Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones

  1. Nickolai V Vysokov
  2. John-Paul Silva
  3. Vera G Lelianova
  4. Jason Suckling
  5. John Cassidy
  6. Jennifer K Blackburn
  7. Natalia Yankova
  8. Mustafa BA Djamgoz
  9. Serguei V Kozlov
  10. Alexander G Tonevitsky
  11. Yuri A Ushkaryov  Is a corresponding author
  1. University of Kent, United Kingdom
  2. Imperial College London, United Kingdom
  3. King’s College London, United Kingdom
  4. BrainPatch Ltd, United Kingdom
  5. UCB-Pharma, United Kingdom
  6. Thomsons Online Benefits, United Kingdom
  7. Arix Bioscience, United Kingdom
  8. Yale University School of Medicine, United States
  9. National Cancer Institute, United States
  10. Higher School of Economics, Russia
  11. Scientific Research Centre Bioclinicum, Russia
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Lasso is cleaved and released into the medium during neuronal development.

(A) Recombinant Lasso constructs used in this work (FS, full size). The three proteolytic cleavage sites and the SS splice site are indicated. The antibody recognition sites/epitopes are shown by …

https://doi.org/10.7554/eLife.37935.003
Figure 1—source data 1

Source data for Figure 1, Panels D and H.

https://doi.org/10.7554/eLife.37935.004
Figure 1—figure supplement 1
Lasso is expressed on dendrites and LPHN1 on axonal growth cones in developing neurons.

(A) Proportional expression of full-size Lasso and its fragments in hippocampal neurons in culture. The data are from Western blots (as in Figure 1C, n = 3), stained using the TN2C antibody. (B) …

https://doi.org/10.7554/eLife.37935.005
Figure 1—figure supplement 1—source data 1

Source data for Figure 1—figure supplement 1, Panels A and G.

https://doi.org/10.7554/eLife.37935.006
Figure 2 with 2 supplements
Soluble Lasso binds to LPHN1 on other cells.

(A) A scheme of LPHN and Lasso constructs used in this experiment. LPH-82 is LPHN1 with the ECD from another adhesion G-protein-coupled receptor, EMR2, used as a negative control. (B) Purification …

https://doi.org/10.7554/eLife.37935.007
Figure 2—figure supplement 1
Soluble Lasso specifically binds to LPHN1-expressing cells.

Interaction between the soluble Lasso species and NB2a cells expressing LPHN1 or Lasso-A. Cells expressing LPHN1 (A), but not Lasso-A (B), are able to bind the soluble Lasso-D. (C) Binding of the …

https://doi.org/10.7554/eLife.37935.008
Figure 2—figure supplement 2
Soluble Lasso specifically binds to LPHN1 on axonal growth cones.

Hippocampal neurons from LPHN1 WT or KO newborn mice were grown in culture for 14 days and then incubated with the medium from NB2a cells stably expressing Lasso-D. The cultures were fixed and …

https://doi.org/10.7554/eLife.37935.010
Using MAIDs to study axonal attraction by soluble chemoattractants.

(A) Left, a photograph of a MAID. Center, a scheme of the experiment: neurons are seeded into the Somal Compartment and their neurites grow into the Axonal Compartment; both compartments are then …

https://doi.org/10.7554/eLife.37935.011
Figure 3—source data 1

Source data for Figure 3, Panels C, E, and H.

https://doi.org/10.7554/eLife.37935.012
Figure 4 with 1 supplement
A gradient of soluble Lasso-D induces axonal attraction via LPHN1.

(A) A scheme of the experiment: hippocampal neurons were cultured in Somal Compartments, purified Lasso was added to Axonal Compartments at 3 DIV. (B) Lasso remains intact in the Axonal Compartment. …

https://doi.org/10.7554/eLife.37935.013
Figure 4—figure supplement 1
Knockout of LPHN1 prevents axonal attraction by soluble Lasso.

(A) Experimental hypothesis: predicted behavior of LPHN1 KO axons in response to a gradient of soluble Lasso. (B) Polymerase chain reaction (PCR)-based genotyping of 6 newborn pups from three …

https://doi.org/10.7554/eLife.37935.015
A spatio-temporal gradient of soluble Lasso induces axonal attraction and fasciculation, but does not increase axonal length.

(A) A scheme of the experiment: HEK293A cells stably transfected with Lasso-D were cultured in the wells of Axonal Compartments; untransfected cells were used as a control. (B) A representative …

https://doi.org/10.7554/eLife.37935.016
Interaction of LPHN1 with soluble Lasso causes LPHN1 aggregation.

