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Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion.

Chance RK, Bashaw GJ - PLoS Genet. (2015)

Bottom Line: Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output.We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo.Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

No MeSH data available.


Slit induces Robo colocalization with Rab5 in cell processes.S2R+ cells expressing Robo and treated with SlitCM were fixed at an earlier timepoint (2’) and stained for endogenous Rab5, a marker of the early endosome, (A, C, E, F, H, J, K, M, O) and either bound Slit ligand (B, G, L), or Robo’s C-terminal tag (D, I, N). Cells with Slit bound to processes show covariance between ligand and early endosome signal (B, P (n = # cells indicated on histogram bar)). This colocalization is reduced either by reducing Slit-binding (ΔIg1 in P), or by inhibiting Clathrin-dependent endocytosis globally with DN-Shibire (F, G), or the Dynamin inhibitor Dynasore (P), or by deleting Robo’s AP-2-binding motifs (K, L). Treatment with Slit CM induces colocalization between Robo and Rab5 in processes as compared to cells treated with Control CM, quantified as a percent increase of thresholded Mander’s Overlap Coefficient between Slit and Control CM (C-E, Q). Inhibiting Clathrin-dependent endocytosis by coexpression with DN-Shibire (H-J), use of Dynasore, or deleting AP-2 adaptor motifs (M-O), or inhibiting Slit-binding by deleting the first Ig domain, causes a loss of Slit-dependent colocalization between Robo C-terminus and the early endosome in processes, quantified as the percent change in colocalization between Slit and Control CM (Q). The percentage change switches from positive to negative (Q (n’s for Ctrl CM on top, Slit CM on bottom). See also S4 Fig.
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pgen.1005402.g005: Slit induces Robo colocalization with Rab5 in cell processes.S2R+ cells expressing Robo and treated with SlitCM were fixed at an earlier timepoint (2’) and stained for endogenous Rab5, a marker of the early endosome, (A, C, E, F, H, J, K, M, O) and either bound Slit ligand (B, G, L), or Robo’s C-terminal tag (D, I, N). Cells with Slit bound to processes show covariance between ligand and early endosome signal (B, P (n = # cells indicated on histogram bar)). This colocalization is reduced either by reducing Slit-binding (ΔIg1 in P), or by inhibiting Clathrin-dependent endocytosis globally with DN-Shibire (F, G), or the Dynamin inhibitor Dynasore (P), or by deleting Robo’s AP-2-binding motifs (K, L). Treatment with Slit CM induces colocalization between Robo and Rab5 in processes as compared to cells treated with Control CM, quantified as a percent increase of thresholded Mander’s Overlap Coefficient between Slit and Control CM (C-E, Q). Inhibiting Clathrin-dependent endocytosis by coexpression with DN-Shibire (H-J), use of Dynasore, or deleting AP-2 adaptor motifs (M-O), or inhibiting Slit-binding by deleting the first Ig domain, causes a loss of Slit-dependent colocalization between Robo C-terminus and the early endosome in processes, quantified as the percent change in colocalization between Slit and Control CM (Q). The percentage change switches from positive to negative (Q (n’s for Ctrl CM on top, Slit CM on bottom). See also S4 Fig.

Mentions: If our receptor manipulations indeed disrupt endocytosis, then we would expect to observe an effect on the intracellular trafficking of internalized Robo in experiments where we track Robo’s C-terminus in vitro following Slit treatment. We find that not only do our C-terminal motif deletions inhibit the Slit-dependent removal of Robo from the surface, but they also reduce Slit-dependent colocalization of Robo with endogenous Rab5, a marker of the early endosome. Immunostaining for Slit and Rab5 reveals colocalization between Slit and the early endosome in cell processes (Fig 5A, 5B and 5P). In response to Slit treatment, we also observe an induction of colocalization between Robo and Rab5, specifically in the varicosities and branchpoints of cell processes (Fig 5C–5E), the same structures that showed Slit-dependent surface Robo turnover (arrowheads in Figs 4B and 5B). We have quantified this response as the percentage change in Manders’ overlap coefficient between Slit and Control CM treatment (Fig 5Q). Expression of DN-Shibire (Fig 5F and 5G), or deletion of Robo’s AP-2-binding motifs (Fig 5K and 5L), prevents the Slit-dependent recruitment of Rab5 in cell processes, resulting in less colocalization of Slit with Rab5 (Fig 5P). There is a concomitant reduction in colocalization of Rab5 with the Robo C-terminal tag in the same endocytosis-deficient conditions (Fig 5H–5O and 5Q). These data provide evidence that Slit stimulates the translocation of Robo to the early endosome, and that this process requires Clathrin-dependent endocytosis specifically from the surface of cells.


Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion.

