1. Cell Biology
  2. Neuroscience

Synaptojanin cooperates in vivo with endophilin through an unexpected mechanism

  1. Yongming Dong
  2. Yueyang Gou
  3. Yi Li
  4. Yan Liu
  5. Jihong Bai  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States
  2. Sichuan University, China
  3. University of Washington, United States
https://doi.org/10.7554/eLife.05660
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Cite this article
  1. Yongming Dong
  2. Yueyang Gou
  3. Yi Li
  4. Yan Liu
  5. Jihong Bai
(2015)
Synaptojanin cooperates in vivo with endophilin through an unexpected mechanism
eLife 4:e05660.
https://doi.org/10.7554/eLife.05660
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9 figures and 1 table

Figures

Figure 1 with 1 supplement
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Synaptojanin UNC-26 lacking the PRD domain fully supports locomotion, endogenous activity, and evoked synaptic currents.

(A) Domain structure of synaptojanin UNC-26. Synaptojanin contains three functional modules: a Sac1 phosphatase domain, a 5-phosphatase domain (5Pase), and a proline-rich domain (PRD). Single-copy …

https://doi.org/10.7554/eLife.05660.003
Figure 1—figure supplement 1
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Mouse synaptojanin ∆PRD is functional in C. elegans neurons.

Truncated version of mouse synaptojanin 1 (1–1045) that lacks the PRD domain was expressed in C. elegans nervous system under control of the pan-neuronal promoter Psnb-1. Electrophysiological …

https://doi.org/10.7554/eLife.05660.004
Figure 2
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Synaptojanin UNC-26∆PRD recovers the recycling vesicle pool and sustains synaptic transmission upon repetitive stimuli.

(A) A schematic diagram is shown to illustrate the FM4-64 loading and unloading procedure. Experimental details are discussed in the ‘Materials and methods’ section. (B) FM4-64 loading and unloading …

https://doi.org/10.7554/eLife.05660.006
Figure 3 with 1 supplement
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The SH3-PRD interaction is dispensable for synaptic activity.

(A) A schematic drawing showing interactions between synaptojanin (UNC-26) PRD and endophilin (UNC-57) SH3. Single-copy transgenes encoding UNC-26∆PRD::GFP and UNC-57∆SH3::mCherry were co-expressed …

https://doi.org/10.7554/eLife.05660.007
Figure 3—figure supplement 1
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UNC-57 and UNC-26 are not overexpressed in transgenic animals.

Monoclonal antibodies against UNC-57BAR and UNC-26 5-phosphatase were developed as described in the ‘Materials and methods’. (A) Immunoblots for UNC-57 detection. 35 µg of total proteins, extracted …

https://doi.org/10.7554/eLife.05660.008
Figure 4
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UNC-26∆PRD and UNC-57∆SH3 restore the number of SVs and recover synaptopHluorin retrieval in unc-57; unc-26 double mutants.

(A) Electron microscopy images of neuromuscular junctions were collected from the ventral nerve cords of adult hermaphrodites. Synaptic profiles of 15 synapses of the wt, 18 synapses of the unc-57; …

https://doi.org/10.7554/eLife.05660.009
Figure 5 with 1 supplement
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Synaptojanin phosphatase domains have distinct functions.

The 5-Pase domain hydrolyzes phosphoinositides, while Sac1 plays an essential but non-catalytic role at synapses. Evoked EPSCs from wt, unc-26(s1710) mutant, and indicated transgenic strains were …

https://doi.org/10.7554/eLife.05660.010
Figure 5—figure supplement 1
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Sac1-inactivated synaptojanin supports synaptic transmission in a PRD independent manner.

Two mutations (C378S and D380N) that inactivate the Sac1 lipid phosphatase activity were introduced into the Sac1 domain of UNC-26∆PRD (residues 1–986). The mutant UNC-26∆PRD (C378S, D380N) was …

https://doi.org/10.7554/eLife.05660.011
Figure 6 with 1 supplement
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Sac1 must be physically linked to UNC-26 5-phosphatase to support synaptic transmission.

