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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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Internalized integrins colocalize with receptor recycling pathway markers in a substratum-dependent manner. Cranial NCCs were cultured on high concentrations of laminin (LM20) and stained for rabs 4 and 11 (red), and either integrin α5 or integrin α6 (green). (A) Confocal z-sections were obtained and the area indicated (white box) zoomed below. Although the whole-cell image represents a compressed z-series, the zoomed portion is one 0.2-μm section. Three-dimensional reconstructions show XZ and YZ cross sections of the white-boxed area within the zoomed portion to aid in determining true colocalization events. (B) Quantification of the amount of rab+ vesicles with integrin colocalization. Integrin α5 is only found in 21% ± 2.0 (mean ± SEM) of rab+ vesicles on LM20, and 18% ± 2.2 of rab+ vesicles on FN20. In contrast, integrin α6 is found in 41% ± 3.2 of rab+ vesicles on LM20, compared with only 17% ± 1.9 of rab+ vesicles on FN20. These results represent four independent experiments (total cells analyzed: n > 10 for each condition). Asterisk indicates significant difference from the other conditions (P < 0.001; t test).
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fig3: Internalized integrins colocalize with receptor recycling pathway markers in a substratum-dependent manner. Cranial NCCs were cultured on high concentrations of laminin (LM20) and stained for rabs 4 and 11 (red), and either integrin α5 or integrin α6 (green). (A) Confocal z-sections were obtained and the area indicated (white box) zoomed below. Although the whole-cell image represents a compressed z-series, the zoomed portion is one 0.2-μm section. Three-dimensional reconstructions show XZ and YZ cross sections of the white-boxed area within the zoomed portion to aid in determining true colocalization events. (B) Quantification of the amount of rab+ vesicles with integrin colocalization. Integrin α5 is only found in 21% ± 2.0 (mean ± SEM) of rab+ vesicles on LM20, and 18% ± 2.2 of rab+ vesicles on FN20. In contrast, integrin α6 is found in 41% ± 3.2 of rab+ vesicles on LM20, compared with only 17% ± 1.9 of rab+ vesicles on FN20. These results represent four independent experiments (total cells analyzed: n > 10 for each condition). Asterisk indicates significant difference from the other conditions (P < 0.001; t test).

Mentions: On high laminin concentrations, both integrin α5 and integrin α6 colocalized with vesicles positive for either rab4 or rab11, or double-positive for both (rab+ vesicles), but a significantly greater percentage of rab+ vesicles contained integrin α6 (40%) compared with integrin α5 (21%) (Fig. 3, A and B; Table I). Integrin α5 appeared to be largely localized in intracellular compartments that were neither rab 4 nor rab11 positive (rab− vesicles), suggesting that the majority of α5 integrin does not participate in receptor recycling. On high fibronectin concentrations, both integrin α5+ and integrin α6+ vesicles colocalized with rab+ vesicles to similar extents (18 and 17%, respectively) (Fig. 3 B; Table I). Thus, in cranial NCCs cultured on high concentrations of laminin, twice as many recycling vesicles contain the relevant receptor, integrin α6, as the nonrelevant receptor, integrin α5. Furthermore, in cranial NCCs cultured on high concentrations of fibronectin, neither receptor appears to be selectively targeted to the receptor recycling pathway (Table I).


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

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

Internalized integrins colocalize with receptor recycling pathway markers in a substratum-dependent manner. Cranial NCCs were cultured on high concentrations of laminin (LM20) and stained for rabs 4 and 11 (red), and either integrin α5 or integrin α6 (green). (A) Confocal z-sections were obtained and the area indicated (white box) zoomed below. Although the whole-cell image represents a compressed z-series, the zoomed portion is one 0.2-μm section. Three-dimensional reconstructions show XZ and YZ cross sections of the white-boxed area within the zoomed portion to aid in determining true colocalization events. (B) Quantification of the amount of rab+ vesicles with integrin colocalization. Integrin α5 is only found in 21% ± 2.0 (mean ± SEM) of rab+ vesicles on LM20, and 18% ± 2.2 of rab+ vesicles on FN20. In contrast, integrin α6 is found in 41% ± 3.2 of rab+ vesicles on LM20, compared with only 17% ± 1.9 of rab+ vesicles on FN20. These results represent four independent experiments (total cells analyzed: n > 10 for each condition). Asterisk indicates significant difference from the other conditions (P < 0.001; t test).
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Related In: Results  -  Collection

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fig3: Internalized integrins colocalize with receptor recycling pathway markers in a substratum-dependent manner. Cranial NCCs were cultured on high concentrations of laminin (LM20) and stained for rabs 4 and 11 (red), and either integrin α5 or integrin α6 (green). (A) Confocal z-sections were obtained and the area indicated (white box) zoomed below. Although the whole-cell image represents a compressed z-series, the zoomed portion is one 0.2-μm section. Three-dimensional reconstructions show XZ and YZ cross sections of the white-boxed area within the zoomed portion to aid in determining true colocalization events. (B) Quantification of the amount of rab+ vesicles with integrin colocalization. Integrin α5 is only found in 21% ± 2.0 (mean ± SEM) of rab+ vesicles on LM20, and 18% ± 2.2 of rab+ vesicles on FN20. In contrast, integrin α6 is found in 41% ± 3.2 of rab+ vesicles on LM20, compared with only 17% ± 1.9 of rab+ vesicles on FN20. These results represent four independent experiments (total cells analyzed: n > 10 for each condition). Asterisk indicates significant difference from the other conditions (P < 0.001; t test).
Mentions: On high laminin concentrations, both integrin α5 and integrin α6 colocalized with vesicles positive for either rab4 or rab11, or double-positive for both (rab+ vesicles), but a significantly greater percentage of rab+ vesicles contained integrin α6 (40%) compared with integrin α5 (21%) (Fig. 3, A and B; Table I). Integrin α5 appeared to be largely localized in intracellular compartments that were neither rab 4 nor rab11 positive (rab− vesicles), suggesting that the majority of α5 integrin does not participate in receptor recycling. On high fibronectin concentrations, both integrin α5+ and integrin α6+ vesicles colocalized with rab+ vesicles to similar extents (18 and 17%, respectively) (Fig. 3 B; Table I). Thus, in cranial NCCs cultured on high concentrations of laminin, twice as many recycling vesicles contain the relevant receptor, integrin α6, as the nonrelevant receptor, integrin α5. Furthermore, in cranial NCCs cultured on high concentrations of fibronectin, neither receptor appears to be selectively targeted to the receptor recycling pathway (Table I).

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