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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

A secreted form of LON-2/glypican that lacks the heparan sulfate chain attachments is functional in axon guidance.(A) The HSPG LON-2/glypican is composed of a core protein and three HS chains. The core protein is predicted to fold into a globular domain on its N-terminal region and to be GPI-anchored. (B) Schematics of the engineered forms of LON-2 that we used: LON-2ΔGAG, in which the HS chain attachment sites are mutated; LON-2ΔGPI, in which the GPI anchor is deleted; N-LON-2, in which the C-terminus is deleted; and C-LON-2, in which the N-terminal globular domain is deleted. Western blot analysis of protein extracts of worms expressing LON-2::GFP or LON-2ΔGAG::GFP confirms that deleting the HS attachment sites on LON-2 affects HS addition on LON-2. Protein extracts from wild type (N2) and an unrelated GFP strain (lqIs4) are negative controls. (C) A form of LON-2/glypican lacking HS chain attachment sites (LON-2ΔGAG) functions in axon guidance. LON-2ΔGAG rescues the AVM guidance defects of double mutants lon-2 slt-1 back to the level of slt-1 single mutants. Secreted globular LON-2/glypican is functional in axon guidance. LON-2/glypican was engineered to be secreted by deleting its GPI anchor (LON-2ΔGPI) or by deleting the C-terminus, thus lacking the GPI anchor and the HS attachment sites (N-LON-2). Both LON-2ΔGPI and N-LON-2 function in axon guidance, as assayed by their ability to rescue axon guidance defects of lon-2 slt-1 back down to the level of slt-1 single mutants. In contrast, a form of LON-2/glypican containing its C-terminus including the three HS attachment sites, but lacking its N-terminal globular domain (C-LON-2), is not functional (see S2 and S3 Tables), indicating that the N-terminal globular domain of the core protein is key to the function of LON-2/glypican in axon guidance. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (D) A form of LON-2/glypican lacking HS chain attachment sites is functional in DTC guidance. The DTC migration of lon-2 mutants carrying the transgene Plon-2::LON-2ΔGAG is rescued back to wild-type levels. Secreted N-terminus globular LON-2/glypican (N-LON-2) is functional in DTC guidance, as DTC guidance defects of lon-2 mutants are rescued by N-LON-2. Transgenic animals were compared to nontransgenic sibling controls (see S7 Table). Data for wild type and lon-2 are the same as in Fig 2B. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001, ** p ≤ 0.01 (z-tests, p-values were corrected by multiplying by the number of comparisons).
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pbio.1002183.g005: A secreted form of LON-2/glypican that lacks the heparan sulfate chain attachments is functional in axon guidance.(A) The HSPG LON-2/glypican is composed of a core protein and three HS chains. The core protein is predicted to fold into a globular domain on its N-terminal region and to be GPI-anchored. (B) Schematics of the engineered forms of LON-2 that we used: LON-2ΔGAG, in which the HS chain attachment sites are mutated; LON-2ΔGPI, in which the GPI anchor is deleted; N-LON-2, in which the C-terminus is deleted; and C-LON-2, in which the N-terminal globular domain is deleted. Western blot analysis of protein extracts of worms expressing LON-2::GFP or LON-2ΔGAG::GFP confirms that deleting the HS attachment sites on LON-2 affects HS addition on LON-2. Protein extracts from wild type (N2) and an unrelated GFP strain (lqIs4) are negative controls. (C) A form of LON-2/glypican lacking HS chain attachment sites (LON-2ΔGAG) functions in axon guidance. LON-2ΔGAG rescues the AVM guidance defects of double mutants lon-2 slt-1 back to the level of slt-1 single mutants. Secreted globular LON-2/glypican is functional in axon guidance. LON-2/glypican was engineered to be secreted by deleting its GPI anchor (LON-2ΔGPI) or by deleting the C-terminus, thus lacking the GPI anchor and the HS attachment sites (N-LON-2). Both LON-2ΔGPI and N-LON-2 function in axon guidance, as assayed by their ability to rescue axon guidance defects of lon-2 slt-1 back down to the level of slt-1 single mutants. In contrast, a form of LON-2/glypican containing its C-terminus including the three HS attachment sites, but lacking its N-terminal globular domain (C-LON-2), is not functional (see S2 and S3 Tables), indicating that the N-terminal globular domain of the core protein is key to the function of LON-2/glypican in axon guidance. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (D) A form of LON-2/glypican lacking HS chain attachment sites is functional in DTC guidance. The DTC migration of lon-2 mutants carrying the transgene Plon-2::LON-2ΔGAG is rescued back to wild-type levels. Secreted N-terminus globular LON-2/glypican (N-LON-2) is functional in DTC guidance, as DTC guidance defects of lon-2 mutants are rescued by N-LON-2. Transgenic animals were compared to nontransgenic sibling controls (see S7 Table). Data for wild type and lon-2 are the same as in Fig 2B. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001, ** p ≤ 0.01 (z-tests, p-values were corrected by multiplying by the number of comparisons).

Mentions: Glypicans are composed of a core protein moiety with covalently linked HS chains attached via a tetrasaccharide linker at specific Serine residues (Fig 5A, [15]). Prior studies on the role of HSPGs in other developmental pathways indicate that both the identity of the HSPG core proteins and the heterogeneity of their HS chains modified by epimerization and sulfations [15] contribute to the specificity of the interactions between particular HSPGs and the proteins that they bind [15,29,30].


Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

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

A secreted form of LON-2/glypican that lacks the heparan sulfate chain attachments is functional in axon guidance.(A) The HSPG LON-2/glypican is composed of a core protein and three HS chains. The core protein is predicted to fold into a globular domain on its N-terminal region and to be GPI-anchored. (B) Schematics of the engineered forms of LON-2 that we used: LON-2ΔGAG, in which the HS chain attachment sites are mutated; LON-2ΔGPI, in which the GPI anchor is deleted; N-LON-2, in which the C-terminus is deleted; and C-LON-2, in which the N-terminal globular domain is deleted. Western blot analysis of protein extracts of worms expressing LON-2::GFP or LON-2ΔGAG::GFP confirms that deleting the HS attachment sites on LON-2 affects HS addition on LON-2. Protein extracts from wild type (N2) and an unrelated GFP strain (lqIs4) are negative controls. (C) A form of LON-2/glypican lacking HS chain attachment sites (LON-2ΔGAG) functions in axon guidance. LON-2ΔGAG rescues the AVM guidance defects of double mutants lon-2 slt-1 back to the level of slt-1 single mutants. Secreted globular LON-2/glypican is functional in axon guidance. LON-2/glypican was engineered to be secreted by deleting its GPI anchor (LON-2ΔGPI) or by deleting the C-terminus, thus lacking the GPI anchor and the HS attachment sites (N-LON-2). Both LON-2ΔGPI and N-LON-2 function in axon guidance, as assayed by their ability to rescue axon guidance defects of lon-2 slt-1 back down to the level of slt-1 single mutants. In contrast, a form of LON-2/glypican containing its C-terminus including the three HS attachment sites, but lacking its N-terminal globular domain (C-LON-2), is not functional (see S2 and S3 Tables), indicating that the N-terminal globular domain of the core protein is key to the function of LON-2/glypican in axon guidance. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (D) A form of LON-2/glypican lacking HS chain attachment sites is functional in DTC guidance. The DTC migration of lon-2 mutants carrying the transgene Plon-2::LON-2ΔGAG is rescued back to wild-type levels. Secreted N-terminus globular LON-2/glypican (N-LON-2) is functional in DTC guidance, as DTC guidance defects of lon-2 mutants are rescued by N-LON-2. Transgenic animals were compared to nontransgenic sibling controls (see S7 Table). Data for wild type and lon-2 are the same as in Fig 2B. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001, ** p ≤ 0.01 (z-tests, p-values were corrected by multiplying by the number of comparisons).
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493048&req=5

pbio.1002183.g005: A secreted form of LON-2/glypican that lacks the heparan sulfate chain attachments is functional in axon guidance.(A) The HSPG LON-2/glypican is composed of a core protein and three HS chains. The core protein is predicted to fold into a globular domain on its N-terminal region and to be GPI-anchored. (B) Schematics of the engineered forms of LON-2 that we used: LON-2ΔGAG, in which the HS chain attachment sites are mutated; LON-2ΔGPI, in which the GPI anchor is deleted; N-LON-2, in which the C-terminus is deleted; and C-LON-2, in which the N-terminal globular domain is deleted. Western blot analysis of protein extracts of worms expressing LON-2::GFP or LON-2ΔGAG::GFP confirms that deleting the HS attachment sites on LON-2 affects HS addition on LON-2. Protein extracts from wild type (N2) and an unrelated GFP strain (lqIs4) are negative controls. (C) A form of LON-2/glypican lacking HS chain attachment sites (LON-2ΔGAG) functions in axon guidance. LON-2ΔGAG rescues the AVM guidance defects of double mutants lon-2 slt-1 back to the level of slt-1 single mutants. Secreted globular LON-2/glypican is functional in axon guidance. LON-2/glypican was engineered to be secreted by deleting its GPI anchor (LON-2ΔGPI) or by deleting the C-terminus, thus lacking the GPI anchor and the HS attachment sites (N-LON-2). Both LON-2ΔGPI and N-LON-2 function in axon guidance, as assayed by their ability to rescue axon guidance defects of lon-2 slt-1 back down to the level of slt-1 single mutants. In contrast, a form of LON-2/glypican containing its C-terminus including the three HS attachment sites, but lacking its N-terminal globular domain (C-LON-2), is not functional (see S2 and S3 Tables), indicating that the N-terminal globular domain of the core protein is key to the function of LON-2/glypican in axon guidance. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (D) A form of LON-2/glypican lacking HS chain attachment sites is functional in DTC guidance. The DTC migration of lon-2 mutants carrying the transgene Plon-2::LON-2ΔGAG is rescued back to wild-type levels. Secreted N-terminus globular LON-2/glypican (N-LON-2) is functional in DTC guidance, as DTC guidance defects of lon-2 mutants are rescued by N-LON-2. Transgenic animals were compared to nontransgenic sibling controls (see S7 Table). Data for wild type and lon-2 are the same as in Fig 2B. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001, ** p ≤ 0.01 (z-tests, p-values were corrected by multiplying by the number of comparisons).
Mentions: Glypicans are composed of a core protein moiety with covalently linked HS chains attached via a tetrasaccharide linker at specific Serine residues (Fig 5A, [15]). Prior studies on the role of HSPGs in other developmental pathways indicate that both the identity of the HSPG core proteins and the heterogeneity of their HS chains modified by epimerization and sulfations [15] contribute to the specificity of the interactions between particular HSPGs and the proteins that they bind [15,29,30].

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