Chance RK, Bashaw GJ - PLoS Genet. (2015)

Slit induces Robo colocalization with Rab5 in cell processes.S2R+ cells expressing Robo and treated with SlitCM were fixed at an earlier timepoint (2’) and stained for endogenous Rab5, a marker of the early endosome, (A, C, E, F, H, J, K, M, O) and either bound Slit ligand (B, G, L), or Robo’s C-terminal tag (D, I, N). Cells with Slit bound to processes show covariance between ligand and early endosome signal (B, P (n = # cells indicated on histogram bar)). This colocalization is reduced either by reducing Slit-binding (ΔIg1 in P), or by inhibiting Clathrin-dependent endocytosis globally with DN-Shibire (F, G), or the Dynamin inhibitor Dynasore (P), or by deleting Robo’s AP-2-binding motifs (K, L). Treatment with Slit CM induces colocalization between Robo and Rab5 in processes as compared to cells treated with Control CM, quantified as a percent increase of thresholded Mander’s Overlap Coefficient between Slit and Control CM (C-E, Q). Inhibiting Clathrin-dependent endocytosis by coexpression with DN-Shibire (H-J), use of Dynasore, or deleting AP-2 adaptor motifs (M-O), or inhibiting Slit-binding by deleting the first Ig domain, causes a loss of Slit-dependent colocalization between Robo C-terminus and the early endosome in processes, quantified as the percent change in colocalization between Slit and Control CM (Q). The percentage change switches from positive to negative (Q (n’s for Ctrl CM on top, Slit CM on bottom). See also S4 Fig.
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Related In: Results  -  Collection

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pgen.1005402.g005: Slit induces Robo colocalization with Rab5 in cell processes.S2R+ cells expressing Robo and treated with SlitCM were fixed at an earlier timepoint (2’) and stained for endogenous Rab5, a marker of the early endosome, (A, C, E, F, H, J, K, M, O) and either bound Slit ligand (B, G, L), or Robo’s C-terminal tag (D, I, N). Cells with Slit bound to processes show covariance between ligand and early endosome signal (B, P (n = # cells indicated on histogram bar)). This colocalization is reduced either by reducing Slit-binding (ΔIg1 in P), or by inhibiting Clathrin-dependent endocytosis globally with DN-Shibire (F, G), or the Dynamin inhibitor Dynasore (P), or by deleting Robo’s AP-2-binding motifs (K, L). Treatment with Slit CM induces colocalization between Robo and Rab5 in processes as compared to cells treated with Control CM, quantified as a percent increase of thresholded Mander’s Overlap Coefficient between Slit and Control CM (C-E, Q). Inhibiting Clathrin-dependent endocytosis by coexpression with DN-Shibire (H-J), use of Dynasore, or deleting AP-2 adaptor motifs (M-O), or inhibiting Slit-binding by deleting the first Ig domain, causes a loss of Slit-dependent colocalization between Robo C-terminus and the early endosome in processes, quantified as the percent change in colocalization between Slit and Control CM (Q). The percentage change switches from positive to negative (Q (n’s for Ctrl CM on top, Slit CM on bottom). See also S4 Fig.
Mentions: If our receptor manipulations indeed disrupt endocytosis, then we would expect to observe an effect on the intracellular trafficking of internalized Robo in experiments where we track Robo’s C-terminus in vitro following Slit treatment. We find that not only do our C-terminal motif deletions inhibit the Slit-dependent removal of Robo from the surface, but they also reduce Slit-dependent colocalization of Robo with endogenous Rab5, a marker of the early endosome. Immunostaining for Slit and Rab5 reveals colocalization between Slit and the early endosome in cell processes (Fig 5A, 5B and 5P). In response to Slit treatment, we also observe an induction of colocalization between Robo and Rab5, specifically in the varicosities and branchpoints of cell processes (Fig 5C–5E), the same structures that showed Slit-dependent surface Robo turnover (arrowheads in Figs 4B and 5B). We have quantified this response as the percentage change in Manders’ overlap coefficient between Slit and Control CM treatment (Fig 5Q). Expression of DN-Shibire (Fig 5F and 5G), or deletion of Robo’s AP-2-binding motifs (Fig 5K and 5L), prevents the Slit-dependent recruitment of Rab5 in cell processes, resulting in less colocalization of Slit with Rab5 (Fig 5P). There is a concomitant reduction in colocalization of Rab5 with the Robo C-terminal tag in the same endocytosis-deficient conditions (Fig 5H–5O and 5Q). These data provide evidence that Slit stimulates the translocation of Robo to the early endosome, and that this process requires Clathrin-dependent endocytosis specifically from the surface of cells.

Bottom Line: Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output.We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo.Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

No MeSH data available.