Andre du Toit

Defining and measuring autophagosome flux—concept and reality

The autophagic system is involved in both bulk degradation of primarily long-lived cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and a number of methods are currently utilized to assess autophagic flux. However, despite major advances in measuring various molecular aspects of the autophagic machinery, we remain less able to express autophagic flux in a highly sensitive, robust, and well-quantifiable manner. Here, we describe a conceptual framework for defining and measuring autophagosome flux at the single-cell level. The concept discussed here is based on the theoretical framework of metabolic control analysis, which distinguishes between the pathway along which there is a flow of material and the quantitative measure of this flow. By treating the autophagic system as a multistep pathway with each step characterized by a particular rate, we are able to provide a single-cell fluorescence live-cell imaging-based approach that describes the accurate assessment of the complete autophagosome pool size, the autophagosome flux, and the transition time required to turn over the intracellular autophagosome pool. In doing so, this perspective provides clarity on whether the system is at steady state or in a transient state moving towards a new steady state. It is hoped that this theoretical account of quantitatively measuring autophagosome flux may contribute towards a new direction in the field of autophagy, a standardized approach that allows the establishment of systematic flux databases of clinically relevant cell and tissue types that serve as important model systems for human pathologies.

Puritans in Africa? Afrikaner “Calvinism” and Kuyperian Neo-Calvinism in Late Nineteenth-Century South Africa

Accounts of South African history and politics have been much influenced by what might be termed the Calvinist paradigm of Afrikaner history. As a model for the historical understanding of modern Afrikaner nationalism and of the ideology of apartheid it has proved persuasive to historians and social scientists alike. In outline, it amounts to the view that the “seventeenth-century Calvinism” which the Afrikaner founding fathers derived from their countries of origin became fixed in the isolated frontier conditions of trekboer society and survived for generations in the form of a kind of “primitive Calvinism”; that in the first part of the nineteenth century, this gave rise to a nascent chosen people ideology among early Afrikaners, which provided much of the motivation for, as well as the self-understanding of, that central event in Afrikaner history, the Great Trek, while simultaneously serving to legitimate the conquest and subordination of indigenous peoples; and that, mediated in this way, an authentic tradition of Afrikaner Calvinism thus constitutes the root source of modern Afrikaner nationalism and the ideology of apartheid. In fact, very little of this purported historical explanation will stand up to rigorous critical scrutiny: in vain will one look for hard evidence, either in the primary sources of early Afrikaner political thinking or in the contemporary secondary literature, of a set of popular beliefs that might be recognised as “primitive Calvinism” or as an ideology of a chosen people with a national mission.

Autophagic flux control in neurodegeneration: Progress and precision targeting—Where do we stand?

Graphical abstract Figure. No caption available. HighlightsNovel methods to assess autophagic flux and discern the dynamics of autophagy in different neuronal types and brain regions.Proposed methods to assess flux so as to discern between molecular defects associated with autophagic cargo and machinery.Understanding of the interplay between flux, autophagosome pool size and aggregate prone protein species.Region specific loss in autophagy proficiency with subsequent protein aggregation and neuronal cell death onset.Autophagy failure in neurodegeneration and autophagy flux control that is aligned with the nature of the molecular defect. Abstract Neurodegenerative diseases are characterised by the presence of cytoplasmic and nuclear protein aggregates that result in toxicity and neuronal cell death. Autophagy is a physiological cellular process that engulfs primarily long‐lived proteins as well as protein aggregates with subsequent cargo delivery for lysosomal degradation. The rate at which the material is degraded through autophagy is referred to as autophagic flux. Although we have progressed substantially in unravelling the role and regulation of the autophagy machinery, its dysfunction in pathology as well as its dynamic changes in the disease progression remains largely unclear. Furthermore, the magnitude of autophagic flux in neuronal subtypes is largely unknown and it is unclear to what extent the flux may be affected in distinct neurodegenerative disease states. In this review, we provide an introduction to autophagy in neuronal homeostasis and indicate how autophagy is currently measured and modulated for therapeutic purposes. We highlight the need not only to develop enhanced methodologies that target and assess autophagic flux precisely, but also to discern the dynamics of autophagy in different neuronal types and brain regions associated with the disease‐specific pathology. Finally, we describe how existing and novel techniques for assessing autophagic flux could be implemented in order to distinguish between molecular defects associated with autophagic cargo and the machinery. In doing so, this review may provide novel insights in the assessment and control of autophagic flux that is aligned with the protein clearance dysfunction in neurodegenerative disorders.

