Midline crossing and Slit responsiveness of commissural axons require USP33 - PubMed (original) (raw)

Midline crossing and Slit responsiveness of commissural axons require USP33

Junichi Yuasa-Kawada et al. Nat Neurosci. 2009 Sep.

Abstract

Commissural axons cross the ventral midline of the neural tube in a Slit-dependent manner. The underlying molecular mechanisms remain unclear. We found that the deubiquitinating enzyme USP33 interacts with the Robo1 receptor. USP33 was essential for midline crossing by commissural axons and for their response to Slit. Our results reveal a previously unknown role for USP33 in vertebrate commissural axon guidance and in Slit signaling.

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Figures

Figure 1

Figure 1

USP33 interacts with Robo1. (a and b) Co-immunoprecipitation of Robo1-HA and GFP-USP33 (a and b) or GFP-USP20 (b) in HEK293 cells. In a, cells were stimulated with control (−) or Slit (+) preparations for 10 min. Immunoprecipitates and total cell lysates (TCL) were immunoblotted with antibodies as indicated. (c) Interaction of the endogenous Robo1 and USP33 proteins in the cell extract prepared from E17 mouse cerebral cortex.

Figure 2

Figure 2

USP33 is required for Slit-induced growth cone collapse in commissural neurons. (a) Time-lapse images of growth cone behavior of E11.5 dorsal spinal cord neurons in response to Slit or control preparations. Scale bar, 5 µm. (b) Quantification of growth cone collapse in response to control or Slit treatment in E9.5 and E11.5 DCC+ commissural neurons (see also panel d; 30 min treatment). The percentage of collapsed growth cones with the mean ± SEM is shown. (c) Slit-induced growth cone collapse in E11.5 siRNA-transfected neurons. (d) Images of Slit-induced growth cone collapse. Growth cones were visualized by staining with anti-DCC (green) and Alexa555–conjugated phalloidin (red). Scale bar, 5 µm. (e) USP33 is not required for response to Sema3F in E11.5 neurons.

Figure 3

Figure 3

USP33 is required for commissural axons to cross the midline. (a) Schematics of axon trajectories (red) of commissural neurons in stage 26 chick spinal cords. Left: a transverse section showing axon midline crossing and anterior turning. Right: anterograde DiI-labeling of the axons in the open-book spinal-cord preparation electroporated with siRNA/YFP (green). A: anterior; P: posterior. (b) Trajectories of DiI-labeled commissural axons in siCtl or siUSP33/_YFP_-electroporated spinal cords at the lumbosacral level. The longitudinal tract is out of focus from the image planes shown. Insets show the overlay of DiI (injection site) and YFP signals. FP: floor plate. Scale bar, 20 µm (200 µm for the insets). (c) Quantification of axon stalling within the midline. ***p < 0.0001 by chi-square test.

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