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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 PIPK pep at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either Mut PIPK pep or PIPK pep. In the presence of the Mut PIPK pep (A), only few coated pits are observed. In contrast, numerous clathrin-coated pits (red circles) and large plasma membrane foldings (arrows) are observed in the presence of PIPK pep (B). (C) Gallery showing unconstricted clathrin-coated pits at periactive zones of synapses within PIPK pep–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of synaptic vesicles (D, SVs), the plasma membrane (PM) cross-sectional profile (E), the total number of clathrin-coated profiles (F), and the percentages of coated profiles at various stages of maturation (G) per synapse. Data represent mean values and SEM for 21 synapses from 2 axons injected with PIPK pep and 20 synapses from 2 axons injected with Mutant PIPK pep.
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fig4: Ultrastructural changes produced by PIPK pep at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either Mut PIPK pep or PIPK pep. In the presence of the Mut PIPK pep (A), only few coated pits are observed. In contrast, numerous clathrin-coated pits (red circles) and large plasma membrane foldings (arrows) are observed in the presence of PIPK pep (B). (C) Gallery showing unconstricted clathrin-coated pits at periactive zones of synapses within PIPK pep–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of synaptic vesicles (D, SVs), the plasma membrane (PM) cross-sectional profile (E), the total number of clathrin-coated profiles (F), and the percentages of coated profiles at various stages of maturation (G) per synapse. Data represent mean values and SEM for 21 synapses from 2 axons injected with PIPK pep and 20 synapses from 2 axons injected with Mutant PIPK pep.

Mentions: Next, we examined the effect of PIPK peptide on synaptic vesicle trafficking using EM. Axons were microinjected with either PIPK or mutant peptide, stimulated (20 Hz for 5 min) to induce exocytosis and compensatory synaptic vesicle recycling, and then fixed (Pieribone et al., 1995). Electron micrographs of synapses within mutant PIPK peptide–injected axons revealed the typical large synaptic vesicle clusters and very few clathrin-coated pits (Fig. 4 A). Under these stimulation conditions, synaptic vesicle recycling is very efficient in control synapses. In contrast, images of synapses from PIPK peptide–injected axons revealed numerous clathrin-coated pits and large folds of the plasma membrane at periactive zones that often extended toward the postsynaptic cell (Fig. 4, B and C). In addition, the average number of synaptic vesicles per synapse in PIPK peptide–injected axons was 33% smaller than in mutant PIPK peptide–injected control axons, indicating that synaptic vesicle recycling was perturbed (Fig. 4 D; P < 0.05; t test). A measurement of the plasma membrane cross-sectional profile within a 1-μm radial distance from the outer edge of the active zone revealed a twofold increase in length relative to mutant PIPK pep, denoting a striking expansion of the plasma membrane (Fig. 4 E; P < 0.05 × 10−6; t test). Further, the total number of clathrin-coated profiles per synapse dramatically increased 10-fold in the presence of PIPK pep (Fig. 4 F; P < 0.05 × 10−8; t test). When the coated profiles were staged according to state of maturation, the greatest increase was observed in unconstricted coated pits (Fig. 4 G).


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 PIPK pep at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either Mut PIPK pep or PIPK pep. In the presence of the Mut PIPK pep (A), only few coated pits are observed. In contrast, numerous clathrin-coated pits (red circles) and large plasma membrane foldings (arrows) are observed in the presence of PIPK pep (B). (C) Gallery showing unconstricted clathrin-coated pits at periactive zones of synapses within PIPK pep–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of synaptic vesicles (D, SVs), the plasma membrane (PM) cross-sectional profile (E), the total number of clathrin-coated profiles (F), and the percentages of coated profiles at various stages of maturation (G) per synapse. Data represent mean values and SEM for 21 synapses from 2 axons injected with PIPK pep and 20 synapses from 2 axons injected with Mutant PIPK pep.
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Related In: Results  -  Collection

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fig4: Ultrastructural changes produced by PIPK pep at lamprey synapses. (A and B) Electron micrographs of stimulated synapses after axonal injection of either Mut PIPK pep or PIPK pep. In the presence of the Mut PIPK pep (A), only few coated pits are observed. In contrast, numerous clathrin-coated pits (red circles) and large plasma membrane foldings (arrows) are observed in the presence of PIPK pep (B). (C) Gallery showing unconstricted clathrin-coated pits at periactive zones of synapses within PIPK pep–injected axons. Bars, 0.2 μm. (D–G) Quantification of the number of synaptic vesicles (D, SVs), the plasma membrane (PM) cross-sectional profile (E), the total number of clathrin-coated profiles (F), and the percentages of coated profiles at various stages of maturation (G) per synapse. Data represent mean values and SEM for 21 synapses from 2 axons injected with PIPK pep and 20 synapses from 2 axons injected with Mutant PIPK pep.
Mentions: Next, we examined the effect of PIPK peptide on synaptic vesicle trafficking using EM. Axons were microinjected with either PIPK or mutant peptide, stimulated (20 Hz for 5 min) to induce exocytosis and compensatory synaptic vesicle recycling, and then fixed (Pieribone et al., 1995). Electron micrographs of synapses within mutant PIPK peptide–injected axons revealed the typical large synaptic vesicle clusters and very few clathrin-coated pits (Fig. 4 A). Under these stimulation conditions, synaptic vesicle recycling is very efficient in control synapses. In contrast, images of synapses from PIPK peptide–injected axons revealed numerous clathrin-coated pits and large folds of the plasma membrane at periactive zones that often extended toward the postsynaptic cell (Fig. 4, B and C). In addition, the average number of synaptic vesicles per synapse in PIPK peptide–injected axons was 33% smaller than in mutant PIPK peptide–injected control axons, indicating that synaptic vesicle recycling was perturbed (Fig. 4 D; P < 0.05; t test). A measurement of the plasma membrane cross-sectional profile within a 1-μm radial distance from the outer edge of the active zone revealed a twofold increase in length relative to mutant PIPK pep, denoting a striking expansion of the plasma membrane (Fig. 4 E; P < 0.05 × 10−6; t test). Further, the total number of clathrin-coated profiles per synapse dramatically increased 10-fold in the presence of PIPK pep (Fig. 4 F; P < 0.05 × 10−8; t test). When the coated profiles were staged according to state of maturation, the greatest increase was observed in unconstricted coated pits (Fig. 4 G).

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