Journal: The Journal of Cell Biology

Article Title: PICK1 regulates AMPA receptor endocytosis via direct interactions with AP2 α-appendage and dynamin

doi: 10.1083/jcb.201701034

Figure Lengend Snippet: PICK1 binds dynamin via BAR and GTPase domains and regulates dynamin polymerization. (A) Endogenous PICK1 interacts with endogenous dynamin 1, 2, and 3 in neurons. Extracts of cortical neurons were immunoprecipitated with PICK1 antibody or control IgG. Proteins were detected by Western blotting. Input is 5% of offered protein. Blots shown are representative of more than five independent experiments. (B) PICK1 interacts directly with dynamin. GST or GST-PICK1 was immobilized on glutathione agarose and incubated with purified HA-dynamin. Proteins were detected by Western blotting. Blots shown are representative of four independent experiments. (C) PICK1 interacts with the GTPase domain of dynamin. HEK293 cells were cotransfected with plasmids expressing Flag-PICK1 and GFP, GFP–dynamin 2, or dynamin domains as indicated. Cells were lysed and incubated with GFP-trap agarose. Blots shown are representative of five independent experiments. (D) Dynamin interacts with the PICK1 BAR domain. HEK cells were cotransfected with plasmids expressing HA-dynamin and GFP, GFP-PICK1, or truncations, as indicated in the top panel. Cells were lysed and incubated with GFP-trap agarose. Blots shown are representative of five independent experiments. (E) PICK1 promotes dynamin polymerization. His 6 -PICK1 at the concentrations indicated was incubated with HA-dynamin. Polymerized dynamin was pelleted by centrifugation, and protein in the supernatant and pellet was analyzed by Western blotting. Graph shows the ratio of dynamin in pellet/supernatant ( n = 4 independent experiments; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s test; values are means ± SEMs). (F) HA-dynamin was incubated with his 6 -PICK1, his 6 -NSF, or his 6 -PICK1 truncations as indicated and processed as in E. Graph shows the ratio of dynamin in pellet/supernatant ( n = 7 independent experiments; *, P < 0.05; one-way ANOVA followed by Tukey’s test; values are means ± SEMs). CTD, C-terminal domain.

Article Snippet: For HEK293 cell experiments, full-length PICK1 was expressed from pcDNA3.1, GFP-tagged truncations of PICK1 and dynamin were expressed from pEGFP-C1 (Clontech), and HA-tagged dynamin 2 was expressed from pcDNA3 (Addgene).

Techniques: Immunoprecipitation, Western Blot, Incubation, Purification, Expressing, Centrifugation

Journal: Cold Spring Harbor Perspectives in Biology

Article Title: Clathrin-Independent Pathways of Endocytosis

doi: 10.1101/cshperspect.a016758

Figure Lengend Snippet: The diversity of endocytic pathways available at the cell surface of metazoan cells. The schematic outlines multiple means by which a cargo located at the plasma membrane or in the extracellular milieu enters the endocytic pathway in metazoan cells. Dynamin-dependent pathways (+; circles) are typically associated with small-scale coat-mediated invaginations, such as clathrin or caveolar pathways. The dynamin-independent pathways reflect a larger diversity of forms, ranging from the small-scale processes to the larger scale membrane invaginations. The main effectors of the CIE pathways are indicated below their primary invaginations. All the dynamin-independent mechanisms appear to use actin filament (red bars) polymerization machinery.

Article Snippet: Here dynamin regulates endocytosis via an endocytic pathway that is cholesterol sensitive, wherein many IL receptor subunits associate with “lipid raft”-like domains ( Lamaze et al. 2001 ).

Techniques: Clinical Proteomics, Membrane