The fast magnetic SUPER template procedure allowed us to test each clone for binding to dynamin-2 loaded with different nucleotides. (49K) DOI:?10.7554/eLife.25197.024 Determine 5source data 1: (panel C) Data and statistical analysis of transferrin internalization in HeLa cells. elife-25197-fig5-data1.docx (397K) DOI:?10.7554/eLife.25197.026 Determine 6source data 1: (panel C) Data and statistical analysis of EGF internalization in HeLa cells. elife-25197-fig6-data1.docx (414K) DOI:?10.7554/eLife.25197.028 Source code 1: postUtrack_1. elife-25197-code1.m (14K) DOI:?10.7554/eLife.25197.029 Source code 2: postUtrack_2_GenerateProfiles. elife-25197-code2.m (20K) DOI:?10.7554/eLife.25197.030 Supplementary file 1: Supplementary File_Manual _Code. elife-25197-supp1.pdf (571K) DOI:?10.7554/eLife.25197.031 Transparent reporting form. elife-25197-transrepform.pdf (689K) DOI:?10.7554/eLife.25197.032 Abstract Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is usually purely dependent on GTP hydrolysis, but how fission is usually mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that this GTP energy is usually primarily spent in constriction. value>0.05). Source files and statistical statement for panel D are available in Physique 1source data 1. Physique 1source data 1.(panel D) Malachite Green Assay.Click here to view.(112K, docx) Physique 1figure product 1. Open in a separate windows Validation of dynamin-2 positive nanobodies.(A)?Immunofluorescence results performed on HeLa cells with different levels MRS1706 of dynamin- two expressions showing three different clones with different abilities to bind dynamin-2; the very strong positive clone (++) and the positive clone (+) bind more cells hSPRY2 overexpressing dynamin-2, the unfavorable clone (-) has a background staining of the cells regardless of the level of dynamin-2. (B) Example of dot blot experiment in which it is possible to observe the difference in dynamin-2 binding of different clones, the plate is usually divided by dynamin-2 without nucleotide, dynamin-2 coupled with GMPPCP and the control without dynamin-2. (C) Magnifications of dot blot spots that confirm the ability of positive and negative clones to MRS1706 bind dynamin-2. Physique 1figure product 2. Open in a separate window SUPER themes pull-down assay control.Western blot of dynamin-1 on magnetic SUPER templates and magnetic silica beads treated with saponin 0.5% showing the strong binding of dynamin on naked MRS1706 beads and the absence of dynamin on SUPER templates when treated with saponin. Physique 1figure product 3. Open in a separate windows Dynamin binding on SUPER themes with and without nucleotides.Western blot of magnetic SUPER templates pull-down assay with dynamin-2 nucleotide-free and GDPAlF4-coupled showing the stronger binding of dynamin to SUPER templates when coupled with nucleotide. Physique 1figure product 4. Open in a separate window Verification of the knock-out procedure for dynamin triple KO cells.Western blot of dynamin TKO fibroblasts whole cell lysates (100 g total proteins loaded) OHT treated or untreated immunoblotted for dynamin and -tubulin as loading control. Physique 1figure product 5. Open in a separate windows Pull-downs of dynamin mutants with dynab.(A)?Pull-down with SUPER templates of dynamin PRD with dynab. (B) Pull-downs of dynab with SUPER themes of dynamin 1 (left panel) dynamin 1 K44A (central panel) and dynamin 1 K142A (right panel). Physique 1figure product 6. Open in a separate window Membrane linens assay with dynamin 1-GDPAlF4 and dynab on fluorescent lipids, level bar: 10 m. As results of the pull-down with GTPase defective mutants were not conclusive, we used a membrane linens assay, in which lipids are dried onto a coverslip and rehydrated to form stacks of lipid bilayers (Roux et al., 2006; Itoh et al., 2005). The addition of Alexa-488-dynamin-1 to the membrane linens resulted in the formation of dynamin tubules, which can be visualized by confocal microscopy (Physique 1B). Atto-565-dynab weakly bound to dynamin-1 tubules. In contrast, in presence of GDPAlF4 we observed a strong binding of dynab to dynamin-1 tubules (Physique 1B). This confirmed that dynab binds preferentially to the transition state of dynamin-1, consistently with pull-down results. As GDPAlF4 can induce formation of membrane-free dynamin oligomers, we further checked that dynab was co-localizing with dynamin 1-coated.