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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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Inhibiting receptor trafficking slows cranial NCC motility on high concentrations of laminin. (A) Internalized cell surface receptors are endocytosed via clathrin-dependent mechanisms to the early endosome. Cargo is then transported either to the late endosome, to the receptor recycling compartments, or rapidly recycled back to the cell surface through a fast recycling pathway. Distinct areas of the pathway are characterized by specific Rab GTPases. The vacuolar proton pump inhibitor bafilomycin A prevents acidification of endocytic structures, thereby impairing transport out of the early endosome. (B) Cumulative distribution plots of cell velocity before drug treatment are shown where each point represents the pre-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs before treatment is 232 μm/h ± 11 (SEM; n = 22) on LM20 (red squares), compared with 196 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.01; t test). (C) Cumulative distribution plots of cell velocity after drug treatment are shown where each point represents the post-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs post-treatment is 174 μm/h ± 10 (SEM; n = 22) on LM20 (red squares), compared with 199 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.05; t test). (D) Cumulative distribution plots of the change in cell velocity after BafA (100 nM) treatment. Each point represents the change in velocity of a single cell after BafA treatment. Cells cultured on LM20 (red squares) and treated with BafA slowed an average of 58 μm/h ± 14 (n = 22) (post-treatment velocities were statistically different from pre-treatment velocities; P < 0.001; paired t test). In contrast, cells cultured on LM1 and treated with BafA (black diamonds; n = 24), and controls, cells cultured on either laminin concentration and treated with vehicle (green triangles; n = 31), did not slow significantly (pre- and post-treatment velocities were not statistically different; P > 0.05, paired t test). Post-treatment velocities of control cells were significantly different from post-treatment velocities of BafA-treated cells on high laminin (P < 0.05; t test). Specific points in each graph are not directly comparable to corresponding points in other graphs, as each point merely reflects a cell's velocity, or change in velocity, in relation to the whole population.
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fig5: Inhibiting receptor trafficking slows cranial NCC motility on high concentrations of laminin. (A) Internalized cell surface receptors are endocytosed via clathrin-dependent mechanisms to the early endosome. Cargo is then transported either to the late endosome, to the receptor recycling compartments, or rapidly recycled back to the cell surface through a fast recycling pathway. Distinct areas of the pathway are characterized by specific Rab GTPases. The vacuolar proton pump inhibitor bafilomycin A prevents acidification of endocytic structures, thereby impairing transport out of the early endosome. (B) Cumulative distribution plots of cell velocity before drug treatment are shown where each point represents the pre-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs before treatment is 232 μm/h ± 11 (SEM; n = 22) on LM20 (red squares), compared with 196 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.01; t test). (C) Cumulative distribution plots of cell velocity after drug treatment are shown where each point represents the post-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs post-treatment is 174 μm/h ± 10 (SEM; n = 22) on LM20 (red squares), compared with 199 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.05; t test). (D) Cumulative distribution plots of the change in cell velocity after BafA (100 nM) treatment. Each point represents the change in velocity of a single cell after BafA treatment. Cells cultured on LM20 (red squares) and treated with BafA slowed an average of 58 μm/h ± 14 (n = 22) (post-treatment velocities were statistically different from pre-treatment velocities; P < 0.001; paired t test). In contrast, cells cultured on LM1 and treated with BafA (black diamonds; n = 24), and controls, cells cultured on either laminin concentration and treated with vehicle (green triangles; n = 31), did not slow significantly (pre- and post-treatment velocities were not statistically different; P > 0.05, paired t test). Post-treatment velocities of control cells were significantly different from post-treatment velocities of BafA-treated cells on high laminin (P < 0.05; t test). Specific points in each graph are not directly comparable to corresponding points in other graphs, as each point merely reflects a cell's velocity, or change in velocity, in relation to the whole population.

Mentions: Bafilomycin A is a specific inhibitor of the vacuolar type H+-ATPase found in all animal cells, plant cells, and microorganisms. BafA does not prevent endocytosis, yet it prevents the acidification of endocytic structures, which impairs the transport of vesicles out of the early endosome (Thomsen et al., 1999). Because internalized receptors destined for both the fast and slow receptor recycling pathways originate in the early endosome, treatment with BafA allows cells to internalize surface receptors but impairs receptor recycling back to the surface (Fig. 5 A). Previous work has shown that BafA treatment retards the rate of transferrin receptor recycling twofold in CHO cells (Presley et al., 1997). We controlled for any nonspecific effects of BafA treatment by comparing the same cell type cultured on two different concentrations of laminin.