(A) A scheme of behavior of LPHN1 fragments at rest (left) and after binding an active agonist (middle) or a non-agonistic antibody (right). (B–D) Distribution of NTF and CTF in NB2a cells stably …

https://doi.org/10.7554/eLife.37935.018
Figure 7 with 1 supplement
Soluble Lasso induces Ca2+signaling in LPHN1-expressing cells and enhances spontaneous exocytosis at neuromuscular junctions.

(A) Changes in intracellular Ca2+ concentration in neuroblastoma cells stably expressing LPHN1 were monitored using a Ca2+ indicator dye, Fluo-4. The scheme of the experiment is shown in Figure …

https://doi.org/10.7554/eLife.37935.020
Figure 7—source data 1

Source data for Figure 7, Panels A-C, E, and F.

https://doi.org/10.7554/eLife.37935.021
Figure 7—figure supplement 1
Design of the experiments testing Lasso induced Ca2+signaling in LPHN1-expressing cells and its presynaptic action at mouse neuromuscular junctions.

(A) Experimental paradigm for testing the effect of Lasso-D on LPHN1-dependent Ca2+ signaling. After 5 min recording of baseline fluorescence of neuroblastoma cells expressing LPHN1, the cells were …

https://doi.org/10.7554/eLife.37935.022
A proposed scheme of the mechanism of axonal attraction by released Lasso ECD.

When Lasso binds the NTF of LPHN1, it causes its re-association with the CTF. This activates Gαq/11 and triggers the PLC signaling cascade. Downstream of this cascade, the local IP3-induced calcium …

