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. 2019 Dec 9;219(1):e201902088. doi: 10.1083/jcb.201902088

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© 2019 Boyer et al.

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Figure 8. — TRIM67 antagonizes TRIM9 in the regulation of VASP and filopodia. (A) Western blot of endogenous VASP immunoprecipitated from denatured cultured embryonic cortical lysate of indicated genotypes, showing VASP⁺ bands that comigrate and colabel with ubiquitin (VASP-Ub, red arrowheads) and run at a heavier apparent molecular weight than unmodified VASP (black arrowhead). Asterisk (*) by ubiquitin immunoblot (IB) marks a nonspecific ubiquitin band in all lanes. A low-intensity (top) and higher-intensity (bottom) scan of anti-ubiquitin blots are included to better reveal bands of interest. (B) Individual data points and box plots of VASP ubiquitination; n (cultures) = 9 +/+ media, 9 +/+ netrin, 7 Trim67^−/− media, 7 Trim67^−/− netrin, 5 Trim9^−/− media, 5 Trim9^−/− netrin, 7 Trim67^−/−:Trim9^−/− media, 5 Trim67^−/−:Trim9^−/− netrin. (C) Individual data points and box plots of FRAP halftime (t_1/2) of GFP-VASP at filopodia tips in embryonic cortical neurons of indicated genotypes treated with netrin-1 or PR-619. Three experiments per condition. n (cells) = 8 +/+ media, 8 +/+ netrin, 10 −/− media, 14 −/− netrin, 8 −/− PR-619. (D) Fluorescent micrographs of growth cones of cortical neurons cultured from Trim67^+/+:Trim9^+/+ or Trim67^−/−:Trim9^−/− embryos, treated with either netrin-1 or a media sham. (E) Individual data points and box plots of filopodia number per growth cone and numerical density in embryonic cortical neurons isolated from brains of indicated genotypes. Three experiments per genotype/treatment. n (cells) = 82 +/+ media, 88 +/+ netrin, 93 −/− media, 92 −/− netrin. (F) Working model: VASP is ubiquitinated by TRIM9, resulting in decreased stability of filopodia on axonal growth cones. This ubiquitination is antagonized by TRIM67. We hypothesize that VASP ubiquitination and the resultant short-lived filopodia allow for efficient filopodial exploration of the extracellular environment. Consequently, when a filopodia encounters netrin, TRIM67 is recruited to filopodia tips, where it antagonizes VASP ubiquitination and increases filopodial lifetime, before axon turning. *, P < 0.05; ***, P < 0.005; n.s., P > 0.05. Box plots are minimum, Q1, Q2, Q3, maximum.