Integrating molecular and network biology to decode endocytosis

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Nature (Impact Factor: 41.46). 09/2007; 448(7156):883-8. DOI: 10.1038/nature06031
Source: PubMed


The strength of network biology lies in its ability to derive cell biological information without a priori mechanistic or molecular knowledge. It is shown here how a careful understanding of a given biological pathway can refine an interactome approach. This permits the elucidation of additional design principles and of spatio-temporal dynamics behind pathways, and aids in experimental design and interpretation.

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    • "We began our analyses with the assumption, based on various reports, that the AP2 adaptor is an integral component of the CME machinery (Boucrot et al., 2010; Cocucci et al., 2012) and assumed that the majority of authentic CME sites contain both AP2 and clathrin. AP2 is an endocytic hub protein whose appendage domains bind numerous endocytic accessory proteins and cargo molecules (Schmid and McMahon, 2007). "
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    ABSTRACT: Cells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes >∼20 s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.
    Cell Reports 09/2015; 12(12). DOI:10.1016/j.celrep.2015.08.048 · 8.36 Impact Factor
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    • "CME is a multi-step process involving the formation, stabilization and maturation of clathrin-coated pits (CCPs), leading to the incorporation of surface receptors and their ligands into clathrin-coated vesicles (CCVs). During CCP maturation, the CME core machinery consisting of clathrin, dynamin and AP2, interacts with a myriad of endocytic accessory proteins (EAPs), many of which are recruited to CCPs via binding to the appendage domain (AD) of the α-adaptin subunit of AP2 (Schmid and McMahon, 2007). Following maturation, membrane scission is catalyzed by the GTPase dynamin, leading to the formation of cargo-containing vesicles. "
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    ABSTRACT: Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this "checkpoint." Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3β signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME. © 2015 The Authors.
    The EMBO Journal 07/2015; 34(16). DOI:10.15252/embj.201591518 · 10.43 Impact Factor
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    • "Clathrin-mediated endocytosis (CME) is an active cellular process for membrane trafficking, which mediates the entry of nutrients and growth factor receptors into the cells [1]. When the target molecule binds to plasma membrane receptor, clathrin and accessory molecules such as AP2, epsin, clathrin assembly lymphoid myeloid leukemia protein (CALM) are recruited from cytoplasm into the membrane, resulting in the formation of clathrin-coated vesicles. "
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    ABSTRACT: CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage-Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM-/- mice. Also, colony forming activity was impaired in CALM-/- LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM-/- LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM-/- murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM-/- MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM-/- MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM-/- MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM-/- MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM-/- KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.
    PLoS ONE 10/2014; 9(10):e109441. DOI:10.1371/journal.pone.0109441 · 3.23 Impact Factor
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