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

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

Inhibiting receptor trafficking slows cranial NCC motility on high concentrations of laminin. (A) Internalized cell surface receptors are endocytosed via clathrin-dependent mechanisms to the early endosome. Cargo is then transported either to the late endosome, to the receptor recycling compartments, or rapidly recycled back to the cell surface through a fast recycling pathway. Distinct areas of the pathway are characterized by specific Rab GTPases. The vacuolar proton pump inhibitor bafilomycin A prevents acidification of endocytic structures, thereby impairing transport out of the early endosome. (B) Cumulative distribution plots of cell velocity before drug treatment are shown where each point represents the pre-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs before treatment is 232 μm/h ± 11 (SEM; n = 22) on LM20 (red squares), compared with 196 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.01; t test). (C) Cumulative distribution plots of cell velocity after drug treatment are shown where each point represents the post-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs post-treatment is 174 μm/h ± 10 (SEM; n = 22) on LM20 (red squares), compared with 199 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.05; t test). (D) Cumulative distribution plots of the change in cell velocity after BafA (100 nM) treatment. Each point represents the change in velocity of a single cell after BafA treatment. Cells cultured on LM20 (red squares) and treated with BafA slowed an average of 58 μm/h ± 14 (n = 22) (post-treatment velocities were statistically different from pre-treatment velocities; P < 0.001; paired t test). In contrast, cells cultured on LM1 and treated with BafA (black diamonds; n = 24), and controls, cells cultured on either laminin concentration and treated with vehicle (green triangles; n = 31), did not slow significantly (pre- and post-treatment velocities were not statistically different; P > 0.05, paired t test). Post-treatment velocities of control cells were significantly different from post-treatment velocities of BafA-treated cells on high laminin (P < 0.05; t test). Specific points in each graph are not directly comparable to corresponding points in other graphs, as each point merely reflects a cell's velocity, or change in velocity, in relation to the whole population.
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Related In: Results  -  Collection

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fig5: Inhibiting receptor trafficking slows cranial NCC motility on high concentrations of laminin. (A) Internalized cell surface receptors are endocytosed via clathrin-dependent mechanisms to the early endosome. Cargo is then transported either to the late endosome, to the receptor recycling compartments, or rapidly recycled back to the cell surface through a fast recycling pathway. Distinct areas of the pathway are characterized by specific Rab GTPases. The vacuolar proton pump inhibitor bafilomycin A prevents acidification of endocytic structures, thereby impairing transport out of the early endosome. (B) Cumulative distribution plots of cell velocity before drug treatment are shown where each point represents the pre-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs before treatment is 232 μm/h ± 11 (SEM; n = 22) on LM20 (red squares), compared with 196 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.01; t test). (C) Cumulative distribution plots of cell velocity after drug treatment are shown where each point represents the post-treatment velocity of a single cell. Cumulative percent on the y-axis refers to the percentage of the population traveling that speed or faster. The average velocity of cranial NCCs post-treatment is 174 μm/h ± 10 (SEM; n = 22) on LM20 (red squares), compared with 199 μm/h ± 8 (n = 24) on LM1 (black diamonds). The difference between the two conditions is statistically significant (P < 0.05; t test). (D) Cumulative distribution plots of the change in cell velocity after BafA (100 nM) treatment. Each point represents the change in velocity of a single cell after BafA treatment. Cells cultured on LM20 (red squares) and treated with BafA slowed an average of 58 μm/h ± 14 (n = 22) (post-treatment velocities were statistically different from pre-treatment velocities; P < 0.001; paired t test). In contrast, cells cultured on LM1 and treated with BafA (black diamonds; n = 24), and controls, cells cultured on either laminin concentration and treated with vehicle (green triangles; n = 31), did not slow significantly (pre- and post-treatment velocities were not statistically different; P > 0.05, paired t test). Post-treatment velocities of control cells were significantly different from post-treatment velocities of BafA-treated cells on high laminin (P < 0.05; t test). Specific points in each graph are not directly comparable to corresponding points in other graphs, as each point merely reflects a cell's velocity, or change in velocity, in relation to the whole population.
Mentions: Bafilomycin A is a specific inhibitor of the vacuolar type H+-ATPase found in all animal cells, plant cells, and microorganisms. BafA does not prevent endocytosis, yet it prevents the acidification of endocytic structures, which impairs the transport of vesicles out of the early endosome (Thomsen et al., 1999). Because internalized receptors destined for both the fast and slow receptor recycling pathways originate in the early endosome, treatment with BafA allows cells to internalize surface receptors but impairs receptor recycling back to the surface (Fig. 5 A). Previous work has shown that BafA treatment retards the rate of transferrin receptor recycling twofold in CHO cells (Presley et al., 1997). We controlled for any nonspecific effects of BafA treatment by comparing the same cell type cultured on two different concentrations of laminin.

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