Newly endocytosed integral cell surface proteins are typically either directed for degradation or subjected to recycling back to the plasma membrane. The sorting of integral cell surface proteins, ...including signalling receptors, nutrient transporters, ion channels, adhesion molecules and polarity markers, within the endolysosomal network for recycling is increasingly recognized as an essential feature in regulating the complexities of physiology at the cell, tissue and organism levels. Historically, endocytic recycling has been regarded as a relatively passive process, where the majority of internalized integral proteins are recycled via a nonspecific sequence-independent 'bulk membrane flow' pathway. Recent work has increasingly challenged this view. The discovery of sequence-specific sorting motifs and the identification of cargo adaptors and associated coat complexes have begun to uncover the highly orchestrated nature of endosomal cargo recycling, thereby providing new insight into the function and (patho)physiology of this process.
Recently identified Parkinson’s disease (PD) genes involved in synaptic vesicle endocytosis, such as DNAJC6 (auxilin), have further implicated synaptic dysfunction in PD pathogenesis. However, how ...synaptic dysfunction contributes to the vulnerability of human dopaminergic neurons has not been previously explored. Here, we demonstrate that commonly mutated, PD-linked leucine-rich repeat kinase 2 (LRRK2) mediates the phosphorylation of auxilin in its clathrin-binding domain at Ser627. Kinase activity-dependent LRRK2 phosphorylation of auxilin led to differential clathrin binding, resulting in disrupted synaptic vesicle endocytosis and decreased synaptic vesicle density in LRRK2 patient-derived dopaminergic neurons. Moreover, impaired synaptic vesicle endocytosis contributed to the accumulation of oxidized dopamine that in turn mediated pathogenic effects such as decreased glucocerebrosidase activity and increased α-synuclein in mutant LRRK2 neurons. Importantly, these pathogenic phenotypes were partially attenuated by restoring auxilin function in mutant LRRK2 dopaminergic neurons. Together, this work suggests that mutant LRRK2 disrupts synaptic vesicle endocytosis, leading to altered dopamine metabolism and dopamine-mediated toxic effects in patient-derived dopaminergic neurons.
Commensal bacteria influence host physiology, without invading host tissues. We show that proteins from segmented filamentous bacteria (SFB) are transferred into intestinal epithelial cells (IECs) ...through adhesion-directed endocytosis that is distinct from the clathrin-dependent endocytosis of invasive pathogens. This process transfers microbial cell wall-associated proteins, including an antigen that stimulates mucosal T helper 17 (T
17) cell differentiation, into the cytosol of IECs in a cell division control protein 42 homolog (CDC42)-dependent manner. Removal of CDC42 activity in vivo led to disruption of endocytosis induced by SFB and decreased epithelial antigen acquisition, with consequent loss of mucosal T
17 cells. Our findings demonstrate direct communication between a resident gut microbe and the host and show that under physiological conditions, IECs acquire antigens from commensal bacteria for generation of T cell responses to the resident microbiota.
Control of membrane traffic: Photoswitchable inhibitors of protein–protein interactions were applied to photoregulate clathrin‐mediated endocytosis (CME) in living cells. Traffic light (TL) peptides ...acting as “stop” and “go” signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation.
The blood–brain barrier (BBB) is an evolutionarily conserved, structural, and functional separation between circulating blood and the central nervous system (CNS). By controlling permeability into ...and out of the nervous system, the BBB has a critical role in the precise regulation of neural processes. Here, we review recent studies demonstrating that permeability at the BBB is dynamically controlled by circadian rhythms and sleep. An endogenous circadian rhythm in the BBB controls transporter function, regulating permeability across the BBB. In addition, sleep promotes the clearance of metabolites along the BBB, as well as endocytosis across the BBB. Finally, we highlight the implications of this regulation for diseases, including epilepsy.
Recent evidence from fly models reveals an endogenous circadian rhythm at the BBB. This rhythm controls function of the permeability-glycoprotein multidrug transporter, which actively pumps both endogenous and exogenous molecules out of the CNS. Function of this transporter is decreased at night, increasing permeability to multiple substrates into the brain overnight.Endocytosis is a newly appreciated important function for sleep. Endocytosis across the BBB is increased during sleep and, when inhibited, increases the need for sleep.Sleep also promotes the clearance of metabolites out of the brain along paravascular spaces. During sleep, the interstitial spaces of the brain are larger, allowing for more robust movement of metabolites from interstitial spaces into the CSF. These solutes can then travel along vessels and out of the brain.
Lung cancer is the leading cause of cancer-related deaths worldwide. As an emerging technology, nanomedicine offers a more effective and biocompatible approach to treating lung cancer by targeting ...and killing uncontrolled cancer cells. This study fabricated a nanocarrier system comprising gold nanoparticles, type I collagen, and alkaline berberine (Au-Col-BB). It then thoroughly evaluated the physical and biological properties of this novel drug delivery system. The material properties were characterized using UV-Vis spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, dynamic light scattering, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. MTT and Calcein AM staining of A549 lung cancer cells demonstrated that treatment with Au-Col-BB significantly decreased cell viability. Annexin V/propidium iodide double staining and cell cycle progression revealed that treatment with Au-Col-BB increased apoptosis 1.82-fold in A549 cells but decreased apoptosis 0.94-cold in BEAS-2B normal lung cells. The percentage of A549 cells in the sub-G1 phase increased from 4.0 % to 11.6 % (p < 0.001), while the percentage of those in the S phase decreased from 14.5 % to 9.6 % (p < 0.010), indicating enhanced apoptosis and reduced proliferation. Treatment with Au-Col-BB significantly reduced the migration distance of A549 cells by 51 %. Au-Col-BB was found to primarily enter cells via clathrin-mediated endocytosis and autophagy, which were inhibited by chlorpromazine and bafilomycin A1, respectively. Retroorbital sinus injection of Au-Col-BB into BALB/c mice demonstrated its integrity and safety in vivo. Overall, this study suggests that the Au-Col-BB nanocarrier system is a promising alternative for antineoplastic drugs, offering effective cancer cell targeting and apoptosis induction while minimizing damage to surrounding healthy tissues.
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Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root ...cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in m subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.
Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-β peptide (Aβ) is associated with AD. However, ...identifying new compounds that antagonize the underlying cellular pathologies caused by Aβ has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Aβ toxicity where the Aβ peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aβ 1—42 peptide forms more oligomers and is more toxic than Aβ 1—40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of ∼140,000 compounds for rescue of Aβ toxicity. Of ∼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aβ burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aβ secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aβ peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Aβ oligomer formation contributes to Aβ toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aβ yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.
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