Carlstrom LP et al. (2011), Bidirectional remodeling of β1-integrin adhesio...

XB-ART-44496

BMC Biol 2011 Nov 30;9:82. doi: 10.1186/1741-7007-9-82.

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Bidirectional remodeling of β1-integrin adhesions during chemotropic regulation of nerve growth.

Carlstrom LP , Hines JH , Henle SJ , Henley JR .

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Chemotropic factors in the extracellular microenvironment guide nerve growth by acting on the growth cone located at the tip of extending axons. Growth cone extension requires the coordination of cytoskeleton-dependent membrane protrusion and dynamic adhesion to the extracellular matrix, yet how chemotropic factors regulate these events remains an outstanding question. We demonstrated previously that the inhibitory factor myelin-associated glycoprotein (MAG) triggers endocytic removal of the adhesion receptor β1-integrin from the growth cone surface membrane to negatively remodel substrate adhesions during chemorepulsion. Here, we tested how a neurotrophin might affect integrin adhesions. We report that brain-derived neurotropic factor (BDNF) positively regulates the formation of substrate adhesions in axonal growth cones during stimulated outgrowth and prevents removal of β1-integrin adhesions by MAG. Treatment of Xenopus spinal neurons with BDNF rapidly triggered β1-integrin clustering and induced the dynamic formation of nascent vinculin-containing adhesion complexes in the growth cone periphery. Both the formation of nascent β1-integrin adhesions and the stimulation of axon extension by BDNF required cytoplasmic calcium ion signaling and integrin activation at the cell surface. Exposure to MAG decreased the number of β1-integrin adhesions in the growth cone during inhibition of axon extension. In contrast, the BDNF-induced adhesions were resistant to negative remodeling by MAG, correlating with the ability of BDNF pretreatment to counteract MAG-inhibition of axon extension. Pre-exposure to MAG prevented the BDNF-induced formation of β1-integrin adhesions and blocked the stimulation of axon extension by BDNF. Altogether, these findings demonstrate the neurotrophin-dependent formation of integrin-based adhesions in the growth cone and reveal how a positive regulator of substrate adhesions can block the negative remodeling and growth inhibitory effects of MAG. Such bidirectional remodeling may allow the growth cone to rapidly adjust adhesiveness to the extracellular matrix as a general mechanism for governing axon extension. Techniques for manipulating integrin internalization and activation state may be important for overcoming local inhibitory factors after traumatic injury or neurodegenerative disease to enhance regenerative nerve growth.

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Species referenced: Xenopus laevis
Genes referenced: actn1 adm alb bdnf blk fxn mag ntrk2 ptk2

