Limits...
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.

Show MeSH

Related in: MedlinePlus

Microinjection of PIPK pep disrupts the actin cytoskeleton at synapses. (A) Confocal image of a lamprey reticulospinal axon injected with Alexa 488–phalloidin (green). Thin, white line denotes axonal border. Phalloidin labels rings of actin at periactive zones of synapses, as indicated by the cartoon (SV, synaptic vesicle). (B and C) Images of axons injected with rhodamine-conjugated (red) Mut PIPK pep or PIPK pep and then with Alexa 488–phalloidin at sites indicated by white asterisks. Although Mut PIPK pep had no effect (B), the fluorescence intensity of phalloidin rings is greatly diminished by PIPK pep (C). Bars, 20 μm. (D) Average fluorescence intensity of the phalloidin rings over several concentration ranges of PIPK peptides as compared with that in the absence of any peptides (Control). Data indicate mean values and SEM from synapses in 31 control, 15 PIPK pep–injected, and 14 Mut PIPK pep–injected axons. Asterisks indicate statistical significance as compared with control (for PIPK pep 10–100 μM, P < 0.05 × 10−13; for PIPK pep 200–700 μM, P < 0.05 × 10−8; t test).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172527&req=5

fig3: Microinjection of PIPK pep disrupts the actin cytoskeleton at synapses. (A) Confocal image of a lamprey reticulospinal axon injected with Alexa 488–phalloidin (green). Thin, white line denotes axonal border. Phalloidin labels rings of actin at periactive zones of synapses, as indicated by the cartoon (SV, synaptic vesicle). (B and C) Images of axons injected with rhodamine-conjugated (red) Mut PIPK pep or PIPK pep and then with Alexa 488–phalloidin at sites indicated by white asterisks. Although Mut PIPK pep had no effect (B), the fluorescence intensity of phalloidin rings is greatly diminished by PIPK pep (C). Bars, 20 μm. (D) Average fluorescence intensity of the phalloidin rings over several concentration ranges of PIPK peptides as compared with that in the absence of any peptides (Control). Data indicate mean values and SEM from synapses in 31 control, 15 PIPK pep–injected, and 14 Mut PIPK pep–injected axons. Asterisks indicate statistical significance as compared with control (for PIPK pep 10–100 μM, P < 0.05 × 10−13; for PIPK pep 200–700 μM, P < 0.05 × 10−8; t test).

Mentions: Lamprey reticulospinal axons form en passant synapses with motor neurons. The large vesicle clusters at these synapses are surrounded by a ring of actin at periactive zones that can be visualized by the microinjection of fluorescently labeled phalloidin (Fig. 3 A) (Shupliakov et al., 2002). To determine whether the talin–PIPK interaction regulates this pool of synaptic actin, individual lamprey axons were first microinjected with rhodamine-conjugated Mut PIPK pep or PIPK pep (red), and immediately after with Alexa 488–phalloidin (green). Although the Mut PIPK peptide did not affect the accumulation of phalloidin fluorescence at synapses, PIPK peptide strongly inhibited this accumulation (Fig. 3, B and C). The PIPK peptide caused an average 64% reduction in the synaptic phalloidin fluorescence at intra-axonal concentrations >10 μM, which is within the range expected to compete the talin–PIPK interaction (Fig. 3 D). This effect is likely an underestimation because the intensity of synaptic phalloidin fluorescence at sites of highest PIPK peptide concentration was often at the limit of detectability, and therefore was not measured. Even at concentrations as high as 200–700 μM, Mut PIPK peptide had no effect on synaptic phalloidin fluorescence (Fig. 3 D).


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)

Microinjection of PIPK pep disrupts the actin cytoskeleton at synapses. (A) Confocal image of a lamprey reticulospinal axon injected with Alexa 488–phalloidin (green). Thin, white line denotes axonal border. Phalloidin labels rings of actin at periactive zones of synapses, as indicated by the cartoon (SV, synaptic vesicle). (B and C) Images of axons injected with rhodamine-conjugated (red) Mut PIPK pep or PIPK pep and then with Alexa 488–phalloidin at sites indicated by white asterisks. Although Mut PIPK pep had no effect (B), the fluorescence intensity of phalloidin rings is greatly diminished by PIPK pep (C). Bars, 20 μm. (D) Average fluorescence intensity of the phalloidin rings over several concentration ranges of PIPK peptides as compared with that in the absence of any peptides (Control). Data indicate mean values and SEM from synapses in 31 control, 15 PIPK pep–injected, and 14 Mut PIPK pep–injected axons. Asterisks indicate statistical significance as compared with control (for PIPK pep 10–100 μM, P < 0.05 × 10−13; for PIPK pep 200–700 μM, P < 0.05 × 10−8; t test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172527&req=5

fig3: Microinjection of PIPK pep disrupts the actin cytoskeleton at synapses. (A) Confocal image of a lamprey reticulospinal axon injected with Alexa 488–phalloidin (green). Thin, white line denotes axonal border. Phalloidin labels rings of actin at periactive zones of synapses, as indicated by the cartoon (SV, synaptic vesicle). (B and C) Images of axons injected with rhodamine-conjugated (red) Mut PIPK pep or PIPK pep and then with Alexa 488–phalloidin at sites indicated by white asterisks. Although Mut PIPK pep had no effect (B), the fluorescence intensity of phalloidin rings is greatly diminished by PIPK pep (C). Bars, 20 μm. (D) Average fluorescence intensity of the phalloidin rings over several concentration ranges of PIPK peptides as compared with that in the absence of any peptides (Control). Data indicate mean values and SEM from synapses in 31 control, 15 PIPK pep–injected, and 14 Mut PIPK pep–injected axons. Asterisks indicate statistical significance as compared with control (for PIPK pep 10–100 μM, P < 0.05 × 10−13; for PIPK pep 200–700 μM, P < 0.05 × 10−8; t test).
Mentions: Lamprey reticulospinal axons form en passant synapses with motor neurons. The large vesicle clusters at these synapses are surrounded by a ring of actin at periactive zones that can be visualized by the microinjection of fluorescently labeled phalloidin (Fig. 3 A) (Shupliakov et al., 2002). To determine whether the talin–PIPK interaction regulates this pool of synaptic actin, individual lamprey axons were first microinjected with rhodamine-conjugated Mut PIPK pep or PIPK pep (red), and immediately after with Alexa 488–phalloidin (green). Although the Mut PIPK peptide did not affect the accumulation of phalloidin fluorescence at synapses, PIPK peptide strongly inhibited this accumulation (Fig. 3, B and C). The PIPK peptide caused an average 64% reduction in the synaptic phalloidin fluorescence at intra-axonal concentrations >10 μM, which is within the range expected to compete the talin–PIPK interaction (Fig. 3 D). This effect is likely an underestimation because the intensity of synaptic phalloidin fluorescence at sites of highest PIPK peptide concentration was often at the limit of detectability, and therefore was not measured. Even at concentrations as high as 200–700 μM, Mut PIPK peptide had no effect on synaptic phalloidin fluorescence (Fig. 3 D).

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