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Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Blanchette CR, Perrat PN, Thackeray A, Bénard CY - PLoS Biol. (2015)

Bottom Line: However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown.Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor.We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.

No MeSH data available.


Related in: MedlinePlus

LON-2/glypican associates with UNC-40/DCC-expressing cells.(A) Experimental design. Each construct was individually and transiently transfected in S2 cells. After 2 d, cells from independent single transfections were mixed and incubated overnight and then immunostained for the corresponding tags. HA::LON-2-conditioned medium was mixed with UNC-40::FLAG-expressing cells. (B) HA::LON-2 is released from cells that produce it and associates with UNC-40-expressing cells. HA::LON-2 fills the cytoplasm of the cells that produce it (indicated by an asterisk, see also S8 Fig). Notably, HA::LON-2 is observed decorating the outline of UNC-40::FLAG-expressing cells (experiments 1, 6, 7, and 8). HA::LON-2ΔGAG also associates with UNC-40::FLAG-expressing cells (experiment 2). Cells expressing UNC-40ΔNt::FLAG that lacks the extracellular domain do not have HA::LON-2 signal, indicating that the association of LON-2 with UNC-40-expressing cells requires the extracellular domain of UNC-40 (experiment 3). HA::LON-2-conditioned medium contains HA::LON-2 that associates with UNC-40::FLAG-expressing cells, indicating that HA::LON-2 is released from the cells that produce into a diffusible form that interacts with UNC-40::FLAG cells (experiment 8). HA::LON-2 does not associate with cells expressing SfGFP::UNC-6 (experiments 4, 6, and 7) or with untransfected cells. UNC-40-FLAG-expressing cells can simultaneously associate with HA::LON-2 and SfGFP::UNC-6 (experiment 6). HA::LON-2 associates with cells expressing a mutant form of UNC-40/DCC that is unable to bind SfGFP::UNC-6, as it lacks the Fn4/5 UNC-6 binding domains (UNC-40ΔFn4/5::FLAG; experiment 7). Scale bars, 10 μm. (C) Quantification of the association of HA::LON-2 (from expressing cells, from medium of expressing cells, or from cells expressing HA::LON-2ΔGAG) with cells expressing UNC-40::FLAG, UNC-40ΔNt::FLAG, SfGFP::UNC-6, or UNC-40ΔFn4/5::FLAG and untransfected cells. Ten different optical fields containing ~300 cells from three independent experiments were quantified and averaged. (D) Cells expressing UNC-40::FLAG can display irregular morphology, which is enhanced by the presence of HA::LON-2. Images of the different morphologies displayed by UNC-40::FLAG-expressing cells: with a smooth edge, with an irregular edge, or with membrane extensions. The morphology of S2 cells expressing mCherry alone or coexpressing UNC-40::FLAG and mCherry, which were mixed with control untransfected cells or with HA::LON-2-expressing cells, were quantified for irregular edges (grey bars) or membrane extensions (black bars). A higher percentage of UNC-40::FLAG-expressing cells show membrane extensions or irregular edges when mixed with HA::LON-2-expressing cells, as compared to when they are mixed with control mCherry cells. Error bars are standard error of the mean. Asterisks denote significant difference: *** p ≤ 0.001, * p ≤ 0.05. ns, not significant. In (D), significant differences in irregular cell shape are indicated by grey asterisks, and significant difference in membrane extensions is indicated by the black asterisk.
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pbio.1002183.g006: LON-2/glypican associates with UNC-40/DCC-expressing cells.(A) Experimental design. Each construct was individually and transiently transfected in S2 cells. After 2 d, cells from independent single transfections were mixed and incubated overnight and then immunostained for the corresponding tags. HA::LON-2-conditioned medium was mixed with UNC-40::FLAG-expressing cells. (B) HA::LON-2 is released from cells that produce it and associates with UNC-40-expressing cells. HA::LON-2 fills the cytoplasm of the cells that produce it (indicated by an asterisk, see also S8 Fig). Notably, HA::LON-2 is observed decorating the outline of UNC-40::FLAG-expressing cells (experiments 1, 6, 7, and 8). HA::LON-2ΔGAG also associates with UNC-40::FLAG-expressing cells (experiment 2). Cells expressing UNC-40ΔNt::FLAG that lacks the extracellular domain do not have HA::LON-2 signal, indicating that the association of LON-2 with UNC-40-expressing cells requires the extracellular domain of UNC-40 (experiment 3). HA::LON-2-conditioned medium contains HA::LON-2 that associates with UNC-40::FLAG-expressing cells, indicating that HA::LON-2 is released from the cells that produce into a diffusible form that interacts with UNC-40::FLAG cells (experiment 8). HA::LON-2 does not associate with cells expressing SfGFP::UNC-6 (experiments 4, 6, and 7) or with untransfected cells. UNC-40-FLAG-expressing cells can simultaneously associate with HA::LON-2 and SfGFP::UNC-6 (experiment 6). HA::LON-2 associates with cells expressing a mutant form of UNC-40/DCC that is unable to bind SfGFP::UNC-6, as it lacks the Fn4/5 UNC-6 binding domains (UNC-40ΔFn4/5::FLAG; experiment 7). Scale bars, 10 μm. (C) Quantification of the association of HA::LON-2 (from expressing cells, from medium of expressing cells, or from cells expressing HA::LON-2ΔGAG) with cells expressing UNC-40::FLAG, UNC-40ΔNt::FLAG, SfGFP::UNC-6, or UNC-40ΔFn4/5::FLAG and untransfected cells. Ten different optical fields containing ~300 cells from three independent experiments were quantified and averaged. (D) Cells expressing UNC-40::FLAG can display irregular morphology, which is enhanced by the presence of HA::LON-2. Images of the different morphologies displayed by UNC-40::FLAG-expressing cells: with a smooth edge, with an irregular edge, or with membrane extensions. The morphology of S2 cells expressing mCherry alone or coexpressing UNC-40::FLAG and mCherry, which were mixed with control untransfected cells or with HA::LON-2-expressing cells, were quantified for irregular edges (grey bars) or membrane extensions (black bars). A higher percentage of UNC-40::FLAG-expressing cells show membrane extensions or irregular edges when mixed with HA::LON-2-expressing cells, as compared to when they are mixed with control mCherry cells. Error bars are standard error of the mean. Asterisks denote significant difference: *** p ≤ 0.001, * p ≤ 0.05. ns, not significant. In (D), significant differences in irregular cell shape are indicated by grey asterisks, and significant difference in membrane extensions is indicated by the black asterisk.

