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, 24 (14), 3711-20

Molecular Determinants of Synapsin Targeting to Presynaptic Terminals

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Molecular Determinants of Synapsin Targeting to Presynaptic Terminals

Daniel Gitler et al. J Neurosci.

Abstract

Although synapsins are abundant synaptic vesicle proteins that are widely used as markers of presynaptic terminals, the mechanisms that target synapsins to presynaptic terminals have not been elucidated. We have addressed this question by imaging the targeting of green fluorescent protein-tagged synapsins in cultured hippocampal neurons. Whereas all synapsin isoforms targeted robustly to presynaptic terminals in wild-type neurons, synapsin Ib scarcely targeted in neurons in which all synapsins were knocked-out. Coexpression of other synapsin isoforms significantly strengthened the targeting of synapsin Ib in knock-out neurons, indicating that heterodimerization is required for synapsin Ib to target. Truncation mutagenesis revealed that synapsin Ia targets via distributed binding sites that include domains B, C, and E. Although domain A was not necessary for targeting, its presence enhanced targeting. Domain D inhibited targeting, but this inhibition was overcome by domain E. Thus, multiple intermolecular and intramolecular interactions are required for synapsins to target to presynaptic terminals.

Figures

Figure 1.
Figure 1.
Targeting of GFP-tagged synapsins in wild-type neurons. A, Domain structure of synapsin isoforms considered in our study. B–F, In 2-week-old cultured neurons, GFP-tagged synapsins are located in punctuate structures.
Figure 2.
Figure 2.
Colocalization of GFP–synapsin Ia with synaptic marker proteins. Neurons expressing GFP–synapsin Ia were stained with antibodies that bind to synaptotagmin I (A), synaptobrevin 2 (B), synaptophysin I (C), or PSD-95 (D). The left column indicates the location of synaptic markers (red), the center column shows the location of GFP–synapsin Ia (green), and mergers of these two images are shown in the right column. Colocalization of synapsin and the synaptic markers is shown by the yellow areas in the merged images.
Figure 3.
Figure 3.
Targeting of GFP-tagged synapsins in synapsin TKO neurons. Although several isoforms of synapsin targeted properly in 2-week-old TKO neurons (A–D), no targeting of synapsin Ib was visible (E). F, Low magnification image of synapsin Ib distribution in a 3-week-old TKO neuron. The neurites of this neuron were substantially brighter than the cell body (arrow), indicating that synapsin Ib is not distributed homogeneously within the neuron. G, H, Axonal distribution of synapsin Ib. Neurons (7 d in vitro) expressing GFP–synapsin Ib were stained with anti-GAP-43 antibodies (red), a marker for axons of mature neurons. Neurites containing the highest levels of synapsin Ib also stained for GAP-43. The arrow in G indicates the cell body of the neuron.
Figure 4.
Figure 4.
Quantification of synaptic targeting by fluorescence ratio imaging. Images of GFP-tagged synapsin Ia (A), DsRed2 (B), and the synaptic targeting factor ratio (C) are shown. The arrows in A indicate synaptic puncta. D, Calculation of the synaptic targeting factor. Top, Intensities of GFP and DsRed2 fluorescence along the axon shown in A and B. Bottom, Targeting factor was calculated for all points along the line, using the average intensity of axonal fluorescence. Note correspondence of the peaks in the targeting factor (arrows) with the puncta indicated in A.
Figure 5.
Figure 5.
Targeting of GFP-tagged synapsin I fragments in TKO neurons. Left, Diagram of the synapsin I fragments that were examined. Right, Mean targeting factor determined for each construct expressed in 2-week-old neurons. The targeting factor of soluble GFP was also measured as a control (bottom). Gray bars correspond to fragments that do not target to synapses, as defined by a targeting factor that is statistically indistinguishable from that of GFP. Error bars represent SEM values.
Figure 6.
Figure 6.
Localization of synapsin Ia fragments to synapses in TKO neurons. GFP-tagged BC (left) and CDE (right) constructs (green) apposed postsynaptic structures containing PSD-95 (red) in TKO neurons.
Figure 7.
Figure 7.
Targeting of domain C of synapsin I in TKO and wild-type neurons. A, GFP-tagged domain C of does not target in 2-week-old cultured TKO neurons. B, The same domain C construct does target in triple wild-type neurons. C, Overlay image of CFP–synapsin IIa (red) and YFP-C domain of synapsin I (green) in a TKO neuron. Synaptic colocalization of these constructs is indicated by yellow regions.
Figure 8.
Figure 8.
Differential effect of coexpression of various synapsin isoforms on targeting of synapsin Ib. Neurons from TKO mice were transfected with CFP–synapsin Ib singly (A) or together with YFP-tagged versions of synapsin Ia (B), synapsin IIa (C), or synapsin IIIa (D). Synapsin Ib colocalized with synapsins Ia and IIa in presynaptic terminals, as evidenced by the yellow color of the terminals. D, Lack of colocalization of synapsin Ib and of synapsin IIIa is evidenced by the localization of synapsin IIIa to synapses (red) and of synapsin Ib to the axon (green). E, Quantification of the targeting of synapsin Ib in the presence of other synapsin isoforms. The asterisks indicate significant (p < 0.05) differences from controls where the synapsin Ib was expressed alone.
Figure 9.
Figure 9.
Targeting of synapsin Ia with phosphorylation site mutations. GFP-tagged synapsin Ia mutants were examined in 2-week-old cultured TKO neurons, and the targeting factor was unaffected by the mutations in the indicated phosphorylation sites.
Figure 10.
Figure 10.
Molecular models of synapsin targeting. Targeting of synapsin Ib (A) and synapsin Ia (B), indicating the role of interactions of protein domains with each other and with SVs. C, Role of dimerization in synapsin targeting and SV cross-linking. Left, Synapsin Ia/Ia homodimers are divalent and can bind two SVs. Center, Synapsin Ia/Ib heterodimers are monovalent and can bind only one SV, allowing synapsin Ib to target in the presence of other synapsins but preventing SV cross-linking. Right, Synapsin Ib/Ib homodimers do not bind to or cross-link SVs.

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