KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Myrlysin (BORCS5/ LOH12CR1), rabbit, IB 1:500 | Proteintech | Cat# 17169–1-AP; RRID:AB_2137150 |
| Diaskedin (BORCS7/C10orf32), rabbit, IB 1:500 | Abnova | Cat# PAB23142; RRID:AB_11122571 |
| GAPDH (0411), HRP-conjugated, IB 1:300 | Santa Cruz | Cat# sc-47724; RRID:AB_627678 |
| LAMP1, rat, IB 1:500, IF 1:500, IHC 1:100 | Developmental Studies Hybridoma Bank | Cat# 1D4B; RRID:AB_2134500 |
| LAMTOR4 (C7orf59) (D6A4V), rabbit, IB 1:500, IF 1:500, IHC 1:500 | Cell Signaling | Cat# 12284; RRID:AB_2797870 |
| Synaptophysin 1 (SYP1) (D-4), mouse, IB 1:1,000 | Santa Cruz | Cat# sc-17750; RRID:AB_628311 |
| Synaptophysin 1 (SYP1) (H-93), rabbit, IF 1:500, IHC 1:500 | Santa Cruz | Cat# sc-9116; RRID:AB_2199007 |
| Synaptogyrin 1 (SYG1), rabbit, IB 1:500 | Synaptic Systems | Cat# 103002; RRID:AB_887818 |
| Vesicular glutamate transporter 1 (VGLUT1), guinea pig, IB 1:500, IF 1:250, IHC 1:500 | Millipore Sigma | Cat# AB5905; RRID:AB_2301751 |
| Synaptic vesicle glycoprotein 2A (SV2), mouse, IB:200, IF: 1:200, IHC 1:500 | Developmental Studies Hybridoma Bank | Cat# SV2; RRID:AB_2315387 |
| Autophagy-related protein LC3B, rabbit, IB 1:10,000 | Sigma-Aldrich | Cat# L7543; RRID:AB_796155 |
| β-tubulin, rabbit, IB 1:1,000 | Cell Signaling | Cat# 2146; RRID:AB_2210545 |
| Cytochrome-C (Cytc) (6H2.B4), mouse, IF 1:1,000 | BD Bioscience | Cat# 556432; RRID:AB_396416 |
| HA (influenza hemagglutinin) epitope tag, chicken, IF 1:250 | Millipore Sigma | Cat# AB3254; RRID:AB_91371 |
| Ankyrin-G (P-20), goat, IF 1:50 | Santa Cruz | Cat# sc-31778; RRID:AB_2289736 |
| Ankyrin-G, mouse, IF 1:20 | NeuroMab | Cat# 73–146; RRID:AB_10697718 |
| Pan-Neurofascin (extracellular), mouse, IF 1:1,000 | UC Davis/NIH NeuroMab Facility | Cat# 75–172, RRID:AB_2282826 |
| GFP polyclonal antibody, chicken, IF 1:500 | Thermo Fisher | Cat# A10262; RRID:AB_2534023 |
| Alexa Fluor 488-conjugated donkey anti-rabbit IgG, IF 1:1,000 | Thermo Fisher | Cat# A21206; RRID:AB_2535792 |
| Alexa Fluor 546-conjugated donkey anti-rabbit IgG, IF 1:1,000, IHC 1:1000 | Thermo Fisher | Cat# A10040; RRID:AB_2534016 |
| Alexa Fluor 488-conjugated donkey anti-mouse IgG, IF 1:1,000 | Thermo Fisher | Cat# A21202; RRID:AB_141607 |
| Alexa Fluor 647-conjugated goat anti-mouse IgG, IHC 1:1,000 | Thermo Fisher | Cat# A21235; RRID:AB_2535804 |
| Alexa Fluor 488-conjugated donkey anti-rat IgG, IF 1:1000, IHC 1:1,000 | Thermo Fisher | Cat# A21208; RRID:AB_2535794 |
| Alexa Fluor 568-conjugated goat anti-rat IgG, IF 1:1000, IHC 1:1,000 | Thermo Fisher | Cat# A11077; RRID:AB_2534121 |
| Alexa Fluor 488-conjugated goat anti-Guinea pig IgG, IHC 1:1,000 | Thermo Fisher | Cat# A11073; RRID:AB_2534117 |
| Alexa Fluor 647-conjugated goat anti-chicken IgY, IF 1:1,000 | Thermo Fisher | Cat# A21449; RRID:AB_2535866 |
| Alexa Fluor 488-conjugated goat anti-chicken IgY, IF 1:1,000 | Thermo Fisher | Cat# A32931; RRID:AB_2762843 |
| Alexa Fluor 647-conjugated donkey anti-goat IgG, IF 1:1,000 | Thermo Fisher | Cat# A21447; RRID:AB_2535864 |
| Alexa Fluor 405-conjugated goat anti-chicken IgY (H+L), IF 1:1,000 | Abcam | Cat# AB175675; RRID:AB_2810980 |