Mentions: Our above observations provide evidence that the HSPG lon-2/glypican functions in the same genetic pathway as unc-6/netrin to guide migrating axons. It has been shown in several models that HSPGs play multifaceted roles across various signaling pathways, such as facilitating ligand-receptor interactions and transporting morphogens, as well as localizing and stabilizing ligands [32,33]. We asked if the LON-2/glypican molecules might interact with either UNC-6/netrin or its receptor UNC-40/DCC, suggesting a potential mechanism of action for LON-2/glypican in unc-6/netrin-mediated guidance. To test these interactions, we generated epitope-tagged versions of LON-2/glypican, UNC-6/netrin, and UNC-40/DCC proteins, with human influenza hemagglutinin (HA), superfolder-GFP (SfGFP), and FLAG, respectively (Fig 6A), and used cell-mixing experiments. We independently expressed each of these labeled proteins in separate populations of Drosophila S2 cells for 2 d, then cocultured them overnight, and detected the tagged proteins by western blot analysis (see S7 Fig) and by immunostaining (Fig 6A).


Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Blanchette CR, Perrat PN, Thackeray A, Bénard CY - PLoS Biol. (2015)

LON-2/glypican associates with UNC-40/DCC-expressing cells.(A) Experimental design. Each construct was individually and transiently transfected in S2 cells. After 2 d, cells from independent single transfections were mixed and incubated overnight and then immunostained for the corresponding tags. HA::LON-2-conditioned medium was mixed with UNC-40::FLAG-expressing cells. (B) HA::LON-2 is released from cells that produce it and associates with UNC-40-expressing cells. HA::LON-2 fills the cytoplasm of the cells that produce it (indicated by an asterisk, see also S8 Fig). Notably, HA::LON-2 is observed decorating the outline of UNC-40::FLAG-expressing cells (experiments 1, 6, 7, and 8). HA::LON-2ΔGAG also associates with UNC-40::FLAG-expressing cells (experiment 2). Cells expressing UNC-40ΔNt::FLAG that lacks the extracellular domain do not have HA::LON-2 signal, indicating that the association of LON-2 with UNC-40-expressing cells requires the extracellular domain of UNC-40 (experiment 3). HA::LON-2-conditioned medium contains HA::LON-2 that associates with UNC-40::FLAG-expressing cells, indicating that HA::LON-2 is released from the cells that produce into a diffusible form that interacts with UNC-40::FLAG cells (experiment 8). HA::LON-2 does not associate with cells expressing SfGFP::UNC-6 (experiments 4, 6, and 7) or with untransfected cells. UNC-40-FLAG-expressing cells can simultaneously associate with HA::LON-2 and SfGFP::UNC-6 (experiment 6). HA::LON-2 associates with cells expressing a mutant form of UNC-40/DCC that is unable to bind SfGFP::UNC-6, as it lacks the Fn4/5 UNC-6 binding domains (UNC-40ΔFn4/5::FLAG; experiment 7). Scale bars, 10 μm. (C) Quantification of the association of HA::LON-2 (from expressing cells, from medium of expressing cells, or from cells expressing HA::LON-2ΔGAG) with cells expressing UNC-40::FLAG, UNC-40ΔNt::FLAG, SfGFP::UNC-6, or UNC-40ΔFn4/5::FLAG and untransfected cells. Ten different optical fields containing ~300 cells from three independent experiments were quantified and averaged. (D) Cells expressing UNC-40::FLAG can display irregular morphology, which is enhanced by the presence of HA::LON-2. Images of the different morphologies displayed by UNC-40::FLAG-expressing cells: with a smooth edge, with an irregular edge, or with membrane extensions. The morphology of S2 cells expressing mCherry alone or coexpressing UNC-40::FLAG and mCherry, which were mixed with control untransfected cells or with HA::LON-2-expressing cells, were quantified for irregular edges (grey bars) or membrane extensions (black bars). A higher percentage of UNC-40::FLAG-expressing cells show membrane extensions or irregular edges when mixed with HA::LON-2-expressing cells, as compared to when they are mixed with control mCherry cells. Error bars are standard error of the mean. Asterisks denote significant difference: *** p ≤ 0.001, * p ≤ 0.05. ns, not significant. In (D), significant differences in irregular cell shape are indicated by grey asterisks, and significant difference in membrane extensions is indicated by the black asterisk.
