GFP-LC3 and receptor puncta count 2 hours after incubation. However, no change was seen in the receptor clearance as was shown by the lack of co-localised puncta clearance. 10 nM Rapamycin on the other hand demonstrated fewer autophagosomes, however; effective receptor turnover, was demonstrated, especially of p62. Spermidine results demonstrated different behaviours, as 20 nM Spermidine showed a slower increase in GFP-LC3 than 1 μM Rapamycin, but demonstrated highly effective p62 clearance at 2 hours, followed by effective NBR1 clearance at the same time and at 8 hours, where p62 turnover was found to be at its lowest. 5 nM Spermidine did not induce the system in the same way as 20 nM Spermidine as was seen by less effective GFP-LC3 turnover. However, 5 nM Spermidine did demonstrate effective p62 clearance at 8 hours as well as effective co-localised puncta clearance at 2 hours and 8 hours of treatment. Conclusion: The means by which autophagy is induced, either by mTOR-dependent or –independent inducers, has an impact on autophagy components expression levels. Furthermore, treatment with higher concentrations of drugs demonstrated a more robust and immediate response of the autophagy components measured as well as their clearance. Conversely, lower drug treatment concentrations demonstrate different times of effectiveness. Taken together this study has shown that the effectiveness of autophagy flux is multifactorial and should be adjusted according to the autophagosome as well as receptor involvement for future research to be successful.
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    The precision control of autophagic flux and vesicle dynamics - a micropattern approach
    (MDPI, 2018) Du Toit, Andre; De Wet, Sholto; Hofmeyr, Jan-Hendrik S.; Muller-Nedebock, Kristian K.; Loos, Ben
    Autophagy failure is implicated in age-related human disease. A decrease in the rate of protein degradation through the entire autophagy pathway, i.e., autophagic flux, has been associated with the onset of cellular proteotoxity and cell death. Although the precision control of autophagy as a pharmacological intervention has received major attention, mammalian model systems that enable a dissection of the relationship between autophagic flux and pathway intermediate pool sizes remain largely underexplored. Here, we make use of a micropattern-based fluorescence life cell imaging approach, allowing a high degree of experimental control and cellular geometry constraints. By assessing two autophagy modulators in a system that achieves a similarly raised autophagic flux, we measure their impact on the pathway intermediate pool size, autophagosome velocity, and motion. Our results reveal a differential effect of autophagic flux enhancement on pathway intermediate pool sizes, velocities, and directionality of autophagosome motion, suggesting distinct control over autophagy function. These findings may be of importance for better understanding the fine-tuning autophagic activity and protein degradation proficiency in different cell and tissue types of age-associated pathologies
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    Spermidine and rapamycin reveal distinct autophagy flux response and cargo receptor clearance profile
    (MDPI, 2021-01-07) De Wet, Sholto; Du Toit, Andre; Loos, Ben
    Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.