Synaptic vesicle recycling is essential for maintaining normal synaptic function. The coupling of exocytosis and endocytosis is assumed to be Ca2+ dependent, but the exact role of Ca2+ and its key ...effector synaptotagmin-1 (syt1) in regulation of endocytosis is poorly understood. Here, we probed the role of syt1 in single- as well as multi-vesicle endocytic events using high-resolution optical recordings. Our experiments showed that the slowed endocytosis phenotype previously reported after syt1 loss of function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss of function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles toward slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin-7. In contrast, after single synaptic vesicle fusion, syt1 acted as an essential determinant of synaptic vesicle endocytosis time course by delaying the kinetics of vesicle retrieval in response to increasing Ca2+ levels.
•Loss of syt1, complexins, or Sr2+ substitution slows endocytosis after activity•Asynchronously released synaptic vesicles are retrieved via slow endocytosis•Link between asynchronous release and slowed endocytosis requires synaptotagmin-7•Whereas, after synchronous single synaptic vesicle fusion, syt1 delays endocytosis
In this study, Li and colleagues demonstrate that synaptic vesicle fusion machinery, comprised of synaptotagmin-1, complexins, and synaptotagmin-7, in addition to determining the timing and Ca2+ dependence of neurotransmitter release, also dictates the properties of subsequent synaptic vesicle endocytosis.
Integration of multiple agent therapy (MAT) into one probe is promising for improving therapeutic efficiency for cancer treatment. However, MAT probe, if entering the cell as a whole, may not be ...optimal for each therapeutic agent (with different physicochemical properties), to achieve their best performance, hindering strategy optimization. A peptide‐conjugated‐AIEgen (FC‐PyTPA) is presented: upon loading with siRNA, it self‐assembles into FCsiRNA‐PyTPA. When approaching the region near tumor cells, FCsiRNA‐PyTPA responds to extracellular MMP‐2 and is cleaved into FCsiRNA and PyTPA. The former enters cells mainly by macropinocytosis and the latter is internalized into cells mainly through caveolae‐mediated endocytosis. This two‐part strategy greatly improves the internalization efficiency of each individual therapeutic agent. Inside the cell, self‐assembly of nanofiber precursor F, gene interference of CsiRNA, and ROS production of PyTPA are activated to inhibit tumor growth.
A multiple‐agent‐therapy probe based on a peptide‐conjugated‐AIEgen was developed with extracellular MMP‐2 and intracellular CB response. When approaching tumor cells, it can be separated into different agents, which then enter the tumor cells by their dominant pathway with high cellular internalization efficiency and effective multimodal cancer therapy.
Clathrin-independent endocytosis (CIE) mediates the cellular uptake of many extracellular ligands, receptors, and pathogens, including several life-threatening bacterial toxins and viruses. So far, ...our understanding of CIE carrier formation has lagged behind that of clathrin-coated vesicles. Impediments have been the imprecise definition of some CIE pathways, the lack of specific cargoes being transported and of exclusive cytosolic markers and regulators. Notwithstanding these limitations, three distinct molecular mechanisms by which CIE carriers form can be defined. Cargo capture by cytosolic proteins is the main mechanism used by fast endophilin-mediated endocytosis (FEME) and interleukin 2 receptor (IL-2R) endocytosis. Acute signaling-induced membrane remodeling drives macropinocytosis. Finally, extracellular lipid or cargo clustering by the glycolipid-lectin (GL-Lect) hypothesis mediates the uptake of Shiga and cholera toxins and receptors by the CLIC/GEEC pathway. Here, we review these mechanisms and highlight current gaps in knowledge that will need to be addressed to complete our understanding of CIE.
CIE is involved in several key cellular processes by mediating the uptake of many receptors and pathogens, although few cargoes use a single pathway exclusively.
CIE pathways remain poorly defined molecularly and their nomenclature is imprecise, with various routes named after carrier morphologies, cargoes, cytosolic markers, or broad regulators.
Some proposed CIE pathways have been reconsidered and their functions in endocytosis have been revised.
CIE carriers form upon cargo capture by cytosolic proteins, acute signaling-induced membrane remodeling, or extracellular lipid or cargo clustering.
Here we show that inhibition of clathrin‐mediated endocytosis does not inhibit clathrin‐independent endocytosis but does alter the endosomal trafficking of cargo proteins that enter cells this way. ...The altered trafficking can be rescued by over‐expression of Rab35 or induced by depletion of Rab35 suggesting that Rab35 activity may serve as an indicator of normal input from clathrin‐mediated endocytosis.
Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.
Emergence of SARS-CoV-2 variants warrants sustainable efforts to upgrade both the diagnostic and therapeutic protocols. Understanding the details of cellular and molecular basis of the virus-host ...cell interaction is essential for developing variant-independent therapeutic options. The internalization of SARS-CoV-2, into lung epithelial cells, is mediated by endocytosis, especially clathrin-mediated endocytosis (CME). Although vaccination is the gold standard strategy against viral infection, selective inhibition of endocytic proteins, complexes, and associated adaptor proteins may present a variant-independent therapeutic strategy. Although clathrin and/or dynamins are the most important proteins involved in CME, other endocytic mechanisms are clathrin and/or dynamin independent and rely on other proteins. Moreover, endocytosis implicates some subcellular structures, like plasma membrane, actin and lysosomes. Also, physiological conditions, such as pH and ion concentrations, represent an additional factor that mediates these events. Accordingly, endocytosis related proteins are potential targets for small molecules that inhibit endocytosis-mediated viral entry. This review summarizes the potential of using small molecules, targeting key proteins, participating in clathrin-dependent and -independent endocytosis, as variant-independent antiviral drugs against SARS-CoV-2 infection. The review takes two approaches. The first outlines the potential role of endocytic inhibitors in preventing endocytosis-mediated viral entry and its mechanism of action, whereas in the second computational analysis was implemented to investigate the selectivity of common inhibitors against endocytic proteins in SARS-CoV-2 endocytosis. The analysis revealed that remdesivir, methyl-β-cyclodextrin, rottlerin, and Bis-T can effectively inhibit clathrin, HMG-CoA reductase, actin, and dynamin I GTPase and are more potent in inhibiting SARS-CoV-2 than chloroquine. CME inhibitors for SARS-CoV-2 infection remain understudied.
β-arrestin1 (or arrestin2) and β-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein-coupled receptor ...(GPCR) signaling through heterotrimeric G proteins. However, further biochemical characterization revealed that β-arrestins do not just "block" the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by β-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted β-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. In addition, it is becoming increasingly evident that β-arrestin-dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by β-arrestins and highlight the physiologic effects of β-arrestin-dependent signaling.
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