Split-intein mediated ligation was used to post-translationally reconnect Sac1 to the remainder of the UNC-26 synaptojanin protein (A). The Sac1 domain (1–493) was linked to the N-terminal half of Np…

https://doi.org/10.7554/eLife.05660.012
Figure 6—figure supplement 1
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Transgenic worms that only express either Sac1::IntN or IntC::UNC-26∆Sac1 did not show functional improvements.

Representative traces (A) and summary data (B) for evoked EPSC amplitude are shown for the indicated genotypes. Transgenes encoding Sac1::IntN and IntC::UNC-26∆Sac1 were designed as described in the …

https://doi.org/10.7554/eLife.05660.013
Figure 7 with 1 supplement
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Sac1 is a synaptic targeting domain.

(A–B) Removal of the Sac1 domain of synaptojanin perturbs synaptic targeting of synaptojanin. Representative images (A) showing various versions of GFP::UNC-26 distribution in the dorsal nerve cord. …

https://doi.org/10.7554/eLife.05660.014
Figure 7—figure supplement 1
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GFP-tagged Sac1 domains localize to synapses.

(A) Representative images showing GFP, GFP::UNC-26Sac1, and GFP::UNC-26Sac1(C378S,D380N) distribution in the dorsal nerve cord axons. Synaptic enrichment of GFP was quantified using ∆F/F = (Fpeak − F…

https://doi.org/10.7554/eLife.05660.015
Figure 8 with 2 supplements
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Endophilin functionally substitutes for the Sac1 domain.

(A) A schematic drawing showing the chimeric UNC-57 endophilin::UNC-26∆Sac1 protein. Other endocytic accessory proteins including DYN-1 dynamin and ITSN-1 intersectin were tethered to UNC-26∆Sac1 …

https://doi.org/10.7554/eLife.05660.016
Figure 8—figure supplement 1
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Targeting UNC-26∆Sac1 to synaptic vesicles, phosphoinositides, and endocytic adaptor protein AP2 does not recover synaptojanin function.

Summary data for evoked EPSC amplitudes are shown for the indicated genotypes in (A–C). Non-enzymatic proteins that are involved in SV cycle and phosphoinositide recognition were tethered to the …

https://doi.org/10.7554/eLife.05660.017
Figure 8—figure supplement 2
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The endophilin tether does not bypass the requirement for a functional synaptojanin 5-phosphatase domain.

The 5-phosphatase activity is required for the UNC-57::UNC-26∆Sac1 chimeric protein. All chimeric proteins were expressed under the control of Prab-3. Representative traces (A) and summary data (B) …

https://doi.org/10.7554/eLife.05660.018
Figure 9 with 3 supplements
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Endophilin BAR domain and its membrane interactions are required for bypassing Sac1.

(A–B) Endophilin BAR is sufficient to bypass the Sac1 requirement. UNC-26∆Sac1 was tethered to worm and rat endophilinA1 BAR (rEndoBAR), respectively. Mutations that disrupt BAR-membrane …

https://doi.org/10.7554/eLife.05660.019
Figure 9—figure supplement 1
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UNC-57 SH3 domain enhances synaptic enrichment of UNC-26∆Sac1.

(A) Distribution of GFP::UNC-57SH3::UNC-26∆Sac1 (domain structure, upper) in the dorsal nerve cord axon is shown (lower). Scale bar: 2 µm. (B) Summary data indicates that UNC-57 SH3 domain enhances …

https://doi.org/10.7554/eLife.05660.020
Figure 9—figure supplement 2
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Specificity in BAR proteins for bypassing the Sac1 requirement.

BAR domains of mouse Nadrin2 (residues 1–248) and mouse amphiphysin (residues 1–250) were tethered to the N-terminus of UNC-26∆Sac1. All chimeric proteins were expressed under the control of Prab-3 …

https://doi.org/10.7554/eLife.05660.021
Figure 9—figure supplement 3
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UNC-26 Sac1 does not bind UNC-57 in solution.

