TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

Supplemental Materials

This article contains the following supporting material:

  • Supplemental Materials
  • Movie 1 - Loss of Trim9 disrupts netrin-dependent DCC clustering at the plasma membrane. Inverted time-lapse TIRF images of Trim9+/+ (left) and Trim9-/- (right) cortical neurons expressing mCherry-DCC before and after netrin-1 application. Time is shown in min:s; images were acquired every 10 s. Netrin-1 addition occurs at 08:10. mCherry-DCC clusters rapidly after addition of netrin-1 in Trim9+/+ neurons, but not Trim9-/- cortical neurons.
  • Movie 2 - The TRIM9 ligase domain, DCC-binding SPRY domain, and TRIM multimerization CC motif are required to fully rescue netrin-dependent clustering of DCC. Inverted time-lapse TIRF images of Trim9-/- cortical neurons expressing mCherry-DCC and either GFP-TRIM9 (top left), GFP-TRIM9RING (top right), GFP-TRIM9SPRY (bottom left), or GFP-TRIM9CC (bottom right), or before and after netrin-1 application. Time is shown in min:s; images were acquired every 10 s.
  • Movie 3 - DCCKR exhibits normal netrin-dependent clustering at the plasma membrane. Inverted time-lapse TIRF images of Trim9+/+ cortical neuron expressing pHluorin-DCCKR before and after netrin-1 application. Time is shown in min:s; images were acquired every 10 s. Netrin-1 addition occurs at 03:20. DCCKR exhibits normal netrin-dependent clustering.
  • Movie 4 - FRNK expression reduces the exuberant axon branching in the corpus callosum of Trim9-/- mice. Inverted confocal z-sections ending in maximum projection through the corpus callosum of Trim9-/-/Thy1-GFP (left) and Trim9-/-/Thy1-GFP/Nex-CRE/FRNKloxSTOPlox (right) littermates. Verified axon branches are noted by red circles. Trim9-/-/Thy1-GFP mice exhibit elevated axon branching in the callosum, which is reduced in Trim9-/-/Thy1-GFP/Nex-CRE/FRNKloxSTOPlox mice.
  • Movie 5 - Pharmacological inhibition of FAK activity decreases netrin-dependent VAMP2-mediated exocytosis. Inverted time-lapse TIRF images of netrin-1 treated (left) or netrin/FAKi treated (right) Trim9+/+ cortical neurons expressing VAMP2-pHlourin. Time is shown in min:s; images were acquired every 0.5 s. The control neuron displays multiple vesicle fusion events (red circles), whereas fusion events are less frequent in the FAKi treated neuron.
  • Movie 6 - : Pharmacological inhibition of FAK activity increases VAMP2-containing vesicle stalling in Trim9+/+ cortical neurons. Inverted time-lapse TIRF images of Trim9+/+ cortical neurons expressing eGFP-VAMP2. Cells were imaged at 110 nm penetration depth to only capture vesicles close to the basal plasma membrane. Time is shown in min:s; images were acquired every 2 s. Control neuron (left) displays active, highly motile vesicles, whereas the FAKi-treated neuron contains a large population of non-motile vesicles.