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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 NCCs recycle internalized integrin α6 back to the cell surface. Cells cultured on high laminin concentrations were allowed to internalize anti-integrin α6 Fab bound to surface receptors for 30 min. The remaining cell surface Fab was blocked and cells were reincubated at 37°C for 20 or 90 min to allow for exocytosis of the integrin α6–Fab complex. (A) Recycled integrin α6 was detected by labeling the unblocked Fab that had reappeared on the surface (red). Cells were also stained for actin (green) as a permeabilization control. Control cells were intentionally permeabilized to show total integrin α6 and actin staining. Bars, 20 μm. (B and C) Average intensities per unit area + SEM (arbitrary units) of integrin α6 and actin fluorescence were determined for at least 20 cells from three independent experiments for each condition. (B) Measurements from permeabilized control cells, as shown in the first column in A, have significantly higher integrin α6 and actin fluorescence than unpermeabilized 0-min controls, as shown in C (P < 0.001; t test). (C) In unpermeabilized cells, actin fluorescence is constant, whereas integrin α6 fluorescence increases at both 20 and 90 min. Asterisk indicates significant difference from 0-min levels (P < 0.0001; t test).
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fig4: Cranial NCCs recycle internalized integrin α6 back to the cell surface. Cells cultured on high laminin concentrations were allowed to internalize anti-integrin α6 Fab bound to surface receptors for 30 min. The remaining cell surface Fab was blocked and cells were reincubated at 37°C for 20 or 90 min to allow for exocytosis of the integrin α6–Fab complex. (A) Recycled integrin α6 was detected by labeling the unblocked Fab that had reappeared on the surface (red). Cells were also stained for actin (green) as a permeabilization control. Control cells were intentionally permeabilized to show total integrin α6 and actin staining. Bars, 20 μm. (B and C) Average intensities per unit area + SEM (arbitrary units) of integrin α6 and actin fluorescence were determined for at least 20 cells from three independent experiments for each condition. (B) Measurements from permeabilized control cells, as shown in the first column in A, have significantly higher integrin α6 and actin fluorescence than unpermeabilized 0-min controls, as shown in C (P < 0.001; t test). (C) In unpermeabilized cells, actin fluorescence is constant, whereas integrin α6 fluorescence increases at both 20 and 90 min. Asterisk indicates significant difference from 0-min levels (P < 0.0001; t test).

Mentions: Next, we investigated the fate of internalized surface integrin α6 in cranial NCCs cultured on high laminin concentrations. In other fast-moving cells and growth cones, internalized receptors are recycled back to the leading edge rather than being degraded (Lawson and Maxfield, 1995; Kamiguchi and Lemmon, 2000). Using an adapted immunohistochemical technique based on that of Kamiguchi and Lemmon (2000) we examined the recycling of integrin α6 in cranial NCCs. Surface α6 was labeled with anti-α6-Fab while cells were allowed to endocytose and traffic receptors for 30 min at 37°C. Cells were cooled to room temperature to prevent further trafficking and Fab remaining at the surface was blocked with unconjugated secondary antibodies. Subsequently, cells were allowed to traffic internalized receptors at 37°C for 20 or 90 min, and any integrin α6-Fab complexes that returned to the surface were detected with labeled secondary antibodies. Intentionally permeabilized cells (Fig. 4 A, first column; Fig. 4 B) could be distinguished from unpermeabilized cells by significantly higher integrin α6 and actin fluorescence. In the 0-min condition, cells fixed at the beginning of the recycling period and reincubated for 90 min had low fluorescence of both integrin α6 and actin (Fig. 4 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 NCCs recycle internalized integrin α6 back to the cell surface. Cells cultured on high laminin concentrations were allowed to internalize anti-integrin α6 Fab bound to surface receptors for 30 min. The remaining cell surface Fab was blocked and cells were reincubated at 37°C for 20 or 90 min to allow for exocytosis of the integrin α6–Fab complex. (A) Recycled integrin α6 was detected by labeling the unblocked Fab that had reappeared on the surface (red). Cells were also stained for actin (green) as a permeabilization control. Control cells were intentionally permeabilized to show total integrin α6 and actin staining. Bars, 20 μm. (B and C) Average intensities per unit area + SEM (arbitrary units) of integrin α6 and actin fluorescence were determined for at least 20 cells from three independent experiments for each condition. (B) Measurements from permeabilized control cells, as shown in the first column in A, have significantly higher integrin α6 and actin fluorescence than unpermeabilized 0-min controls, as shown in C (P < 0.001; t test). (C) In unpermeabilized cells, actin fluorescence is constant, whereas integrin α6 fluorescence increases at both 20 and 90 min. Asterisk indicates significant difference from 0-min levels (P < 0.0001; t test).
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fig4: Cranial NCCs recycle internalized integrin α6 back to the cell surface. Cells cultured on high laminin concentrations were allowed to internalize anti-integrin α6 Fab bound to surface receptors for 30 min. The remaining cell surface Fab was blocked and cells were reincubated at 37°C for 20 or 90 min to allow for exocytosis of the integrin α6–Fab complex. (A) Recycled integrin α6 was detected by labeling the unblocked Fab that had reappeared on the surface (red). Cells were also stained for actin (green) as a permeabilization control. Control cells were intentionally permeabilized to show total integrin α6 and actin staining. Bars, 20 μm. (B and C) Average intensities per unit area + SEM (arbitrary units) of integrin α6 and actin fluorescence were determined for at least 20 cells from three independent experiments for each condition. (B) Measurements from permeabilized control cells, as shown in the first column in A, have significantly higher integrin α6 and actin fluorescence than unpermeabilized 0-min controls, as shown in C (P < 0.001; t test). (C) In unpermeabilized cells, actin fluorescence is constant, whereas integrin α6 fluorescence increases at both 20 and 90 min. Asterisk indicates significant difference from 0-min levels (P < 0.0001; t test).
Mentions: Next, we investigated the fate of internalized surface integrin α6 in cranial NCCs cultured on high laminin concentrations. In other fast-moving cells and growth cones, internalized receptors are recycled back to the leading edge rather than being degraded (Lawson and Maxfield, 1995; Kamiguchi and Lemmon, 2000). Using an adapted immunohistochemical technique based on that of Kamiguchi and Lemmon (2000) we examined the recycling of integrin α6 in cranial NCCs. Surface α6 was labeled with anti-α6-Fab while cells were allowed to endocytose and traffic receptors for 30 min at 37°C. Cells were cooled to room temperature to prevent further trafficking and Fab remaining at the surface was blocked with unconjugated secondary antibodies. Subsequently, cells were allowed to traffic internalized receptors at 37°C for 20 or 90 min, and any integrin α6-Fab complexes that returned to the surface were detected with labeled secondary antibodies. Intentionally permeabilized cells (Fig. 4 A, first column; Fig. 4 B) could be distinguished from unpermeabilized cells by significantly higher integrin α6 and actin fluorescence. In the 0-min condition, cells fixed at the beginning of the recycling period and reincubated for 90 min had low fluorescence of both integrin α6 and actin (Fig. 4 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