α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can ...be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.
Although several reports suggest that the entry of infectious bronchitis virus (IBV) depends on lipid rafts and low pH, the endocytic route and intracellular trafficking are unclear. In this study, ...we aimed to shed greater light on early steps in IBV infection. By using chemical inhibitors, RNA interference, and dominant negative mutants, we observed that lipid rafts and low pH was indeed required for virus entry; IBV mainly utilized the clathrin mediated endocytosis (CME) for entry; GTPase dynamin 1 was involved in virus containing vesicle scission; and the penetration of IBV into cells led to active cytoskeleton rearrangement. By using R18 labeled virus, we found that virus particles moved along with the classical endosome/lysosome track. Functional inactivation of Rab5 and Rab7 significantly inhibited IBV infection. Finally, by using dual R18/DiOC labeled IBV, we observed that membrane fusion was induced after 1 h.p.i. in late endosome/lysosome.
•Intact lipid rafts is involved in IBV entry.•Low pH in intracyplasmic vesicles is required for IBV entry.•IBV penetrates cells via clathrin mediated endocytosis.•IBV moves along with the classical endosome/lysosome track, finally fuses with late endosome/lysosome.
PI3Ks are a family of lipid kinases that phosphorylate intracellular inositol lipids to regulate signalling and intracellular vesicular traffic. Mammals have eight isoforms of PI3K, divided into ...three classes. The class I PI3Ks generate 3-phosphoinositide lipids, which directly activate signal transduction pathways. In addition to being frequently genetically activated in cancer, similar mutations in class I PI3Ks have now also been found in a human non-malignant overgrowth syndrome and a primary immune disorder that predisposes to lymphoma. The class II and class III PI3Ks are regulators of membrane traffic along the endocytic route, in endosomal recycling and autophagy, with an often indirect effect on cell signalling. Here, we summarize current knowledge of the different PI3K classes and isoforms, focusing on recently uncovered biological functions and the mechanisms by which these kinases are activated. Deeper insight into the PI3K isoforms will undoubtedly continue to contribute to a better understanding of fundamental cell biological processes and, ultimately, of human disease.
Candida albicans interactions with epithelial cells are critical for commensal growth, fungal pathogenicity and host defence. This review will outline our current understanding of
C. ...albicans–epithelial interactions and will discuss how this may lead to the induction of a protective mucosal immune response.
The emergence of SARS-CoV-2 variants of concern with progressively increased transmissibility between humans is a threat to global public health. The Omicron variant of SARS-CoV-2 also evades ...immunity from natural infection or vaccines
, but it is unclear whether its exceptional transmissibility is due to immune evasion or intrinsic virological properties. Here we compared the replication competence and cellular tropism of the wild-type virus and the D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) variants in ex vivo explant cultures of human bronchi and lungs. We also evaluated the dependence on TMPRSS2 and cathepsins for infection. We show that Omicron replicates faster than all other SARS-CoV-2 variants studied in the bronchi but less efficiently in the lung parenchyma. All variants of concern have similar cellular tropism compared to the wild type. Omicron is more dependent on cathepsins than the other variants of concern tested, suggesting that the Omicron variant enters cells through a different route compared with the other variants. The lower replication competence of Omicron in the human lungs may explain the reduced severity of Omicron that is now being reported in epidemiological studies, although determinants of severity are multifactorial. These findings provide important biological correlates to previous epidemiological observations.
How the cell converts graded signals into threshold‐activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor ...(EGFR)‐activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose–response curve of EGFR ubiquitination correlate precisely with the non‐clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR‐NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re‐engineered on a phosphorylation/ubiquitination‐incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.
The amount of EGF present for binding to its receptor governs an on–off switch of EGFR ubiquitination and hence ligand‐controlled non‐clathrin‐mediated endocytosis and EGFR degradation.
DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset ...neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.
We used the nematode Caenorhabditis elegans to study the roles of endocytosis and lysosomal function in uptake and subsequent toxicity of silver nanoparticles (AgNP) in vivo. To focus on AgNP uptake ...and effects rather than silver ion (AgNO
3
) effects, we used a minimally dissolvable AgNP, citrate-coated AgNPs (CIT-AgNPs). We found that the clathrin-mediated endocytosis inhibitor chlorpromazine reduced the toxicity of CIT-AgNPs but not AgNO
3
. We also tested the sensitivity of three endocytosis-deficient mutants (rme-1, rme-6 and rme-8) and two lysosomal function deficient mutants (cup-5 and glo-1) as compared to wild-type (N2 strain). One of the endocytosis-deficient mutants (rme-6) took up less silver and was resistant to the acute toxicity of CIT-AgNPs compared to N2s. None of those mutants showed altered sensitivity to AgNO
3
. Lysosome and lysosome-related organelle mutants were more sensitive to the growth-inhibiting effects of both CIT-AgNPs and AgNO
3
. Our study provides mechanistic evidence suggesting that early endosome formation is necessary for AgNP-induced toxicity in vivo, as rme-6 mutants were less sensitive to the toxic effects of AgNPs than C. elegans with mutations involved in later steps in the endocytic process.
Growing evidence demonstrated that bioaccumulation of polystyrene nanoplastics (PS-NPs) in various organisms including human beings caused destructive effects on health. Nanoplastics may adversely ...affect fetal development potentially since they can pass through the placental barrier. However, very little has been known about the embryonic toxicity of polystyrene nanoplastics, especially in embryonic neurulation, the early developmental stage of the fetus, as well as the corresponding mechanisms. In this study, we first observed that 60- or 900-nm PS-NPs (especially 60-nm PS-NPs) could cross mouse placentas and affect developing mice fetuses. To avoid the indirect adverse effects derived from the restricted placenta, we employed early chick embryos as a developmental model to evaluate direct adverse effects of PS-NPs on embryo/fetal development, revealing suppressive effects on embryo development and an increased frequency of congenital abnormalities (especially in the nervous system), including neural tube defects. Thus, we focused on the potential negative effects of PS-NPs on neurulation, the earliest stage of nervous system development. Using caveolin-1 immunofluorescent staining of SH-SY5Y cells exposed to PS-NPs-GFP, we demonstrated that PS-NPs were internalized by SH-SY5Y cells via caveolae-mediated endocytosis. Transmission electron microscopy; LC3B immunofluorescent staining; and Atg7, Atg5, p62 and LC3B western blot results revealed that autophagy was activated in SH-SY5Y cells exposed to PS-NPs. However, PS-NPs were not degraded by the autophagic-lysosomal system given the lack of LAMP1 changes and minimal PS-NPs-GFP and LAMP1 colocalization. Furthermore, the cytoplasmic accumulation of PS-NPs caused faulty apoptotic cell death in SH-SY5Y cells and the developing neural tube as revealed by c-caspase3 immunofluorescent staining. Thus, defective neural tube morphogenesis, as demonstrated by neural tube defects, occurred during embryogenesis in the context of PS-NP exposure.
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