New Insights Into Autophagy Dysfunction Related to Amyloid Beta Toxicity and Neuropathology in Alzheimer's Disease

The fine control of neuronal proteostasis is an essential element that preserves cell viability. Advancing age is a major risk factor for Alzheimers disease (AD), and autophagy is thought to dictate normal and pathological aging through intricate molecular machinery controlling protein aggregation. Although the role of autophagy dysfunction in AD is known, the dynamic changes during the progression of the disease remain unclear. Recent studies have provided new insight into the molecular mechanisms that link defective autophagy and cellular fate, underscoring the pathogenic events associated with AD. Here, we will focus on recent studies that underpin a distinct role for autophagy deficits and highly localized autophagic defects, impacting primarily the amyloidogenic pathway activity. By uniquely assessing the dynamic changes in key proteins during the disease progression in the context of the autophagy machinery function and amyloid beta toxicity, specifically, a connect between protein degradation failure and cell death susceptibility is revealed which may suggest new avenues for the development of better targeted therapeutic interventions.

The Precision Control of Autophagic Flux and Vesicle Dynamics—A Micropattern Approach

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.

Measuring Autophagosome Flux

ABSTRACT Macroautophagy/autophagy is a proteolytic pathway that is involved in both bulk degradation of cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and many techniques exist to assess autophagic flux. Although these techniques have generated invaluable information about the autophagic system, the quest continues for developing methods that not only enhance sensitivity and provide a means of quantification, but also accurately reflect the dynamic character of the pathway. Based on the theoretical framework of metabolic control analysis, where the autophagosome flux is the quantitative description of the rate a flow along a pathway, here we treat the autophagy system as a multi-step pathway. We describe a single-cell fluorescence live-cell imaging-based approach that allows the autophagosome flux to be accurately measured. This method characterizes autophagy in terms of its complete autophagosome and autolysosome pool size, the autophagosome flux, J, and the transition time, τ, for autophagosomes and autolysosomes at steady state. This approach provides a sensitive quantitative method to measure autophagosome flux, pool sizes and transition time in cells and tissues of clinical relevance. Abbreviations: ATG5/APG5, autophagy-related 5; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; J, flux; MEF, mouse embryonic fibroblast; MTOR, mechanistic target of rapamycin kinase; nA, number of autophagosomes; nAL, number of autolysosomes; nL, number of lysosomes; p-MTOR, phosphorylated mechanistic target of rapamycin kinase; RFP, red fluorescent protein; siRNA, small interfering RNA; τ, transition time; TEM, transmission electron microscopy.

Methods for Measuring Autophagosome Flux—Impact and Relevance

Abstract Autophagy is an essential protein degradative pathway that maintains cellular and metabolic homeostasis. Autophagy dysfunction is associated with many human pathologies and impacts directly on the susceptibility of the cell to undergo cell death. Although we have learnt a great deal about the molecular machinery that governs the autophagic process, an accurate capturing of especially the dynamic rearrangement of membranes and degradation of cargo that form part of the autophagy machinery remains challenging, and consequently the translation of autophagy control to the clinical environment has remained poor. In this chapter we highlight the recent advances in the fields of measuring and modeling autophagic flux and the entire autophagy system. We emphasize the dynamic nature of the autophagy system itself, the connection between autophagy and the metabolic status of the cell, and we suggest an assessment approach that allows the integration of cargo and machinery fluxes. Finally, by discussing control analysis and a systems approach as uniquely positioned tools, we underscore the current knowledge base of the autophagy system and its models as well as key-associated metabolic parameters and molecular checkpoints that intricately link to cellular fate. In doing so, we hope to bring about clarity on required measuring approaches, to better assess and interpret autophagic flux dysfunction and systems failure, yielding future advances in precisely controlling and manipulating the autophagic process for therapeutic purposes.

On South Africa's options

South Africas Options: Strategies for Sharing Power by F. van Zyl Slabbert and David Welsh. Cape Town: David Philip, 1979