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New tools for "hot-wiring" clathrin-mediated endocytosis with temporal and spatial precision.

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is difficult because the initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. A clathrin-binding protein fragment ("hook") is inducibly attached to an "anchor" protein at the plasma membrane, which triggers the formation of new clathrin-coated vesicles. Our design incorporates temporal and spatial control by the use of chemical and optogenetic methods for inducing hook-anchor attachment. Moreover, the cargo is defined. Because several steps in vesicle creation are bypassed, we term it "hot-wiring." We use hot-wired endocytosis to describe the functional interactions between clathrin and AP2. Two distinct sites on the β2 subunit, one on the hinge and the other on the appendage, are necessary and sufficient for functional clathrin engagement.

Pubmed ID: 28954824 RIS Download

Mesh terms: Adaptor Protein Complex 2 | Cell Line | Clathrin | Clathrin-Coated Vesicles | Coated Pits, Cell-Membrane | Endocytosis | Epithelial Cells | Gene Expression Regulation | Genes, Reporter | Green Fluorescent Proteins | HeLa Cells | Humans | Luminescent Proteins | Metabolic Engineering | Optogenetics | Protein Binding | Protein Subunits | Retinal Pigment Epithelium | Signal Transduction | Tacrolimus | Tacrolimus Binding Proteins

Addgene (Reagent, Plasmid)

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Addgene

A non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Addgene’s mission is to accelerate research and discovery by improving access to useful research materials and information. We facilitate the sharing of high-quality scientific materials, research reproducibility, and open science by archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Our repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.

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Fiji

Software package as distribution of ImageJ and ImageJ2 together with Java, Java3D and plugins organized into coherent menu structure. Used to assist research in life sciences.

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Adobe Illustrator

Vector graphics software to create digital graphics, illustrations, and typography for several types of media: print, web, interactive, video, and mobile.

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Adobe Photoshop

Software for image processing, analysis, and editing. The software includes features such as touch capabilities, a customizable toolbar, 2D and 3D image merging, and Cloud access and options.

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