| HRP-conjugated goat anti-rabbit IgG (H+L), IB 1:5,000 | Jackson ImmunoResearch | Cat# 111–035-144; RRID:AB_2307391 |
| HRP-conjugated goat anti-rat IgG (H+L), IB 1:5,000 | Jackson ImmunoResearch | Cat# 112–035-143; RRID:AB_2338138 |
| HRP-conjugated donkey anti-mouse IgG (H+L), IB 1:5,000 | Jackson ImmunoResearch | Cat# 715–035-150; RRID:AB_2340770 |
| HRP-conjugated donkey anti-Guinea pig IgG (H+L), IB 1:5,000 | Jackson ImmunoResearch | Cat# 706–035-148; RRID:AB_2340447 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Lipofectamine 2000 | Thermo Fisher | Cat# 11668019 |
| Alexa Fluor 594-conjugated α-Bungarotoxin | Thermo Fisher | Cat# B35451 |
| CF®640R Mix-n-Stain antibody labeling kit | Biotium | Cat# 92245 |
| Alexa Fluor 647-Phalloidin | Thermo Fisher | Cat# A22287 |
| Janelia Fluor®−549 Halo Tag Ligand | Gift from Luke Lavis, HHMI Janelia, USA | N/A |
| NeutrAvidin Protein | Thermo Fisher | Cat# 31050 |
| D-biotin | Sigma-Aldrich | Cat# 47868 |
| Experimental Models: Cell Lines | ||
| Primary hippocampal neuron cultures from rat E18 brains | N/A | N/A |
| Primary hippocampal neuron cultures from WT and myrlysin-KO mouse E18 brains | N/A | N/A |
| Mouse embryonic fibroblasts (MEFs) from WT and myrlysin-KO E15 mice | N/A | N/A |
| Experimental Models: Organisms/Strains | ||
| Mice: myrlysin-KO 10 bp deletion | Made by The Jackson Laboratory | N/A |
| Mice: C57BL/6J | The Jackson Laboratory | Stock No: 000664 | Black 6 |
| Rats: Sprague Dawley® outbred | ENVIGO | Stock No: Sprague Dawley® SD® |
| Oligonucleotides | ||
| sgRNA #1: 5ʹ-ATCTTGGCCCGATGTTTAGC-3ʹ | This study | N/A |
| sgRNA #2: 5ʹ-GGCTAAACATCGGGCCAAGA-3ʹ | This study | N/A |
| Primer: transgenic N0 genotyping forward: 5ʹ-ACTACAAACTCCTCTTTCTAATGACTG-3ʹ | This study | N/A |
| Primer: transgenic N0 genotyping reverse: 5ʹ-CTTCAGACAGGGCATTCAGG-3ʹ | This study | N/A |
| Primer: 10 bp deletion genotyping forward: 5ʹ-CATCTCCGGCTAAACATC GGGC —3ʹ | Eurofins Genomics | N/A |
| Primer: 10 bp deletion genotyping reverse: 5ʹ- CTGAGCTACCACCACGATATC-3ʹ | Eurofins Genomics | N/A |
| Recombinant DNA | ||
| Modified Clontech plasmid-LAMP1-RFP | Addgene; Sherer et al., 2003; deposited by Walther Mothes, Yale, USA | Cat# 1817 |
| pEGFP-N1-LAMP1-GFP | Farías et al., 2017 | N/A |
| pEGFP-N1-SYG1-GFP | Zhao and Nonet, 2001 | N/A |
| pEGFP-N1-SYG1-Halo | This study | N/A |
| pEGFP-N1-SYP1-YFP | Gift from Ann Marie Craig, UBC, Canada | N/A |
| pmCherry-N1-SYP1-mCherry | Gift from Ann Marie Craig, UBC, Canada | N/A |
| pEGFP-N1-VMAT2-GFP | This study | N/A |
| pmCherry-N1-ARL8B-mCherry | Farías et al., 2017 | N/A |
| pcDNA3.1/CT-GFP-ARL8B-GFP | Kaniuk et al., 2011 | N/A |
| Modified Clontech Plasmid-LAMP1-SBP-RFP | Guardia et al., 2019 | N/A |
| Modified Clontech pmCherry-N1-Strep-KIFC1*-HA | Guardia et al., 2019; deposited in Addgene | Cat# 120171 |
| pEGFP-N1-CD63-GFP | Farías et al., 2017 | N/A |
| Software and Algorithms | ||
| Fiji/ImageJ | NIH | https://fiji.sc/ |
| TIDE software | Free online software | https://tide.nki.nl/ |
IF: immunofluorescence, IB: immunoblotting, IHC: immunohistochemistry.