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A role for talin in presynaptic function.

Morgan JR, Di Paolo G, Werner H, Shchedrina VA, Pypaert M, Pieribone VA, De Camilli P - J. Cell Biol. (2004)

Bottom Line: To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin-PIP kinase interaction and then examined their effects on synaptic structure.A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed.Thus, the interaction of PIP kinase with talin in presynaptic compartments provides a mechanism to coordinate PI(4,5)P(2) synthesis, actin dynamics, and endocytosis, and further supports a functional link between actin and clathrin-mediated endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06519, USA.

ABSTRACT
Talin, an adaptor between integrin and the actin cytoskeleton at sites of cell adhesion, was recently found to be present at neuronal synapses, where its function remains unknown. Talin interacts with phosphatidylinositol-(4)-phosphate 5-kinase type Igamma, the major phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)]-synthesizing enzyme in brain. To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin-PIP kinase interaction and then examined their effects on synaptic structure. A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed. The endocytic defect included an accumulation of clathrin-coated pits with wide necks, as previously observed after perturbing actin at these synapses. Thus, the interaction of PIP kinase with talin in presynaptic compartments provides a mechanism to coordinate PI(4,5)P(2) synthesis, actin dynamics, and endocytosis, and further supports a functional link between actin and clathrin-mediated endocytosis.

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Ultrastructural changes produced by talin head at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either GST or GST-talin head. In the presence of GST (A), only few coated pits are observed. In contrast, more clathrin-coated pits (red circles) and large membrane foldings (arrows) are observed in the presence of GST-talin head (B). (C) Gallery showing unconstricted clathrin-coated pits in periactive zones within GST-talin head–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of SVs (D), the PM cross-sectional profile (E), the total number of clathrin-coated profiles (F), and percentages of coated profiles at various stages of maturation (G) per synapse cross section. Data represent mean values and SEM of 10 synapses from 4 axons injected with GST controls and 11 synapses from 4 axons injected with GST-talin head.
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fig5: Ultrastructural changes produced by talin head at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either GST or GST-talin head. In the presence of GST (A), only few coated pits are observed. In contrast, more clathrin-coated pits (red circles) and large membrane foldings (arrows) are observed in the presence of GST-talin head (B). (C) Gallery showing unconstricted clathrin-coated pits in periactive zones within GST-talin head–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of SVs (D), the PM cross-sectional profile (E), the total number of clathrin-coated profiles (F), and percentages of coated profiles at various stages of maturation (G) per synapse cross section. Data represent mean values and SEM of 10 synapses from 4 axons injected with GST controls and 11 synapses from 4 axons injected with GST-talin head.

Mentions: To further assess the specificity of the effects on synaptic vesicle recycling, we injected GST-talin head. This reagent is expected to compete the interactions of the FERM domain of endogenous talin with its binding partners. Electron micrographs of stimulated synapses within axons injected with GST-talin head revealed a perturbation of clathrin-mediated endocytosis at synapses similar to that observed with PIPK pep (Fig. 5). Compared with GST controls, GST-talin head caused an increase in the length of the plasma membrane cross-sectional profile around synapses (Fig. 5, A, B, and E; P < 0.05 t test), and a twofold increase in the number of clathrin-coated profiles (Fig. 5 F). Like the PIPK peptide, GST-talin head specifically increased the number of unconstricted coated pits (Fig. 5, B, C, and G; P < 0.05 t test), further supporting a presynaptic role for talin in coated vesicle maturation. Only a modest, nonstatistically significant decrease in the number of synaptic vesicles was observed in the presence of GST-talin head (Fig. 5 D). The less dramatic phenotype observed with GST-talin head, rather than with the PIPK peptide, likely reflects the lower axonal concentration that was achieved by microinjection of this fusion protein, which was estimated to be in the range of 0.5–2.5 μM (i.e., in the same range of the KD of the talin–PIPK interaction). However, the similarity to the PIPK peptide phenotype indicates that GST-talin head targeted the same process.


A role for talin in presynaptic function.

