Limits...
Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM.

Chen MS, Almeida EA, Huovila AP, Takahashi Y, Shaw LM, Mercurio AM, White JM - J. Cell Biol. (1999)

Bottom Line: In Ca2+-containing media, laminin E8 beads did not bind to eggs.Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding.Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.

ABSTRACT
Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). In the present study we tested the hypothesis that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin beta or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin beta beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

Show MeSH

Related in: MedlinePlus

Effect of latrunculin  on staining of egg actin with  FITC-phalloidin and binding of  fertilin- and laminin E8–coated  beads to eggs. Eggs were collected in M199 medium and  treated with acidic Tyrode's solution to remove the zonae and  allowed to recover for 3 h at  37°C in M199 as described in  Materials and Methods. Where  indicated, eggs were pretreated  for 1 h at 37°C with 20 μg/ml latrunculin A in M199. For actin  staining (top) eggs were fixed  and treated with FITC-phalloidin as described in Materials  and Methods. For bead binding  (middle and bottom) eggs were  washed into Puck's saline A  containing 1 mM CaCl2 (CT and latrunculin) or 0.5 mM MnCl2 (Mn2+) and incubated with fertilin β– (middle) or laminin E8– (bottom)  coated fluorescent beads. Activation of laminin E8 binding by Mn2+ is shown for comparison (bottom, far right). After 1 h at 37°C, the  eggs were washed and observed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown. Latrunculin did not inhibit binding of laminin E8 beads to Mn2+-treated eggs (Chen, M., and J. White, unpublished  data).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132920&req=5

Figure 11: Effect of latrunculin on staining of egg actin with FITC-phalloidin and binding of fertilin- and laminin E8–coated beads to eggs. Eggs were collected in M199 medium and treated with acidic Tyrode's solution to remove the zonae and allowed to recover for 3 h at 37°C in M199 as described in Materials and Methods. Where indicated, eggs were pretreated for 1 h at 37°C with 20 μg/ml latrunculin A in M199. For actin staining (top) eggs were fixed and treated with FITC-phalloidin as described in Materials and Methods. For bead binding (middle and bottom) eggs were washed into Puck's saline A containing 1 mM CaCl2 (CT and latrunculin) or 0.5 mM MnCl2 (Mn2+) and incubated with fertilin β– (middle) or laminin E8– (bottom) coated fluorescent beads. Activation of laminin E8 binding by Mn2+ is shown for comparison (bottom, far right). After 1 h at 37°C, the eggs were washed and observed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown. Latrunculin did not inhibit binding of laminin E8 beads to Mn2+-treated eggs (Chen, M., and J. White, unpublished data).

Mentions: Integrin function and clustering have been shown to be dependent upon cytoskeletal linkages (Kucik et al., 1996; Lub et al., 1997; Yauch et al., 1997). Therefore, we examined the effects of latrunculin, an agent that inhibits actin polymerization and sequesters actin monomers (Spector et al., 1989; Lamaze et al., 1997), on the binding of fertilin β– and laminin E8–coated beads to eggs. Fertilization-competent mouse eggs normally display dense cortical actin, as visualized by phalloidin staining, particularly concentrated over the meiotic spindle (Reima and Lehtonen, 1985; Longo, 1987). Treatment of eggs with latrunculin significantly decreased the amount of polymerized actin stained with phalloidin (Fig. 11, top). Parallel treatment with latrunculin decreased fertilin β bead binding to eggs (Fig. 11, middle), but did not increase laminin E8 bead binding (Fig. 11, bottom).


Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM.

Chen MS, Almeida EA, Huovila AP, Takahashi Y, Shaw LM, Mercurio AM, White JM - J. Cell Biol. (1999)

Effect of latrunculin  on staining of egg actin with  FITC-phalloidin and binding of  fertilin- and laminin E8–coated  beads to eggs. Eggs were collected in M199 medium and  treated with acidic Tyrode's solution to remove the zonae and  allowed to recover for 3 h at  37°C in M199 as described in  Materials and Methods. Where  indicated, eggs were pretreated  for 1 h at 37°C with 20 μg/ml latrunculin A in M199. For actin  staining (top) eggs were fixed  and treated with FITC-phalloidin as described in Materials  and Methods. For bead binding  (middle and bottom) eggs were  washed into Puck's saline A  containing 1 mM CaCl2 (CT and latrunculin) or 0.5 mM MnCl2 (Mn2+) and incubated with fertilin β– (middle) or laminin E8– (bottom)  coated fluorescent beads. Activation of laminin E8 binding by Mn2+ is shown for comparison (bottom, far right). After 1 h at 37°C, the  eggs were washed and observed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown. Latrunculin did not inhibit binding of laminin E8 beads to Mn2+-treated eggs (Chen, M., and J. White, unpublished  data).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132920&req=5

Figure 11: Effect of latrunculin on staining of egg actin with FITC-phalloidin and binding of fertilin- and laminin E8–coated beads to eggs. Eggs were collected in M199 medium and treated with acidic Tyrode's solution to remove the zonae and allowed to recover for 3 h at 37°C in M199 as described in Materials and Methods. Where indicated, eggs were pretreated for 1 h at 37°C with 20 μg/ml latrunculin A in M199. For actin staining (top) eggs were fixed and treated with FITC-phalloidin as described in Materials and Methods. For bead binding (middle and bottom) eggs were washed into Puck's saline A containing 1 mM CaCl2 (CT and latrunculin) or 0.5 mM MnCl2 (Mn2+) and incubated with fertilin β– (middle) or laminin E8– (bottom) coated fluorescent beads. Activation of laminin E8 binding by Mn2+ is shown for comparison (bottom, far right). After 1 h at 37°C, the eggs were washed and observed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown. Latrunculin did not inhibit binding of laminin E8 beads to Mn2+-treated eggs (Chen, M., and J. White, unpublished data).
Mentions: Integrin function and clustering have been shown to be dependent upon cytoskeletal linkages (Kucik et al., 1996; Lub et al., 1997; Yauch et al., 1997). Therefore, we examined the effects of latrunculin, an agent that inhibits actin polymerization and sequesters actin monomers (Spector et al., 1989; Lamaze et al., 1997), on the binding of fertilin β– and laminin E8–coated beads to eggs. Fertilization-competent mouse eggs normally display dense cortical actin, as visualized by phalloidin staining, particularly concentrated over the meiotic spindle (Reima and Lehtonen, 1985; Longo, 1987). Treatment of eggs with latrunculin significantly decreased the amount of polymerized actin stained with phalloidin (Fig. 11, top). Parallel treatment with latrunculin decreased fertilin β bead binding to eggs (Fig. 11, middle), but did not increase laminin E8 bead binding (Fig. 11, bottom).

Bottom Line: In Ca2+-containing media, laminin E8 beads did not bind to eggs.Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding.Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.

ABSTRACT
Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). In the present study we tested the hypothesis that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin beta or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin beta beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

Show MeSH
Related in: MedlinePlus