Research Student The Hospital for Sick Children (SickKids) Toronto, Ontario, Canada
Background: The transmembrane receptor Roundabout (ROBO) and its extracellular ligand SLIT was first discovered to be important in inhibiting axonal migration across the midline during nervous system development. Since then, it’s been shown to promote/inhibit angiogenesis as well as inhibit neutrophil recruitment and macropinocytosis largely through actin cytoskeletal remodeling. A recent study found that clathrin-mediated endocytosis (CME) of ROBO1 is required for SLIT2-ROBO1 signaling. However, it remains unclear if these effects are specifically SLIT2-mediated, or if inducing CME through other means can result in similar morphological effects.
Objective: To determine if ROBO1-mediated inhibition of cell migration and spreading is SLIT2-specific or if crosslinking of ROBO1 with a specific antibody at the surface can induce the same phenotypic effect.
Design/Methods: RAW264.7 cells were serum-starved for 30 minutes and treated with either no antibody, IgG control, anti-ROBO1, or N-SLIT2. After spreading for 1 hour, cells were examined via confocal microscopy and cell surface area was measured using the image analysis software Volocity®.
Results: RAW264.7 cells exposed to N-SLIT2 saw a decrease in cell spreading. Antibody-crosslinking at the cell surface resulted in cell spreading similar to N-SLIT2.
Conclusion(s): The effects of ROBO1 on cellular architecture requires CME that is not specifically triggered by N-SLIT2. Limitations of the study: in vitro models cannot capture the inherent complexity of in vivo systems. Future directions: repeat experiments to further validate the significance of results. Identify if CME of ROBO1 is necessary to induce similar effects by using pharmacological inhibitors of CME (e.g. dyngo-4a).
