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.

  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Sep 2015 · Cell Reports
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jul 2015 · The EMBO Journal
  • Source
    • "This analysis revealed changes in the expression of several CME pathway constituents (Appendix: Supplementary Table S3). The CME pathway is composed by two main hubs the CLTC and the AP2 (Schmid and McMahon, 2007). The AP2-β subunit was revealed as decreased in VaD (AP2B1, 0.59; p < 1.0·10 −3 ) while the AP2-α subunit did not show any difference in expression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Progressive synaptic failure precedes the loss of neurons and decline in cognitive function in neurodegenerative disorders, but the specific proteins and posttranslational modifications that promote synaptic failure in vascular dementia (VaD) remain largely unknown. We therefore used an isobaric tag for relative and absolute proteomic quantitation (iTRAQ) to profile the synapse-associated proteome of post-mortem human cortex from vascular dementia patients and age-matched controls. Brain tissue from VaD patients exhibited significant down-regulation of critical synaptic proteins including clathrin (0.29; p < 1.0⋅10(-3)) and GDI1 (0.51; p = 3.0⋅10(-3)), whereas SNAP25 (1.6; p = 5.5⋅10(-3)), bassoon (1.4; p = 1.3⋅10(-3)), excitatory amino acid transporter 2 (2.6; p = 9.2⋅10(-3)) and Ca(2+)/calmodulin dependent kinase II (1.6; p = 3.0⋅10(-2)) were substantially up-regulated. Our analyses further revealed divergent patterns of protein modification in the dementia patient samples, including a specific deamidation of synapsin1 predicted to compromise protein structure. Our results reveal potential molecular targets for intervention in synaptic failure and prevention of cognitive decline in VaD. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Full-text · Article · Dec 2014 · Neurochemistry International
Show more