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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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Interaction of PIPK with talin and synaptic localization of talin in the lamprey spinal cord. (A) Western blot with an anti-talin antibody of rat brain extract (load) and the material affinity purified from such extract by either GST or GST-PIPKIγ tail in pull-down experiments (S, supernatant; P, pellet). The top talin band is pulled down selectively by GST-PIPKIγ tail. (B) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from rat brain extracts (ext.) showing specificity of the talin–PIPKIγ interaction. (C) Western blot with an anti-talin antibody of lamprey extract (load) and of the material purified by either GST or GST-PIPKIγ tail. (D) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from lamprey spinal cord extracts. (E) Western blot with an anti-PIPKIγ antibody of lamprey extract (load) and of the material purified by either GST or GST-talin head. The 90-kD doublet is specifically affinity purified by GST-talin head. (F) TLC demonstrating the generation of phosphoinositides from lamprey extracts by immunoprecipitates of PIPKIγ antibody (α-PIPK IP) or control IgGs (ctrl IgGs). Note the prominent [32P]PIP2 band produced by the α-PIPK IP. (G, top panels) Western blots of anti-talin immunoprecipitates from rat brain extracts showing that coprecipitation of PIPKIγ (“α-talin IP” lane) is prevented by PIPK pep but not mutant PIPK pep (100 μM). (G, bottom panels) TLC and quantification of [32P]PIP2 showing correlation between PIPKIγ coprecipitation and PIP2-synthesizing activity. (cpm, counts per minute). (H) Western blot of anti-talin immunoprecipitates from lamprey spinal cord extracts showing that the PIPK pep (300 μM) prevents coprecipitation of PIPK. (I) Double immunofluorescence of cross sections of lamprey spinal cord stained for talin (red) and for the presynaptic marker, synapsin (green). Arrows indicate synapses in the neuropil. Asterisks indicate reticulospinal axons. Note the colocalization of talin with synapsin at the reticulospinal synapses (insets).
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fig1: Interaction of PIPK with talin and synaptic localization of talin in the lamprey spinal cord. (A) Western blot with an anti-talin antibody of rat brain extract (load) and the material affinity purified from such extract by either GST or GST-PIPKIγ tail in pull-down experiments (S, supernatant; P, pellet). The top talin band is pulled down selectively by GST-PIPKIγ tail. (B) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from rat brain extracts (ext.) showing specificity of the talin–PIPKIγ interaction. (C) Western blot with an anti-talin antibody of lamprey extract (load) and of the material purified by either GST or GST-PIPKIγ tail. (D) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from lamprey spinal cord extracts. (E) Western blot with an anti-PIPKIγ antibody of lamprey extract (load) and of the material purified by either GST or GST-talin head. The 90-kD doublet is specifically affinity purified by GST-talin head. (F) TLC demonstrating the generation of phosphoinositides from lamprey extracts by immunoprecipitates of PIPKIγ antibody (α-PIPK IP) or control IgGs (ctrl IgGs). Note the prominent [32P]PIP2 band produced by the α-PIPK IP. (G, top panels) Western blots of anti-talin immunoprecipitates from rat brain extracts showing that coprecipitation of PIPKIγ (“α-talin IP” lane) is prevented by PIPK pep but not mutant PIPK pep (100 μM). (G, bottom panels) TLC and quantification of [32P]PIP2 showing correlation between PIPKIγ coprecipitation and PIP2-synthesizing activity. (cpm, counts per minute). (H) Western blot of anti-talin immunoprecipitates from lamprey spinal cord extracts showing that the PIPK pep (300 μM) prevents coprecipitation of PIPK. (I) Double immunofluorescence of cross sections of lamprey spinal cord stained for talin (red) and for the presynaptic marker, synapsin (green). Arrows indicate synapses in the neuropil. Asterisks indicate reticulospinal axons. Note the colocalization of talin with synapsin at the reticulospinal synapses (insets).