https://doi.org/10.7554/eLife.37935.024

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-FLAG M2
affinity gel
Sigma-AldrichA2220
AntibodyChicken anti-mycMilliporeAB3252
RRID:AB_2235702
(Immunocytochemistry
1:1,000)
AntibodyMouse anti-actininSigma-AldrichA7811(Western blot 1:1,500)
AntibodyMouse anti-FLAG M2Sigma-AldrichF3165
RRID:AB_259529
(Immunocytochemistry
1:1,000)
AntibodyMouse anti-Lasso/
teneurin-2 C-terminus
(Silva et al., 2011)dmAbTN2C
(Immunocytochemistry
1:300; Western blot
1:1,000)
AntibodyMouse anti-MAP-2NeuromicsMO22116(Immunocytochemistry
1:1,000)
AntibodyMouse anti-synapsinSanta-Cruz
Biotechnology
sc-376623
RRID:AB_11150313
(Immunocytochemistry
1:1,000)
AntibodyMouse monoclonal
anti-myc
Millipore05–419
RRID:AB_309725
clone 9E10
(Immunocytochemistry
1:1000; Western blot 1:)
AntibodyMouse monoclonal
anti-V5
AbD Serotec/Bio-RadMCA1360clone SV5-Pk1
(Immunocytochemistry
1:2,000)
AntibodyRabbit anti-GFPThermo Fisher
Scientific
A-11122
RRID: AB_221569
(Immunocytochemistry
1:1,000)
AntibodyRabbit anti-NF-HNeuromicsRA22116(Immunocytochemistry
1:1,000; Western blot
1:10,000)
AntibodyRabbit anti-PSD-95MilliporeAB9708
RRID:AB_11212529
(Immunocytochemistry
1:2,000)
AntibodyRabbit anti-TauSynaptic Systems314 002
RRID:AB_993042
(Immunocytochemistry
1:1,000)
AntibodyRabbit anti-V5Thermo Fisher
Scientific
PA1-29324
RRID:AB_1961277
(Immunocytochemistry
1:2,000)
AntibodyRabbit polyclonal
anti-LPHN1 NTF
(Davletov et al., 1998)RL1(Immunocytochemistry
1:1,000)
AntibodyRabbit polyclonal
anti-LPHN1-peptide
(Davydov et al., 2009)PAL1(Immunocytochemistry;
Western blot 3 ng/mL)
AntibodySheep anti-teneurin-2
N-terminus
R and D systemsAF4578
RRID:AB_10719438
TN2N
(Western blot 1 μg/mL)
Cell line
(Homo sapiens)
HEK293AECCCRRID:CVCL_6910
Cell line
(Mus musculus)
Neuroblastoma 2aATCCRRID:CVCL_0470
Chemical
compound
B27 SupplementLife Technologies17504044
Chemical
compound
Ca-free Hibernate-A
medium
BrainBits UKHE-Ca
Chemical
compound
Fluo-4 acetomethoxy
ester
Thermo Fisher
Scientific
F14201
Chemical
compound
Insulin Transferrin
Selenium Supplement
Life Technologies41400045
Chemical
compound
Neurobasal-A mediumThermo Fisher
Scientific
21103049
Chemical
compound
Purified protein:
BSA-TRITC
Thermo Fisher
Scientific
A23016
Chemical
compound
Vybrant DiOThermo Fisher
Scientific
V22886
Commercial
assay or kit
Amaxa Rat Neuron
Nucleofector Kit
LonzaVAPG-1003
Commercial
assay or kit
SuperSignal West
Femto Maximum
Sensitivity Substrate
Thermo Fisher
Scientific
34094
OtherMicrofluidic Axon
Isolation Devices (MAIDs)
Xona MicrofluidicsSND150
Recombinant
DNA reagent
BLOCK-iT Lentiviral Pol
II miR RNAi Expression
System pLenti6/V5-GW/
EmGFP-miR
Life TechnologiesK4938-00
Recombinant
DNA reagent
Bottom pre-miRNA
oligo targeting LPHN1
mRNA
This paperLPHN1miR14BSequence provided
under Methods
Recombinant
DNA reagent
Lasso-A(Silva et al., 2011)GenBank: JF784341
Recombinant
DNA reagent
Lasso-D(Silva et al., 2011)GenBank: JF784344
Recombinant
DNA reagent
Lasso-FS(Silva et al., 2011)GenBank: JF784340
Recombinant
DNA reagent
Lasso-G(Silva et al., 2011)GenBank: JF784347GST-Lasso
Recombinant
DNA reagent
LPH-42(Volynski et al., 2004)GenBank:MF966512V5-LPH-A
Recombinant
DNA reagent
pLenti6.2-GW/EmGFP-miR
negative control
Thermo Fisher
Scientific
K4938-00
Recombinant
DNA reagent
Primer: N255: Neo
Forward
This paperSequence provided
under Methods
Recombinant
DNA reagent
Primer: N424: Neo/
LPHN1 Reverse
This paperSequence provided
under Methods
Recombinant
DNA reagent
Primer: N425:
LPHN1 Forward
This paperSequence provided
under Methods
Recombinant
DNA reagent
Top pre-miRNA
oligo targeting
LPHN1 mRNA
This paperLPHN1miR14TSequence provided
under Methods
Peptide,
recombinant
protein
Purified protein:
Alexa Fluor
647-labeled LTXN4C
(Volynski et al., 2004)N/A
Peptide,
recombinant
protein
Purified protein:
Human BDNF
R and D Systems248-BD
Peptide,
recombinant
protein
Purified protein:
Lasso-D
(Silva et al., 2011)N/A
Peptide,
recombinant
protein
Purified protein:
Lasso-G
(Silva et al., 2011)N/AGST-Lasso
Peptide,
recombinant
protein
Purified protein:
LTXN4C
(Volynski et al., 2003)N/A
SoftwareAxoScope 10Axon Instruments
SoftwareFIJI, ImageJNIMH, Bethesda,
Maryland, USA
RRID:SCR_002285
RRID:SCR_003070
SoftwareLSM 510 Software
(for image acquisition)
Carl Zeiss
Microimaging GmbH
LSM 510
SoftwareLSM Image Browser
(for image
archiving and
measurements)
Carl Zeiss
Microimaging GmbH
RRID:SCR_014344
SoftwareMATLABMathworksRRID:SCR_001622
Software, algorithmMATLABMathworkshttps://github.com/artificialbrain-tech/Axon-Guidance-ScriptsAxonal guidance scripts
SoftwareMiniAnalysisSynaptosoft
SoftwareVolocity (for image
acquisition and
stitching)
Perkin-ElmerRRID:SCR_002668
Strain (Escherichia coli)E. coli: K12 JM109Promega
Corporation
L2005
Strain (Mus musculus)Mouse: C57BL/6J,
Adgrl1-/-, LPHN1 KO
This paperAG148/2P0 hippocampus
Strain (Mus musculus)Mouse: C57BL/6J,
Adgrl1-/-, LPHN1 KO
This paperAG148/2P21 flexor digitorum
brevis muscle
Strain (Rattus norvegicus)Rat: E18
hippocampus
BrainBits UKRhp

Additional files

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