Journal: Molecular Biology of the Cell

Article Title: Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking

doi: 10.1091/mbc.E15-04-0245

Figure Lengend Snippet: Phenotype of epsinR knocksideways. (A) Schematic diagram of FKBP-tagged epsinR. (B) Western blots of epsinR-FKBP–expressing cells. The cells were treated with siRNA to deplete endogenous epsinR, and the blots were probed with anti-epsinR, using anti-clathrin as a loading control. The epsinR-FKBP construct is expressed at a similar level to endogenous epsinR and is siRNA resistant. (C) Addition of rapamycin (rapa) to the epsinR-FKBP–expressing cells causes the construct to accumulate in a mitochondrial fraction (isolated using magnetic beads) and become depleted from our CCV fraction. (D) Relocation to mitochondria demonstrated by immunofluorescence. A mixed population of epsinR-FKBP–expressing cells, from which endogenous epsinR had been depleted using siRNA, and wild-type cells (asterisks) were treated with rapamycin and labeled for total epsinR. Scale bar, 20 μm. (E) Electron micrographs of epsinR-FKBP–expressing cells, depleted of endogenous epsinR, either with or without rapamycin treatment. In the rapamycin-treated cells, enlarged clathrin-coated structures can be seen in the Golgi region (arrows). (F) The diameters of clathrin-coated budding profiles, associated with either the plasma membrane or the Golgi region, were measured in electron micrographs of control and rapamycin-treated cells. Data were collected from two separate experiments, analyzing at least five separate cells for each experiment. At least 20 profiles were measured for each condition. Rapamycin treatment causes an increase in the diameter of clathrin-coated structures in the Golgi region from 85.3 ± 27.6 to 108.6 ± 29.1 nm (SD; p = 0.0059 for the Golgi region; p = 0.2246 for the plasma membrane; ns, not significant).

Article Snippet: The mCherry-tagged clathrin light-chain plasmid was obtained from Addgene. pLXIN(epsinR-FKBP) and pLXIN(gadkin-FKBP) were constructed synthetically, as this substantially reduced the manipulations.

Techniques: Western Blot, Expressing, Construct, Isolation, Magnetic Beads, Immunofluorescence, Labeling

Journal: Molecular Biology of the Cell

Article Title: Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking

doi: 10.1091/mbc.E15-04-0245

Figure Lengend Snippet: Estimated copy number for all major CCV proteins.

Article Snippet: The mCherry-tagged clathrin light-chain plasmid was obtained from Addgene. pLXIN(epsinR-FKBP) and pLXIN(gadkin-FKBP) were constructed synthetically, as this substantially reduced the manipulations.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking

doi: 10.1091/mbc.E15-04-0245

Figure Lengend Snippet: Expression of a gadkin-FKBP construct. (A) Schematic diagram of FKBP-tagged gadkin. (B) Expression and fractionation of gadkin-FKBP. The construct is highly overexpressed and enriched in CCVs. Unlike clathrin (CHC) and AP-1, it cannot be detected in a high-speed supernatant, indicating that it has no cytosolic pool but is entirely membrane associated. (C) Homogenates and CCV fractions from control and gadkin-FKBP–expressing cells in the absence of rapamycin. Gadkin, clathrin, and AP-1 are all enriched similarly in the CCV fraction, whether or not the cells are expressing gadkin-FKBP. (D) Cells expressing gadkin-FKBP and Mitotrap were mixed with nonexpressing HeLa cells and labeled for either gadkin or AP-1. Gadkin-FKBP localizes in a punctate pattern in the perinuclear region of the cell and is also concentrated at the cell periphery. In wild-type cells (marked with asterisks), AP-1 is mainly localized in the perinuclear region of the cell, but in gadkin-FKBP–expressing cells (which coexpress Mitotrap), there is a second pool at the cell periphery (arrows). Scale bar, 20 μm.

Article Snippet: The mCherry-tagged clathrin light-chain plasmid was obtained from Addgene. pLXIN(epsinR-FKBP) and pLXIN(gadkin-FKBP) were constructed synthetically, as this substantially reduced the manipulations.

Techniques: Expressing, Construct, Fractionation, Labeling

Journal: Molecular Biology of the Cell

Article Title: Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking

doi: 10.1091/mbc.E15-04-0245

Figure Lengend Snippet: Schematic diagram of how the gadkin knocksideways might affect trafficking. (A) In control cells, gadkin associates with membranes via its palmitic acids and interacts with the appendage of AP-1; however, there is a large excess of AP-1 over gadkin. After vesicle budding, the gadkin uses its kinesin-1–binding site to pull the vesicles along microtubules and its Arp2/3 binding site to anchor the vesicles in the actin cortex. The inset is modified from . (B) In the gadkin knocksideways situation, gadkin-FKBP binds essentially all of the membrane-associated AP-1 and also binds to mitochondria, cross-linking the mitochondria to vesicles, with AP-1 and other machinery (but not clathrin) still attached. This may then deplete the machinery from the TGN and endosomes, causing a block in CCV formation.

Article Snippet: The mCherry-tagged clathrin light-chain plasmid was obtained from Addgene. pLXIN(epsinR-FKBP) and pLXIN(gadkin-FKBP) were constructed synthetically, as this substantially reduced the manipulations.

Techniques: Binding Assay, Modification, Blocking Assay