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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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Binding of a PIPK peptide to talin head. (A) Diagram showing the domain structure of human PIPKIγ and the sequences of the PIPK peptides. Underlined residues indicate the talin binding region. (B–E) ITC traces showing the responses when either PIPK pep (B) or Mutant PIPK pep (D) was injected (arrow) into a chamber containing talin head (Tal H). Decreased magnitude of the response indicates talin saturation by PIPK pep. Dose–response curves show that PIPK pep (C), but not Mutant PIPK pep (E), binds to talin head. Data points were derived from B and D and were fit with a nonlinear least squares function (solid line).
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fig2: Binding of a PIPK peptide to talin head. (A) Diagram showing the domain structure of human PIPKIγ and the sequences of the PIPK peptides. Underlined residues indicate the talin binding region. (B–E) ITC traces showing the responses when either PIPK pep (B) or Mutant PIPK pep (D) was injected (arrow) into a chamber containing talin head (Tal H). Decreased magnitude of the response indicates talin saturation by PIPK pep. Dose–response curves show that PIPK pep (C), but not Mutant PIPK pep (E), binds to talin head. Data points were derived from B and D and were fit with a nonlinear least squares function (solid line).

Mentions: Next, we characterized a 14-mer PIPK peptide (PIPK pep) centered around the minimal talin-binding site in human PIPKIγ, WVYSPL (Fig. 2 A; Di Paolo et al., 2002). Pre-incubation of rat brain extracts with PIPK pep nearly completely abolished the ability of anti-talin antibodies to coprecipitate PIP kinase both from rat brain (Fig. 1 G, fourth lane) and lamprey spinal cord extracts (Fig. 1 H; right lane). In contrast, a mutant PIPK peptide (Mut PIPK pep; Fig. 2 A) in which the critical tryptophan was replaced by an alanine had negligible effects (Fig. 1 G, fifth lane). Isothermal titration calorimetry (ITC) revealed that PIPK pep binds to the head of talin in a saturable manner (Fig. 2, B and C) with a KD of 0.65 μM, as determined by fitting to a nonlinear least squares function. This value is roughly in agreement with previous measurements made by nuclear magnetic resonance spectroscopy using a slightly longer PIPKIγ peptide (Barsukov et al., 2003). Mut PIPK pep demonstrated only negligible binding to talin head (Fig. 2, D and E).


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)

Binding of a PIPK peptide to talin head. (A) Diagram showing the domain structure of human PIPKIγ and the sequences of the PIPK peptides. Underlined residues indicate the talin binding region. (B–E) ITC traces showing the responses when either PIPK pep (B) or Mutant PIPK pep (D) was injected (arrow) into a chamber containing talin head (Tal H). Decreased magnitude of the response indicates talin saturation by PIPK pep. Dose–response curves show that PIPK pep (C), but not Mutant PIPK pep (E), binds to talin head. Data points were derived from B and D and were fit with a nonlinear least squares function (solid line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172527&req=5

fig2: Binding of a PIPK peptide to talin head. (A) Diagram showing the domain structure of human PIPKIγ and the sequences of the PIPK peptides. Underlined residues indicate the talin binding region. (B–E) ITC traces showing the responses when either PIPK pep (B) or Mutant PIPK pep (D) was injected (arrow) into a chamber containing talin head (Tal H). Decreased magnitude of the response indicates talin saturation by PIPK pep. Dose–response curves show that PIPK pep (C), but not Mutant PIPK pep (E), binds to talin head. Data points were derived from B and D and were fit with a nonlinear least squares function (solid line).
Mentions: Next, we characterized a 14-mer PIPK peptide (PIPK pep) centered around the minimal talin-binding site in human PIPKIγ, WVYSPL (Fig. 2 A; Di Paolo et al., 2002). Pre-incubation of rat brain extracts with PIPK pep nearly completely abolished the ability of anti-talin antibodies to coprecipitate PIP kinase both from rat brain (Fig. 1 G, fourth lane) and lamprey spinal cord extracts (Fig. 1 H; right lane). In contrast, a mutant PIPK peptide (Mut PIPK pep; Fig. 2 A) in which the critical tryptophan was replaced by an alanine had negligible effects (Fig. 1 G, fifth lane). Isothermal titration calorimetry (ITC) revealed that PIPK pep binds to the head of talin in a saturable manner (Fig. 2, B and C) with a KD of 0.65 μM, as determined by fitting to a nonlinear least squares function. This value is roughly in agreement with previous measurements made by nuclear magnetic resonance spectroscopy using a slightly longer PIPKIγ peptide (Barsukov et al., 2003). Mut PIPK pep demonstrated only negligible binding to talin head (Fig. 2, D and E).

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