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Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

Strachan LR, Condic ML - J. Cell Biol. (2004)

Bottom Line: NCCs showed both ligand- and receptor-specific integrin regulation in vitro.On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min.Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

ABSTRACT
Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

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Cranial neural crest modulate surface integrin levels in a substratum-dependent manner. Cell surface–labeled integrins were immunoprecipitated and analyzed on Western blot. (A and B) Average band intensities (relative to the high substratum condition) are given below each lane. (A) Cranial neural crest down-regulate surface levels of the laminin receptor integrin α6, but not the fibronectin receptor integrin α5, when cultured on high (LM20) compared with low (LM1) concentrations of laminin. (B) Surface levels of both integrin α5 and α6 are equivalent in cranial neural crest cultured on low (FN1) and high (FN20) concentrations of fibronectin. (C) The average relative band intensity ratio (LM1:LM20 or FN1:FN20) for both integrin α5 and α6; y-axis is a log scale. Means and 95% confidence intervals from at least five independent experiments are as follows: LMα5 1.07x (1.01–1.13), LMα6 2.04x (1.79–2.34), FNα5 1.02x (0.95–1.11), and FNα6 0.95x (0.90–1.01). Asterisk indicates that the average band intensity of integrin α6 on LM1 is significantly greater than on LM20 (P < 0.002; Mann-Whitney U-test). Average band intensities for all other conditions did not significantly differ. Equal protein amounts were used for immunoprecipitations.
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fig1: Cranial neural crest modulate surface integrin levels in a substratum-dependent manner. Cell surface–labeled integrins were immunoprecipitated and analyzed on Western blot. (A and B) Average band intensities (relative to the high substratum condition) are given below each lane. (A) Cranial neural crest down-regulate surface levels of the laminin receptor integrin α6, but not the fibronectin receptor integrin α5, when cultured on high (LM20) compared with low (LM1) concentrations of laminin. (B) Surface levels of both integrin α5 and α6 are equivalent in cranial neural crest cultured on low (FN1) and high (FN20) concentrations of fibronectin. (C) The average relative band intensity ratio (LM1:LM20 or FN1:FN20) for both integrin α5 and α6; y-axis is a log scale. Means and 95% confidence intervals from at least five independent experiments are as follows: LMα5 1.07x (1.01–1.13), LMα6 2.04x (1.79–2.34), FNα5 1.02x (0.95–1.11), and FNα6 0.95x (0.90–1.01). Asterisk indicates that the average band intensity of integrin α6 on LM1 is significantly greater than on LM20 (P < 0.002; Mann-Whitney U-test). Average band intensities for all other conditions did not significantly differ. Equal protein amounts were used for immunoprecipitations.

Mentions: To determine whether cranial NCCs modulate their surface integrin levels in a substratum-dependent manner, we examined the steady-state surface expression of two integrin receptors, α6 (a laminin receptor) and α5 (a fibronectin receptor), in cranial NCCs cultured on two different concentrations of laminin and fibronectin. Mesencephalic neural tubes were cultured on glass coated with low (1 μg/ml) and high (20 μg/ml) concentrations of either laminin or fibronectin. Application of laminin at 1 and 20 μg/ml resulted in a density of 45 and 430 ng/cm2 bound laminin, respectively, and application of fibronectin at 1 and 20 μg/ml resulted in 35 and 700 ng/cm2 bound fibronectin, respectively (Strachan and Condic, 2003). After labeling surface proteins with biotin, integrins α5 and α6 were immunoprecipitated, and the surface pools were detected with avidin on Western blots. As we previously observed (Strachan and Condic, 2003), cranial NCCs cultured on varying concentrations of laminin regulated surface levels of the laminin receptor, integrin α6, but not the fibronectin receptor, integrin α5 (Fig. 1, A and C). On low concentrations of laminin (LM1), cells expressed greater amounts of integrin α6 on their surface compared with cells cultured on high laminin concentrations (LM20). Quantification of band intensities from both laminin concentrations indicates an average twofold increase in surface α6 protein on LM1 relative to LM20 (n = 6; P < 0.002, Mann-Whitney U-test). On both concentrations of laminin, surface levels of the fibronectin receptor, integrin α5, were equivalent, suggesting that integrin regulation is receptor specific (Fig. 1, A and C). In contrast to cells cultured on laminin, cranial NCCs cultured on varying concentrations of fibronectin did not modulate surface levels of either integrin α5 or α6 (Fig. 1, B and C).


Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

Strachan LR, Condic ML - J. Cell Biol. (2004)

Cranial neural crest modulate surface integrin levels in a substratum-dependent manner. Cell surface–labeled integrins were immunoprecipitated and analyzed on Western blot. (A and B) Average band intensities (relative to the high substratum condition) are given below each lane. (A) Cranial neural crest down-regulate surface levels of the laminin receptor integrin α6, but not the fibronectin receptor integrin α5, when cultured on high (LM20) compared with low (LM1) concentrations of laminin. (B) Surface levels of both integrin α5 and α6 are equivalent in cranial neural crest cultured on low (FN1) and high (FN20) concentrations of fibronectin. (C) The average relative band intensity ratio (LM1:LM20 or FN1:FN20) for both integrin α5 and α6; y-axis is a log scale. Means and 95% confidence intervals from at least five independent experiments are as follows: LMα5 1.07x (1.01–1.13), LMα6 2.04x (1.79–2.34), FNα5 1.02x (0.95–1.11), and FNα6 0.95x (0.90–1.01). Asterisk indicates that the average band intensity of integrin α6 on LM1 is significantly greater than on LM20 (P < 0.002; Mann-Whitney U-test). Average band intensities for all other conditions did not significantly differ. Equal protein amounts were used for immunoprecipitations.
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Related In: Results  -  Collection

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fig1: Cranial neural crest modulate surface integrin levels in a substratum-dependent manner. Cell surface–labeled integrins were immunoprecipitated and analyzed on Western blot. (A and B) Average band intensities (relative to the high substratum condition) are given below each lane. (A) Cranial neural crest down-regulate surface levels of the laminin receptor integrin α6, but not the fibronectin receptor integrin α5, when cultured on high (LM20) compared with low (LM1) concentrations of laminin. (B) Surface levels of both integrin α5 and α6 are equivalent in cranial neural crest cultured on low (FN1) and high (FN20) concentrations of fibronectin. (C) The average relative band intensity ratio (LM1:LM20 or FN1:FN20) for both integrin α5 and α6; y-axis is a log scale. Means and 95% confidence intervals from at least five independent experiments are as follows: LMα5 1.07x (1.01–1.13), LMα6 2.04x (1.79–2.34), FNα5 1.02x (0.95–1.11), and FNα6 0.95x (0.90–1.01). Asterisk indicates that the average band intensity of integrin α6 on LM1 is significantly greater than on LM20 (P < 0.002; Mann-Whitney U-test). Average band intensities for all other conditions did not significantly differ. Equal protein amounts were used for immunoprecipitations.
Mentions: To determine whether cranial NCCs modulate their surface integrin levels in a substratum-dependent manner, we examined the steady-state surface expression of two integrin receptors, α6 (a laminin receptor) and α5 (a fibronectin receptor), in cranial NCCs cultured on two different concentrations of laminin and fibronectin. Mesencephalic neural tubes were cultured on glass coated with low (1 μg/ml) and high (20 μg/ml) concentrations of either laminin or fibronectin. Application of laminin at 1 and 20 μg/ml resulted in a density of 45 and 430 ng/cm2 bound laminin, respectively, and application of fibronectin at 1 and 20 μg/ml resulted in 35 and 700 ng/cm2 bound fibronectin, respectively (Strachan and Condic, 2003). After labeling surface proteins with biotin, integrins α5 and α6 were immunoprecipitated, and the surface pools were detected with avidin on Western blots. As we previously observed (Strachan and Condic, 2003), cranial NCCs cultured on varying concentrations of laminin regulated surface levels of the laminin receptor, integrin α6, but not the fibronectin receptor, integrin α5 (Fig. 1, A and C). On low concentrations of laminin (LM1), cells expressed greater amounts of integrin α6 on their surface compared with cells cultured on high laminin concentrations (LM20). Quantification of band intensities from both laminin concentrations indicates an average twofold increase in surface α6 protein on LM1 relative to LM20 (n = 6; P < 0.002, Mann-Whitney U-test). On both concentrations of laminin, surface levels of the fibronectin receptor, integrin α5, were equivalent, suggesting that integrin regulation is receptor specific (Fig. 1, A and C). In contrast to cells cultured on laminin, cranial NCCs cultured on varying concentrations of fibronectin did not modulate surface levels of either integrin α5 or α6 (Fig. 1, B and C).

Bottom Line: NCCs showed both ligand- and receptor-specific integrin regulation in vitro.On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min.Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

ABSTRACT
Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Show MeSH
Related in: MedlinePlus