GST pull-down assays were performed as described in the ‘Materials and methods’. Briefly, GST::UNC-57 (10 µg) was immobilized on glutathione beads. Maltose-binding protein (MBP)::UNC-26 fragments …

https://doi.org/10.7554/eLife.05660.022

Tables

Table 1

Summary of data from electrophysiological recordings and locomotion analyses

https://doi.org/10.7554/eLife.05660.005
Evoked EPSC Amp. (nA)Endogenous EPSCLocomotion speed (µm/s)
Frequency (Hz)Amp. (pA)
Wild type (N2)3.2 ± 0.2 (n = 23)49.8 ± 2.5 (n = 30)22.4 ± 1.0140 ± 8 (n = 65)
unc-26 (s1710)–0.9 ± 0.1 (n = 15)†12.0 ± 1.5 (n = 16)†20.7 ± 0.628 ± 3 (n = 65)†
Si[Prab-3::unc-26::gfp]3.1 ± 0.2 (n = 16)#50.8 ± 5.4 (n = 16)#23.2 ± 1.4139 ± 8 (n = 60)#
Si[Prab-3::unc-26∆PRD::gfp]3.4 ± 0.3 (n = 16)#47.4 ± 5.0 (n = 17)#24.8 ± 1.3141 ± 10 (n = 60)#
Ex[Psnb-1::mSYJ1∆PRD]3.1 ± 0.3 (n = 7)#49.6 ± 6.1 (n = 11)#21.5 ± 0.6143 ± 10 (n = 37)#
Ex[Prab-3::gfp::unc-26(C378S,D380N)]3.5 ± 0.2 (n = 10)#53.1 ± 6.6 (n = 10)23.8 ± 1.3135 ± 9 (n = 60)#
Ex[Prab-3::gfp::unc-26∆PRD(C378S,D380N)]3.2 ± 0.3 (n = 10)#50.7 ± 5.7 (n = 10)24.2 ± 0.9130 ± 9 (n = 60)#
Ex[Prab-3::gfp::unc-26(D716A)]0.8 ± 0.1 (n = 14)10.0 ± 1.8 (N = 15)20.9 ± 0.828 ± 3 (n = 60)
Ex[Prab-3::gfp::unc-26∆Sac1]1.2 ± 0.2 (n = 10)7.2 ± 1.9 (N = 10)21.3 ± 1.231 ± 3 (n = 60)
Ex[Prab-3::unc-26Sac1 + Prab-3::unc-26∆Sac1]1.0 ± 0.2 (n = 10)16.1 ± 1.8 (n = 10)20.4 ± 0.532 ± 3 (n = 60)
Ex[Prab-3::unc-26Sac1::IntN + Prab-3::IntC::unc-26∆Sac1]3.2 ± 0.4 (n = 10)#53.1 ± 7.1 (n = 10)#24.8 ± 1.2100 ± 7 (n = 60)#
Ex[Prab-3::unc26∆Sac1::rab-3]1.0 ± 0.2 (n = 10)13.2 ± 2.3 (n = 10)20.4 ± 0.9
Ex[Prab-3::unc-26∆Sac1::snb-1]1.4 ± 0.2 (n = 11)17.9 ± 2.6 (n = 11)21.1 ± 0.8
Ex[Prab-3::bem1PX::unc-26∆Sac1]0.8 ± 0.1 (n = 7)6.3 ± 0.6 (n = 7)18.8 ± 1.2
Ex[Prab-3::plc∂PH::unc-26∆Sac1]1.0 ± 0.2 (n = 7)8.5 ± 1.8 (n = 7)18.7 ± 1.1
Ex[Prab-3::btkPH::unc-26∆Sac1]1.2 ± 0.2 (n = 11)11.4 ± 1.2 (n = 11)18.4 ± 0.8
Ex[Prab-3::apa-2::unc-26∆Sac1]1.1 ± 0.1 (n = 11)12.8 ± 1.8 (n = 11)19.4 ± 0.8
Ex[Prab-3::apb-1::unc-26∆Sac1]0.9 ± 0.1 (n = 11)13.2 ± 2.5 (n = 11)18.2 ± 1.5
Ex[Prab-3::apm-2::unc-26∆Sac1]1.1 ± 0.1 (n = 10)15.3 ± 2.7 (n = 10)21.0 ± 0.9
