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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 accumulate internalized surface integrin receptors in a substratum-dependent manner. Surface receptors were labeled with biotin, cells were incubated at 37°C for the indicated time points, then cooled to 25°C and treated with the reducing agent MesNa to remove biotin from receptors remaining on the surface. Cells were lysed, the receptors were immunoprecipitated, and the internalized portion revealed on Western blots with HRP-conjugated streptavidin. Bottom panels show internal loading control (α-tubulin). (A) On both low (LM1) and high (LM20) concentrations of laminin, there is an accumulation of internalized integrin α6 over 20 min. (B) On both LM1 and LM20, accumulation of internalized integrin α5 reaches steady-state levels by 2 min. (C) On high (FN20) fibronectin concentrations there is no accumulation of internalized integrin α5 or integrin α6 above the level observed at 2 min. (D) The average increase in band intensity from 2 to 20 min for each condition from three independent experiments is shown. Asterisks indicate significant increases in internalized α6 over time on both LM1 and LM20 (P < 0.05; Wilcoxon signed-rank test).
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fig2: Cranial neural crest accumulate internalized surface integrin receptors in a substratum-dependent manner. Surface receptors were labeled with biotin, cells were incubated at 37°C for the indicated time points, then cooled to 25°C and treated with the reducing agent MesNa to remove biotin from receptors remaining on the surface. Cells were lysed, the receptors were immunoprecipitated, and the internalized portion revealed on Western blots with HRP-conjugated streptavidin. Bottom panels show internal loading control (α-tubulin). (A) On both low (LM1) and high (LM20) concentrations of laminin, there is an accumulation of internalized integrin α6 over 20 min. (B) On both LM1 and LM20, accumulation of internalized integrin α5 reaches steady-state levels by 2 min. (C) On high (FN20) fibronectin concentrations there is no accumulation of internalized integrin α5 or integrin α6 above the level observed at 2 min. (D) The average increase in band intensity from 2 to 20 min for each condition from three independent experiments is shown. Asterisks indicate significant increases in internalized α6 over time on both LM1 and LM20 (P < 0.05; Wilcoxon signed-rank test).

Mentions: To analyze the kinetics of surface integrin regulation in cranial NCCs, we followed the internalization of labeled receptors during a short time period. We found a difference between the accumulation of internalized integrin α6 and integrin α5 (Fig. 2). On both low and high laminin concentrations, internalized integrin α6 reached steady-state levels by 20 min; i.e., there was no greater accumulation after 30 or 60 min (unpublished data). In contrast to integrin α6, low levels of integrin α5 accumulated rapidly (within 2 min) in cells cultured on both laminin concentrations, and the internalized pool did not increase over time (Fig. 2, B and D). Thus, cranial NCCs on laminin internalize and accumulate laminin, but not fibronectin receptors.


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 accumulate internalized surface integrin receptors in a substratum-dependent manner. Surface receptors were labeled with biotin, cells were incubated at 37°C for the indicated time points, then cooled to 25°C and treated with the reducing agent MesNa to remove biotin from receptors remaining on the surface. Cells were lysed, the receptors were immunoprecipitated, and the internalized portion revealed on Western blots with HRP-conjugated streptavidin. Bottom panels show internal loading control (α-tubulin). (A) On both low (LM1) and high (LM20) concentrations of laminin, there is an accumulation of internalized integrin α6 over 20 min. (B) On both LM1 and LM20, accumulation of internalized integrin α5 reaches steady-state levels by 2 min. (C) On high (FN20) fibronectin concentrations there is no accumulation of internalized integrin α5 or integrin α6 above the level observed at 2 min. (D) The average increase in band intensity from 2 to 20 min for each condition from three independent experiments is shown. Asterisks indicate significant increases in internalized α6 over time on both LM1 and LM20 (P < 0.05; Wilcoxon signed-rank test).
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Related In: Results  -  Collection

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fig2: Cranial neural crest accumulate internalized surface integrin receptors in a substratum-dependent manner. Surface receptors were labeled with biotin, cells were incubated at 37°C for the indicated time points, then cooled to 25°C and treated with the reducing agent MesNa to remove biotin from receptors remaining on the surface. Cells were lysed, the receptors were immunoprecipitated, and the internalized portion revealed on Western blots with HRP-conjugated streptavidin. Bottom panels show internal loading control (α-tubulin). (A) On both low (LM1) and high (LM20) concentrations of laminin, there is an accumulation of internalized integrin α6 over 20 min. (B) On both LM1 and LM20, accumulation of internalized integrin α5 reaches steady-state levels by 2 min. (C) On high (FN20) fibronectin concentrations there is no accumulation of internalized integrin α5 or integrin α6 above the level observed at 2 min. (D) The average increase in band intensity from 2 to 20 min for each condition from three independent experiments is shown. Asterisks indicate significant increases in internalized α6 over time on both LM1 and LM20 (P < 0.05; Wilcoxon signed-rank test).
Mentions: To analyze the kinetics of surface integrin regulation in cranial NCCs, we followed the internalization of labeled receptors during a short time period. We found a difference between the accumulation of internalized integrin α6 and integrin α5 (Fig. 2). On both low and high laminin concentrations, internalized integrin α6 reached steady-state levels by 20 min; i.e., there was no greater accumulation after 30 or 60 min (unpublished data). In contrast to integrin α6, low levels of integrin α5 accumulated rapidly (within 2 min) in cells cultured on both laminin concentrations, and the internalized pool did not increase over time (Fig. 2, B and D). Thus, cranial NCCs on laminin internalize and accumulate laminin, but not fibronectin receptors.

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