Morgan JR, Di Paolo G, Werner H, Shchedrina VA, Pypaert M, Pieribone VA, De Camilli P - J. Cell Biol. (2004)

Ultrastructural changes produced by talin head at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either GST or GST-talin head. In the presence of GST (A), only few coated pits are observed. In contrast, more clathrin-coated pits (red circles) and large membrane foldings (arrows) are observed in the presence of GST-talin head (B). (C) Gallery showing unconstricted clathrin-coated pits in periactive zones within GST-talin head–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of SVs (D), the PM cross-sectional profile (E), the total number of clathrin-coated profiles (F), and percentages of coated profiles at various stages of maturation (G) per synapse cross section. Data represent mean values and SEM of 10 synapses from 4 axons injected with GST controls and 11 synapses from 4 axons injected with GST-talin head.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172527&req=5

fig5: Ultrastructural changes produced by talin head at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either GST or GST-talin head. In the presence of GST (A), only few coated pits are observed. In contrast, more clathrin-coated pits (red circles) and large membrane foldings (arrows) are observed in the presence of GST-talin head (B). (C) Gallery showing unconstricted clathrin-coated pits in periactive zones within GST-talin head–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of SVs (D), the PM cross-sectional profile (E), the total number of clathrin-coated profiles (F), and percentages of coated profiles at various stages of maturation (G) per synapse cross section. Data represent mean values and SEM of 10 synapses from 4 axons injected with GST controls and 11 synapses from 4 axons injected with GST-talin head.
Mentions: To further assess the specificity of the effects on synaptic vesicle recycling, we injected GST-talin head. This reagent is expected to compete the interactions of the FERM domain of endogenous talin with its binding partners. Electron micrographs of stimulated synapses within axons injected with GST-talin head revealed a perturbation of clathrin-mediated endocytosis at synapses similar to that observed with PIPK pep (Fig. 5). Compared with GST controls, GST-talin head caused an increase in the length of the plasma membrane cross-sectional profile around synapses (Fig. 5, A, B, and E; P < 0.05 t test), and a twofold increase in the number of clathrin-coated profiles (Fig. 5 F). Like the PIPK peptide, GST-talin head specifically increased the number of unconstricted coated pits (Fig. 5, B, C, and G; P < 0.05 t test), further supporting a presynaptic role for talin in coated vesicle maturation. Only a modest, nonstatistically significant decrease in the number of synaptic vesicles was observed in the presence of GST-talin head (Fig. 5 D). The less dramatic phenotype observed with GST-talin head, rather than with the PIPK peptide, likely reflects the lower axonal concentration that was achieved by microinjection of this fusion protein, which was estimated to be in the range of 0.5–2.5 μM (i.e., in the same range of the KD of the talin–PIPK interaction). However, the similarity to the PIPK peptide phenotype indicates that GST-talin head targeted the same process.

Bottom Line: To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin-PIP kinase interaction and then examined their effects on synaptic structure.A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed.Thus, the interaction of PIP kinase with talin in presynaptic compartments provides a mechanism to coordinate PI(4,5)P(2) synthesis, actin dynamics, and endocytosis, and further supports a functional link between actin and clathrin-mediated endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06519, USA.

ABSTRACT
Talin, an adaptor between integrin and the actin cytoskeleton at sites of cell adhesion, was recently found to be present at neuronal synapses, where its function remains unknown. Talin interacts with phosphatidylinositol-(4)-phosphate 5-kinase type Igamma, the major phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)]-synthesizing enzyme in brain. To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin-PIP kinase interaction and then examined their effects on synaptic structure. A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed. The endocytic defect included an accumulation of clathrin-coated pits with wide necks, as previously observed after perturbing actin at these synapses. Thus, the interaction of PIP kinase with talin in presynaptic compartments provides a mechanism to coordinate PI(4,5)P(2) synthesis, actin dynamics, and endocytosis, and further supports a functional link between actin and clathrin-mediated endocytosis.

Show MeSH
Related in: MedlinePlus