Mentions: In Western blots of rat brain protein extracts, talin immunoreactivity migrates as a 230-kD band (Fig. 1 A) and a 190-kD proteolytic fragment lacking the head domain (Bolton et al., 1997; Fig. 1 A). Affinity chromatography using a GST fusion protein of the 28-aa COOH-terminal tail of human PIPKIγ (GST-PIPKIγ tail) resulted in the purification of the upper talin band, as expected (Fig. 1 A; Di Paolo et al., 2002). The proteolytic fragment, which lacks the PIPKIγ binding site, remained in the supernatant. Talin was the only major band retained by GST-PIPKIγ tail, as shown by Coomassie blue staining of the affinity-purified material (Fig. 1 B). Similarly, anti-talin antibodies recognized two proteins in lamprey spinal cord extracts of the appropriate size for talin and its proteolytic fragment (Fig. 1 C), and the larger protein, which corresponded to a major band visible by Coomassie blue (Fig. 1 D), was pulled down by GST-PIPKIγ tail. Western blots of lamprey extracts with an anti-PIPKIγ antibody raised against the 28-aa COOH-terminal tail revealed a 90-kD protein doublet that was affinity purified by a GST fusion protein of human talin head (GST-talin head; Fig. 1 E). Immunoprecipitates generated by this PIPKIγ antibody from lamprey extracts contained a much higher PIP2-synthesizing activity than did the control, as demonstrated by TLC separation of 32P-labeled phosphoinositides generated by in vitro incubation with brain lipids and γ-[32P] ATP (Fig. 1 F). Further, using anti-talin antibodies, PIPKIγ was coimmunoprecipitated from both rat brain (Fig. 1 G, middle lane) and lamprey extracts (Fig. 1 H, left lane) (Di Paolo et al., 2002). Thus, lamprey contains a PIP kinase that interacts with talin. Immunostaining of lamprey spinal cord cross sections demonstrated that talin is concentrated at synapses, including those of the large reticulospinal axons, as shown by its colocalization with the synaptic protein synapsin (Fig. 1 I) (De Camilli et al., 1983; Pieribone et al., 1995).


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)

Interaction of PIPK with talin and synaptic localization of talin in the lamprey spinal cord. (A) Western blot with an anti-talin antibody of rat brain extract (load) and the material affinity purified from such extract by either GST or GST-PIPKIγ tail in pull-down experiments (S, supernatant; P, pellet). The top talin band is pulled down selectively by GST-PIPKIγ tail. (B) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from rat brain extracts (ext.) showing specificity of the talin–PIPKIγ interaction. (C) Western blot with an anti-talin antibody of lamprey extract (load) and of the material purified by either GST or GST-PIPKIγ tail. (D) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from lamprey spinal cord extracts. (E) Western blot with an anti-PIPKIγ antibody of lamprey extract (load) and of the material purified by either GST or GST-talin head. The 90-kD doublet is specifically affinity purified by GST-talin head. (F) TLC demonstrating the generation of phosphoinositides from lamprey extracts by immunoprecipitates of PIPKIγ antibody (α-PIPK IP) or control IgGs (ctrl IgGs). Note the prominent [32P]PIP2 band produced by the α-PIPK IP. (G, top panels) Western blots of anti-talin immunoprecipitates from rat brain extracts showing that coprecipitation of PIPKIγ (“α-talin IP” lane) is prevented by PIPK pep but not mutant PIPK pep (100 μM). (G, bottom panels) TLC and quantification of [32P]PIP2 showing correlation between PIPKIγ coprecipitation and PIP2-synthesizing activity. (cpm, counts per minute). (H) Western blot of anti-talin immunoprecipitates from lamprey spinal cord extracts showing that the PIPK pep (300 μM) prevents coprecipitation of PIPK. (I) Double immunofluorescence of cross sections of lamprey spinal cord stained for talin (red) and for the presynaptic marker, synapsin (green). Arrows indicate synapses in the neuropil. Asterisks indicate reticulospinal axons. Note the colocalization of talin with synapsin at the reticulospinal synapses (insets).
