Robo3.1A suppresses slit-mediated repulsion by triggering degradation of Robo2

Lingyong Li, Shengbing Liu, Yun Lei, Ye Cheng, Changqun Yao, Xuechu Zhen

Research output: Contribution to journalArticle

Abstract

Slits and Robos control the midline crossing of commissural axons, which are not sensitive to the midline repellent Slit before crossing but gain Slit responsiveness to exit the midline and avoid recrossing. Robo3.1A promotes midline crossing of commissural axons by suppressing the axonal responsiveness to the midline repellent Slit, but the underlying mechanism remains unclear. By using a cell surface binding assay and immunoprecipitation, we observed that Robo3.1A did not bind Slit on its own but prevented the specific binding of Slit to the cell surface when it was coexpressed with its close homologue Robo1 or Robo2 (Robo1/2), which are known to mediate the Slit repulsion. Cotransfection with Robo3.1A significantly reduced the protein level of Robo2 in HEK293 cells, and overexpression of Robo3.1A also significantly decreased Robo2 protein level in cerebellar granule cells. Downregulation of endogenous Robo3 by specific small interference RNA (siRNA) significantly increased Robo1 protein level, Slit binding to the cell surface was significantly elevated, and Slit-triggered growth cone collapse appeared after downregulation of Robo3 in cultured cortical neurons. Immunocytochemical staining showed that Robo2 and Robo3 colocalized in intracellular vesicles positive for the marker of late endosomes and lysosomes, but not trans-Golgi apparatus and early endosomes. Thus Robo3.1A may prevent the Slit responsiveness by recruiting Robo1/2 into a late endosome- and lysosome-dependent degradation pathway.

Original languageEnglish (US)
Pages (from-to)835-846
Number of pages12
JournalJournal of Neuroscience Research
Volume92
Issue number7
DOIs
StatePublished - Jul 2014
Externally publishedYes

Fingerprint

Endosomes
Lysosomes
Axons
Down-Regulation
Growth Cones
HEK293 Cells
Golgi Apparatus
RNA Interference
Immunoprecipitation
Staining and Labeling
Neurons
roundabout protein

Keywords

  • Axon guidance
  • Degradation
  • Robo2
  • Robo3
  • Slit

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

Cite this

Robo3.1A suppresses slit-mediated repulsion by triggering degradation of Robo2. / Li, Lingyong; Liu, Shengbing; Lei, Yun; Cheng, Ye; Yao, Changqun; Zhen, Xuechu.

In: Journal of Neuroscience Research, Vol. 92, No. 7, 07.2014, p. 835-846.

Research output: Contribution to journalArticle

Li, Lingyong ; Liu, Shengbing ; Lei, Yun ; Cheng, Ye ; Yao, Changqun ; Zhen, Xuechu. / Robo3.1A suppresses slit-mediated repulsion by triggering degradation of Robo2. In: Journal of Neuroscience Research. 2014 ; Vol. 92, No. 7. pp. 835-846.
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