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	Figure 1. BDNF stimulates β1-integrin clustering in the nerve growth cone. (A) Xenopus spinal neuron growth cone immunolabeled for surface β1-integrin after treatment with vehicle alone (Control), or 5 min and 20 min BDNF (50 ng/mL) bath application. Arrowheads denote β1-integrin clusters. Scale bar, 5 μm. (B, C) Quantification of β1-integrin clustering after 5 min and 20 min BDNF treatment expressed as mean number of clusters per growth cone (B) and the percentage of growth cone filopodia that contain at least one β1-integrin cluster (C; see Methods). (D) Quantification of β1-integrin surface levels in the growth cone after 5 min and 20 min BDNF treatment (see Methods). Data are the mean ± standard error of the mean. (n > 200, n/s P > 0.05, P < 0.01, *P < 0.001, ANOVA with Tukey's post hoc analysis.) BDNF: brain-derived neurotrophic factor.
	Figure 2. BDNF triggers the formation of nascent growth cone adhesions. (A) Representative confocal images of dual immunolabeled growth cones showing β1-integrin (red) and known cytoplasmic adhesion components (green: FAK; PY; vinculin; talin; α-actinin) after BDNF treatment (50 ng/mL; 20 min). Arrowheads in the merged images denote spots of co-localization. Scale bar, 5 μm. (B) Quantification of the percentage of total immunostaining for adhesion components, α5-integrin, and TrkB receptors that overlapped with β1-integrin clusters. Data are the mean ± standard error of the mean. (n > 40, n/s P > 0.05, **P < 0.001, ANOVA with Tukey's post hoc analysis.) BDNF: brain-derived neurotrophic factor; FAK: focal adhesion kinase; PY: phosphotyrosine.
	Figure 3. BDNF-induced β1-integrin clustering requires intact lipid microenvironments and functional β1-integrin. (A) Representative immunolabeled images showing β1-integrin after vehicle (BSA), BDNF (50 ng/mL; 20 min), (B) L-t-LacCer (20 μM) and L-t-LacCer plus BDNF, and (C) β1-integrin function blocking antibody (Fxn Blk Ab, 5 μg/mL) alone and plus BDNF treatments. Arrowheads designate clustered β1-integrins. Scale bar, 5 μm. (D) Quantification of β1-integrin clustering according to the treatment groups. Data are the mean ± standard error of the mean. (n > 150, **P < 0.001, ANOVA with Tukey's post hoc analysis.) BDNF: brain-derived neurotrophic factor; BSA: bovine serum albumin; L-t-LacCer: β-D-lactosyl-N-octanoyl-L-threo-sphingosine.
	Figure 4. Disrupting β1-integrin clustering impedes BDNF-dependent axon outgrowth. (A) Axon growth assays with BDNF (50 ng/mL) or L-t-LacCer (20 μM) plus BDNF treatments demonstrating representative growth rates during a 60-min period. Scale bar, 10 μm. (B) Quantification of the axon growth rates of vehicle (BSA), BDNF, L-t-LacCer alone, L-t-LacCer plus BDNF, D-e-LacCer (20 μM), D-e-LacCer plus BDNF, β1-integrin function blocking antibody 2999 (Fxn Blk Ab; 5 μg/mL) alone and plus BDNF, control antibody (Control Ab, 5 μg/mL) and Control Ab plus BDNF treatments. Data are the mean ± standard error of the mean. (n > 150, **p < 0.001, ANOVA with Tukey's post hoc analysis). BDNF: brain-derived neurotrophic factor; BSA: bovine serum albumin; D-e-LacCer: D-lactosyl-β1-1'-N-octanoyl-D-erythro-sphingosine; L-t-LacCer: β-D-lactosyl-N-octanoyl-L-threo-sphingosine.
	Figure 5. BDNF-induced β1-integrin clustering and stimulated axon outgrowth is Ca2+-dependent. (A) Representative immunolabeled growth cones showing the distribution of surface β1-integrin after treatments with vehicle (BSA), BDNF (50 ng/mL), or BAPTA-AM (1 μM; 30 nM [Ca2+]e) plus BDNF. Arrowheads denote β1-integrin clustering. Scale bar, 5 μm. (B) Quantification of β1-integrin clustering according to treatments with vehicle (BSA), BDNF (50 ng/mL), BAPTA-AM (1 μM; 30 nM [Ca2+]e) alone, BAPTA-AM (1 μM; 30 nM [Ca2+]e) plus BDNF, CdCl2 (50 μM; 20 min) alone, and CdCl2 plus BDNF. (C) Quantification of the mean axon growth rate after treatments with vehicle (BSA), BDNF (50 ng/mL), BAPTA-AM (1 μM; 30 nM [Ca2+]e), and BAPTA-AM (1 μM; 30 nM [Ca2+]e) plus BDNF. Data are the mean ± standard error of the mean. (n > 100, P < 0.05, *P < 0.001, ANOVA with Tukey's post hoc analysis.) BDNF: brain-derived neurotrophic factor; BSA: bovine serum albumin; [Ca2+]e: extracellular Ca2+ concentration.
	Figure 6. BDNF priming counteracts inhibitory MAG-effects on β1-integrin clustering and growth inhibition. (A) Representative immunolabeled images showing β1-integrin after control (BSA), MAG (1 μg/mL; 5 min), or combination treatments with BDNF (50 ng/mL; 20 min) and MAG. Detailed time course graphical representation of the combination treatments located in Additional file 8. Arrowheads designate clustered β1-integrins. Scale bar, 5 μm. (B) Quantification of β1-integrin clustering after vehicle (BSA), BDNF (50 ng/mL), and MAG (1 μg/mL) treatments alone or followed by secondary exposure to BDNF or MAG. (C) Quantification of β1-integrin surface levels after vehicle (BSA), BDNF (50 ng/mL), and MAG (1 μg/mL) treatments alone or followed by secondary exposure to BDNF or MAG. (D) Quantification of the mean axon growth rate according to the treatment groups. Data are the mean ± standard error of the mean. (n > 150, P < 0.01, *P < 0.001, ANOVA with Tukey's post hoc analysis.) BDNF: brain-derived neurotrophic factor; BSA: bovine serum albumin; MAG: myelin-associated glycoprotein.
	Figure 7. Comparison of bidirectional β1-integrin remodeling during nerve growth. Summary figure showing the clustering and global surface levels of β1-integrin in the growth cone correlated with the mean axon growth rate for the experimental treatments in Figure 6. Superscripts denote order of treatment. Symbols (+/-) denote positive and negative effects relative to controls.

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