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fig1: Interaction of PIPK with talin and synaptic localization of talin in the lamprey spinal cord. (A) Western blot with an anti-talin antibody of rat brain extract (load) and the material affinity purified from such extract by either GST or GST-PIPKIγ tail in pull-down experiments (S, supernatant; P, pellet). The top talin band is pulled down selectively by GST-PIPKIγ tail. (B) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from rat brain extracts (ext.) showing specificity of the talin–PIPKIγ interaction. (C) Western blot with an anti-talin antibody of lamprey extract (load) and of the material purified by either GST or GST-PIPKIγ tail. (D) Coomassie blue–stained protein gel of a pull-down with GST-PIPKIγ tail from lamprey spinal cord extracts. (E) Western blot with an anti-PIPKIγ antibody of lamprey extract (load) and of the material purified by either GST or GST-talin head. The 90-kD doublet is specifically affinity purified by GST-talin head. (F) TLC demonstrating the generation of phosphoinositides from lamprey extracts by immunoprecipitates of PIPKIγ antibody (α-PIPK IP) or control IgGs (ctrl IgGs). Note the prominent [32P]PIP2 band produced by the α-PIPK IP. (G, top panels) Western blots of anti-talin immunoprecipitates from rat brain extracts showing that coprecipitation of PIPKIγ (“α-talin IP” lane) is prevented by PIPK pep but not mutant PIPK pep (100 μM). (G, bottom panels) TLC and quantification of [32P]PIP2 showing correlation between PIPKIγ coprecipitation and PIP2-synthesizing activity. (cpm, counts per minute). (H) Western blot of anti-talin immunoprecipitates from lamprey spinal cord extracts showing that the PIPK pep (300 μM) prevents coprecipitation of PIPK. (I) Double immunofluorescence of cross sections of lamprey spinal cord stained for talin (red) and for the presynaptic marker, synapsin (green). Arrows indicate synapses in the neuropil. Asterisks indicate reticulospinal axons. Note the colocalization of talin with synapsin at the reticulospinal synapses (insets).
Mentions: In Western blots of rat brain protein extracts, talin immunoreactivity migrates as a 230-kD band (Fig. 1 A) and a 190-kD proteolytic fragment lacking the head domain (Bolton et al., 1997; Fig. 1 A). Affinity chromatography using a GST fusion protein of the 28-aa COOH-terminal tail of human PIPKIγ (GST-PIPKIγ tail) resulted in the purification of the upper talin band, as expected (Fig. 1 A; Di Paolo et al., 2002). The proteolytic fragment, which lacks the PIPKIγ binding site, remained in the supernatant. Talin was the only major band retained by GST-PIPKIγ tail, as shown by Coomassie blue staining of the affinity-purified material (Fig. 1 B). Similarly, anti-talin antibodies recognized two proteins in lamprey spinal cord extracts of the appropriate size for talin and its proteolytic fragment (Fig. 1 C), and the larger protein, which corresponded to a major band visible by Coomassie blue (Fig. 1 D), was pulled down by GST-PIPKIγ tail. Western blots of lamprey extracts with an anti-PIPKIγ antibody raised against the 28-aa COOH-terminal tail revealed a 90-kD protein doublet that was affinity purified by a GST fusion protein of human talin head (GST-talin head; Fig. 1 E). Immunoprecipitates generated by this PIPKIγ antibody from lamprey extracts contained a much higher PIP2-synthesizing activity than did the control, as demonstrated by TLC separation of 32P-labeled phosphoinositides generated by in vitro incubation with brain lipids and γ-[32P] ATP (Fig. 1 F). Further, using anti-talin antibodies, PIPKIγ was coimmunoprecipitated from both rat brain (Fig. 1 G, middle lane) and lamprey extracts (Fig. 1 H, left lane) (Di Paolo et al., 2002). Thus, lamprey contains a PIP kinase that interacts with talin. Immunostaining of lamprey spinal cord cross sections demonstrated that talin is concentrated at synapses, including those of the large reticulospinal axons, as shown by its colocalization with the synaptic protein synapsin (Fig. 1 I) (De Camilli et al., 1983; Pieribone et al., 1995).

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