The endocytic pathway is a system of dynamically communicating vesicles, known as early endosomes, that internalize, sort, and traffic nutrients, trophic factors, and signaling molecules to sites throughout the cell. In all eukaryotic cells, early endosome functions are regulated by Rab5 activity, dependent upon its binding to GTP, whereas Rab5 bound to GDP represents the biologically inactive form. An increasing number of neurodegenerative diseases are associated with endocytic dysfunction and, in the case of Alzheimer's disease (AD) and Down syndrome (DS), an early appearing highly characteristic reflection of endocytic pathway dysfunction is an abnormal enlargement of Rab5 positive endosomes. In AD and DS, endosome enlargement accompanying accelerated endocytosis and fusion, upregulated transcription of endocytosis-related genes, and aberrant signaling by endosomes are caused by pathological Rab5 overactivation. In this chapter, we describe a battery of methods that have been used to assess Rab5 activation in models of AD/DS and are applicable to other cell and animal disease models. These methods include (1) fluorescence recovery after photobleaching (FRAP) assay; (2) quantitative measurement of endosome size by light, fluorescence and electron microscopy; (3) detection of GTP-Rab5 by in situ immunocytochemistry in vitro and ex vivo; (4) immunoprecipitation and GTP-agarose pull-down assay; (5) biochemical detection of Rab5 in endosome-enriched subcellular fractions obtained by OptiPrep™ density gradient centrifugation of mouse brain.
© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Phagocytosis is a complex cellular uptake process involving multiple distinct steps of cargo recognition, uptake, phagosome maturation and eventual phagolysosome resolution. Emerging literature shows that heterogeneity of phagocytosis at multiple steps at a single cell level influences the population outcome. However, the determinants of phagocytic heterogeneity are not clear. Here we show that the variance in the endocytic capacity of individual cells in a macrophage population determines subsequent phagocytic uptake and trafficking. Our results document the extensive heterogeneity in the endocytic uptake of individual macrophages, and show that cells with higher endocytic capacity preferentially phagocytose diverse cargo, including pathogenic Mycobacterium tuberculosis. Interestingly, M. tuberculosis infected cells sustain the higher endocytic capacity following infection. Modulating endocytic capacity by inhibiting endocytosis reduces phagocytic uptake. Differential uptake of M. tuberculosis into cells with different endocytic capacities correlates with the efficiency of phagocytic delivery to lysosomes, thus contributing further to phagocytic as well as mycobacterial heterogeneity. Thus, variance in endocytic capacity is a determinant of generating heterogeneity in phagocytosis at multiple steps.
© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multiple mitochondrial quality control pathways exist to maintain the health of mitochondria and ensure cell homeostasis. Here, we investigate the role of the endosomal adaptor Tollip during the mitochondrial stress response and identify its interaction and colocalisation with the Parkinson's disease-associated E3 ubiquitin ligase Parkin. The interaction between Tollip and Parkin is dependent on the ubiquitin-binding CUE domain of Tollip, but independent of Tom1 and mitophagy. Interestingly, this interaction is independent of Parkin mitochondrial recruitment and ligase activity but requires an intact ubiquitin-like (UBL) domain. Importantly, Tollip regulates Parkin-dependent endosomal trafficking of a discrete subset of mitochondrial-derived vesicles (MDVs) to facilitate delivery to lysosomes. Retromer function and an interaction with Tom1 allow Tollip to facilitate late endosome/lysosome trafficking in response to mitochondrial stress. We find that upregulation of TOM20-positive MDVs upon mitochondrial stress requires Tollip interaction with ubiquitin, endosomal membranes and Tom1 to ensure their trafficking to the lysosomes. Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial-derived cargo to the lysosomes.
© 2020 The Authors. Published under the terms of the CC BY 4.0 license.

The most common disease-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, F508del, leads to cystic fibrosis (CF), by arresting CFTR processing and trafficking to the plasma membrane. The FDA-approved modulators partially restore CFTR function and slow down the progression of CF lung disease by increasing processing and delivery to the plasma membrane and improving activity of F508del-CFTR Cl- channels. However, the modulators do not correct compromised membrane stability of rescued F508del-CFTR. Transforming growth factor (TGF)-β1 is a well-established gene modifier of CF associated with worse lung disease in F508del-homozygous patients, by inhibiting CFTR biogenesis and blocking the functional rescue of F508del-CFTR. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein localized at the apical and basolateral membrane domain of human bronchial epithelial cells. Phosphorylation of the apical membrane CFTR by LMTK2 triggers its endocytosis and reduces the abundance of membrane-associated CFTR, impairing the CFTR-mediated Cl- transport. We have previously shown that LMTK2 knockdown improves the pharmacologically rescued F508del-CFTR abundance and function. Thus, reducing the LMTK2 recruitment to the plasma membrane may provide a useful strategy to potentiate the pharmacological rescue of F508del-CFTR. Here, we elucidate the mechanism of LMTK2 recruitment to the apical plasma membrane in polarized CFBE41o- cells. TGF-β1 increased LMTK2 abundance selectively at the apical membrane by accelerating its recycling in Rab11-positive vesicles without affecting LMTK2 mRNA levels, protein biosynthesis, or endocytosis. Our data suggest that controlling TGF-β1 signaling may attenuate recruitment of LMTK2 to the apical membrane thereby improving stability of pharmacologically rescued F508del-CFTR.
Copyright © 2020 Cruz, Mitash, Farinha and Swiatecka-Urban.

The insulin family of growth factors plays an important role in development and function of the nervous system. Reduced insulin and insulin-growth-factor signaling (IIS), however, can improve symptoms of neurodegenerative diseases in laboratory model organisms and protect against age-associated decline in neuronal function. Recently, we showed that chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber escape response circuit. Here, we expand our initial findings by demonstrating that reduced functional output in the giant fiber system of aging flies can be prevented by increasing proteasomal activity within the circuit. Manipulations of IIS in neurons can also affect longevity, underscoring the relevance of the nervous system for aging.
Copyright © 2018 Elsevier Inc. All rights reserved.