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pbio.1002183.g006: LON-2/glypican associates with UNC-40/DCC-expressing cells.(A) Experimental design. Each construct was individually and transiently transfected in S2 cells. After 2 d, cells from independent single transfections were mixed and incubated overnight and then immunostained for the corresponding tags. HA::LON-2-conditioned medium was mixed with UNC-40::FLAG-expressing cells. (B) HA::LON-2 is released from cells that produce it and associates with UNC-40-expressing cells. HA::LON-2 fills the cytoplasm of the cells that produce it (indicated by an asterisk, see also S8 Fig). Notably, HA::LON-2 is observed decorating the outline of UNC-40::FLAG-expressing cells (experiments 1, 6, 7, and 8). HA::LON-2ΔGAG also associates with UNC-40::FLAG-expressing cells (experiment 2). Cells expressing UNC-40ΔNt::FLAG that lacks the extracellular domain do not have HA::LON-2 signal, indicating that the association of LON-2 with UNC-40-expressing cells requires the extracellular domain of UNC-40 (experiment 3). HA::LON-2-conditioned medium contains HA::LON-2 that associates with UNC-40::FLAG-expressing cells, indicating that HA::LON-2 is released from the cells that produce into a diffusible form that interacts with UNC-40::FLAG cells (experiment 8). HA::LON-2 does not associate with cells expressing SfGFP::UNC-6 (experiments 4, 6, and 7) or with untransfected cells. UNC-40-FLAG-expressing cells can simultaneously associate with HA::LON-2 and SfGFP::UNC-6 (experiment 6). HA::LON-2 associates with cells expressing a mutant form of UNC-40/DCC that is unable to bind SfGFP::UNC-6, as it lacks the Fn4/5 UNC-6 binding domains (UNC-40ΔFn4/5::FLAG; experiment 7). Scale bars, 10 μm. (C) Quantification of the association of HA::LON-2 (from expressing cells, from medium of expressing cells, or from cells expressing HA::LON-2ΔGAG) with cells expressing UNC-40::FLAG, UNC-40ΔNt::FLAG, SfGFP::UNC-6, or UNC-40ΔFn4/5::FLAG and untransfected cells. Ten different optical fields containing ~300 cells from three independent experiments were quantified and averaged. (D) Cells expressing UNC-40::FLAG can display irregular morphology, which is enhanced by the presence of HA::LON-2. Images of the different morphologies displayed by UNC-40::FLAG-expressing cells: with a smooth edge, with an irregular edge, or with membrane extensions. The morphology of S2 cells expressing mCherry alone or coexpressing UNC-40::FLAG and mCherry, which were mixed with control untransfected cells or with HA::LON-2-expressing cells, were quantified for irregular edges (grey bars) or membrane extensions (black bars). A higher percentage of UNC-40::FLAG-expressing cells show membrane extensions or irregular edges when mixed with HA::LON-2-expressing cells, as compared to when they are mixed with control mCherry cells. Error bars are standard error of the mean. Asterisks denote significant difference: *** p ≤ 0.001, * p ≤ 0.05. ns, not significant. In (D), significant differences in irregular cell shape are indicated by grey asterisks, and significant difference in membrane extensions is indicated by the black asterisk.
Mentions: Our above observations provide evidence that the HSPG lon-2/glypican functions in the same genetic pathway as unc-6/netrin to guide migrating axons. It has been shown in several models that HSPGs play multifaceted roles across various signaling pathways, such as facilitating ligand-receptor interactions and transporting morphogens, as well as localizing and stabilizing ligands [32,33]. We asked if the LON-2/glypican molecules might interact with either UNC-6/netrin or its receptor UNC-40/DCC, suggesting a potential mechanism of action for LON-2/glypican in unc-6/netrin-mediated guidance. To test these interactions, we generated epitope-tagged versions of LON-2/glypican, UNC-6/netrin, and UNC-40/DCC proteins, with human influenza hemagglutinin (HA), superfolder-GFP (SfGFP), and FLAG, respectively (Fig 6A), and used cell-mixing experiments. We independently expressed each of these labeled proteins in separate populations of Drosophila S2 cells for 2 d, then cocultured them overnight, and detected the tagged proteins by western blot analysis (see S7 Fig) and by immunostaining (Fig 6A).

Bottom Line: However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown.Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor.We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.

No MeSH data available.


Related in: MedlinePlus