(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 …
Source data for Figure 1, Panels D and H.
(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) …
Source data for Figure 1—figure supplement 1, Panels A and G.
(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 …
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 …
Source data for Figure 2—figure supplement 2, Panel C.
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 …
(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 …
Source data for Figure 3, Panels C, E, and H.
(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. …
Source data for Figure 1—figure supplement 1, Panels D and E.
(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 …
(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 …
Source data for Figure 5, Panels D-G.
(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 …
Source data for Figure 6, Panel F.
(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 …
Source data for Figure 7, Panels A-C, E, and F.
(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 …
Source data for Figure 7—figure supplement 1, Panel C.
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 …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Anti-FLAG M2 affinity gel | Sigma-Aldrich | A2220 | |
Antibody | Chicken anti-myc | Millipore | AB3252 RRID:AB_2235702 | (Immunocytochemistry 1:1,000) |
Antibody | Mouse anti-actinin | Sigma-Aldrich | A7811 | (Western blot 1:1,500) |
Antibody | Mouse anti-FLAG M2 | Sigma-Aldrich | F3165 RRID:AB_259529 | (Immunocytochemistry 1:1,000) |
Antibody | Mouse anti-Lasso/ teneurin-2 C-terminus | (Silva et al., 2011) | dmAb | TN2C (Immunocytochemistry 1:300; Western blot 1:1,000) |
Antibody | Mouse anti-MAP-2 | Neuromics | MO22116 | (Immunocytochemistry 1:1,000) |
Antibody | Mouse anti-synapsin | Santa-Cruz Biotechnology | sc-376623 RRID:AB_11150313 | (Immunocytochemistry 1:1,000) |
Antibody | Mouse monoclonal anti-myc | Millipore | 05–419 RRID:AB_309725 | clone 9E10 (Immunocytochemistry 1:1000; Western blot 1:) |
Antibody | Mouse monoclonal anti-V5 | AbD Serotec/Bio-Rad | MCA1360 | clone SV5-Pk1 (Immunocytochemistry 1:2,000) |
Antibody | Rabbit anti-GFP | Thermo Fisher Scientific | A-11122 RRID: AB_221569 | (Immunocytochemistry 1:1,000) |
Antibody | Rabbit anti-NF-H | Neuromics | RA22116 | (Immunocytochemistry 1:1,000; Western blot 1:10,000) |
Antibody | Rabbit anti-PSD-95 | Millipore | AB9708 RRID:AB_11212529 | (Immunocytochemistry 1:2,000) |
Antibody | Rabbit anti-Tau | Synaptic Systems | 314 002 RRID:AB_993042 | (Immunocytochemistry 1:1,000) |
Antibody | Rabbit anti-V5 | Thermo Fisher Scientific | PA1-29324 RRID:AB_1961277 | (Immunocytochemistry 1:2,000) |
Antibody | Rabbit polyclonal anti-LPHN1 NTF | (Davletov et al., 1998) | RL1 | (Immunocytochemistry 1:1,000) |
Antibody | Rabbit polyclonal anti-LPHN1-peptide | (Davydov et al., 2009) | PAL1 | (Immunocytochemistry; Western blot 3 ng/mL) |
Antibody | Sheep anti-teneurin-2 N-terminus | R and D systems | AF4578 RRID:AB_10719438 | TN2N (Western blot 1 μg/mL) |
Cell line (Homo sapiens) | HEK293A | ECCC | RRID:CVCL_6910 | |
Cell line (Mus musculus) | Neuroblastoma 2a | ATCC | RRID:CVCL_0470 | |
Chemical compound | B27 Supplement | Life Technologies | 17504044 | |
Chemical compound | Ca-free Hibernate-A medium | BrainBits UK | HE-Ca | |
Chemical compound | Fluo-4 acetomethoxy ester | Thermo Fisher Scientific | F14201 | |
Chemical compound | Insulin Transferrin Selenium Supplement | Life Technologies | 41400045 | |
Chemical compound | Neurobasal-A medium | Thermo Fisher Scientific | 21103049 | |
Chemical compound | Purified protein: BSA-TRITC | Thermo Fisher Scientific | A23016 | |
Chemical compound | Vybrant DiO | Thermo Fisher Scientific | V22886 | |
Commercial assay or kit | Amaxa Rat Neuron Nucleofector Kit | Lonza | VAPG-1003 | |
Commercial assay or kit | SuperSignal West Femto Maximum Sensitivity Substrate | Thermo Fisher Scientific | 34094 | |
Other | Microfluidic Axon Isolation Devices (MAIDs) | Xona Microfluidics | SND150 | |
Recombinant DNA reagent | BLOCK-iT Lentiviral Pol II miR RNAi Expression System pLenti6/V5-GW/ EmGFP-miR | Life Technologies | K4938-00 | |
Recombinant DNA reagent | Bottom pre-miRNA oligo targeting LPHN1 mRNA | This paper | LPHN1miR14B | Sequence 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: JF784347 | GST-Lasso |
Recombinant DNA reagent | LPH-42 | (Volynski et al., 2004) | GenBank:MF966512 | V5-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 paper | Sequence provided under Methods | |
Recombinant DNA reagent | Primer: N424: Neo/ LPHN1 Reverse | This paper | Sequence provided under Methods | |
Recombinant DNA reagent | Primer: N425: LPHN1 Forward | This paper | Sequence provided under Methods | |
Recombinant DNA reagent | Top pre-miRNA oligo targeting LPHN1 mRNA | This paper | LPHN1miR14T | Sequence 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 Systems | 248-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/A | GST-Lasso |
Peptide, recombinant protein | Purified protein: LTXN4C | (Volynski et al., 2003) | N/A | |
Software | AxoScope 10 | Axon Instruments | ||
Software | FIJI, ImageJ | NIMH, Bethesda, Maryland, USA | RRID:SCR_002285 RRID:SCR_003070 | |
Software | LSM 510 Software (for image acquisition) | Carl Zeiss Microimaging GmbH | LSM 510 | |
Software | LSM Image Browser (for image archiving and measurements) | Carl Zeiss Microimaging GmbH | RRID:SCR_014344 | |
Software | MATLAB | Mathworks | RRID:SCR_001622 | |
Software, algorithm | MATLAB | Mathworks | https://github.com/artificialbrain-tech/Axon-Guidance-Scripts | Axonal guidance scripts |
Software | MiniAnalysis | Synaptosoft | ||
Software | Volocity (for image acquisition and stitching) | Perkin-Elmer | RRID:SCR_002668 | |
Strain (Escherichia coli) | E. coli: K12 JM109 | Promega Corporation | L2005 | |
Strain (Mus musculus) | Mouse: C57BL/6J, Adgrl1-/-, LPHN1 KO | This paper | AG148/2 | P0 hippocampus |
Strain (Mus musculus) | Mouse: C57BL/6J, Adgrl1-/-, LPHN1 KO | This paper | AG148/2 | P21 flexor digitorum brevis muscle |
Strain (Rattus norvegicus) | Rat: E18 hippocampus | BrainBits UK | Rhp |