Ex[Prab-3::aps-2::unc-26∆Sac1]1.3 ± 0.2 (n = 9)12.7 ± 1.4 (n = 9)20.1 ± 0.9
Ex[Prab-3::unc-57::unc-26∆Sac1]2.5 ± 0.3 (n = 11)#28.0 ± 4.1 (n = 11)§23.0 ± 1.4
Ex[Prab-3::dyn-1::unc-26∆Sac1]1.3 ± 0.3 (n = 10)14.4 ± 2.9 (n = 10)19.4 ± 0.9
Ex[Prab-3::itsn-1::unc-26∆Sac1]0.9 ± 0.1 (n = 9)13.9 ± 1.4 (n = 9)21.5 ± 1.4
Ex[Prab-3::unc-57::unc-26∆Sac1∆PRD]3.0 ± 0.3 (n = 11)#36.7 ± 5.4 (n = 11)§21.7 ± 1.3
Ex[Prab-3::unc-57::unc-26∆Sac1(D716A)]0.8 ± 0.2 (n = 9)13.0 ± 2.3 (n = 9)21.0 ± 0.7
Ex[Prab-3::unc-57BAR::unc-26∆Sac1]2.9 ± 0.2 (n = 12)#23.9 ± 2.5 (n = 12)#21.3 ± 0.8
Ex[Prab-3::rEndoBAR::unc-26∆Sac1]3.1 ± 0.4 (n = 13)#28.5 ± 4.6 (n = 13)#24.1 ± 1.4
Ex[Prab-3::mAmphBAR::unc-26∆Sac1]1.4 ± 0.2 (n = 10)19.2 ± 2.3 (n = 10)20.8 ± 0.8
Ex[Prab-3::mNadrin2BAR::unc-26∆Sac1]1.7 ± 0.2 (n = 11)‡16.1 ± 3.1 (n = 11)23.8 ± 1.9
Ex[Prab-3::rEndoBAR∆N::unc-26∆Sac1]1.3 ± 0.2 (n = 12)9.1 ± 0.9 (n = 12)18.9 ± 0.4
Ex[Prab-3::rEndoBAR(K76E,K78E)::unc-26∆Sac1]1.5 ± 0.2 (n = 10)13.5 ± 1.3 (n = 10)19.3 ± 0.7
N2Prab-3::unc-26∆PRD(D716A) overexpression1.5 ± 0.3 (n = 9)*24.6 ± 3.8 (n = 10)*25.7 ± 0.9
Prab-3::unc-26∆PRD overexpression2.9 ± 0.3 (n = 9)53.5 ± 4.6 (n = 9)25.4 ± 1.3
Prab-3::unc-26∆Sac1∆PRD(D716A) overexpression3.5 ± 0.3 (n = 10)49.0 ± 7.5 (n = 10)25.9 ± 1.9
unc-57(e406); unc-26(s1710)–0.8 ± 0.2 (n = 9)†8.6 ± 0.8 (n = 10)†21.9 ± 1.127 ± 3 (n = 60)†
Si[Psnb-1::unc-57∆SH3::mCherry]; Si[Prab-3::unc-26∆PRD::gfp]3.2 ± 0.2 (n = 9)50.3 ± 4.1 (n = 9)23.1 ± 1.0142 ± 9 (n = 62)
Si[Psnb-1::rEndoBAR::unc-26∆Sac1∆PRD]3.0 ± 0.3 (n = 10)50.9 ± 4.1 (n = 10)26.5 ± 1.1109 ± 4 (n = 68)
  1. *

    p < 0.001 when compared with N2.

  2. †

    p < 0.0001 when compared with N2.

  3. ‡

    p < 0.05 when compared with unc-26 mutant.

  4. §

    p < 0.001 when compared with unc-26 mutant.

  5. #

    p < 0.0001 when compared with unc-26 mutant.

  6. p < 0.0001 when compared with unc-57; unc-26 double mutants.

  7. Si: single-copy transgene (MosSci insertion).

  8. Ex: extrachromosomal array.

  9. ‘Amp.’ indicates amplitude.

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