Reagents and antibodies
All antibodies and reagents used in this study are listed in Additional file 2: Tables S1 and S2.
Plasmid constructions and Lentivirus production
The SPAST and FBXL17 expression constructs were amplified using RT-PCR using cDNA and incorporated into various plasmid vectors. Detailed information on the plasmid constructs is provided in Additional file 2: Table S3. The 3xHA-FBXL17 clone was purchased from GeneCopoeia Inc. (EX-H4410-M06, Rockville, MD, US). For lentiviral vector construction and production, the lenti-FBXL17_shRNA clone was purchased from OriGene (#TL304572, Rockville, MD, US). For the production of Y52C-SPAST expressing lentivirus, 293FT cells were transfected with pLVx-Y52C SPAST along with psPAX2 (Genome), PMD2.G (Envelope), and then media was harvested 48 h post-transfection. For generation of CK2β-Cas9 stable cell, the CK2β CRISPR/Cas9 (sc-401684) and CK2β homology-induced repair (HDR, sc-401684-HDR) plasmids were purchased from Santa Cruz Biothechnology (Dallas, TX, US).
Identification of the SPAST binding proteins through HuProt™ microarray
To identify SPAST binding proteins, His-tagged SPAST protein was induced and purified in an Escherichia coli expression system and then analyzed at Gene On Biotech (Daejeon, Republic of Korea) using Human Huprot™ protein microarray (CDI Labs, Mayaguez, PR, US). Human protein microarray, which contains over 20,000 full-length recombinant human proteins, was used. Briefly, the protein microarray was incubated with blocking buffer (2% BSA in 1 × PBS with 0.1% tween-20) for 2 h, and 3 μg of biotinylated His-SPAST was treated onto the array for 8 h at 4 ℃. Subsequently, the array was incubated with 1 μg of streptavidin-fluorescence (Alexa-Fluor 635 nm) for 1 h at 4 ℃. The microarray result was detected using a GenePix4100A microarray laser scanner (Molecular Devices, San Jose, CA, US).
HEK293T and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, welgene, Gyeongsan, Republic of Korea) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, US) and antibiotics (Thermo Fisher Scientific) in a humidified incubator with 5% CO2 at 37 °C. For proliferation, ReNcell CX was cultured in ReN NSC maintenance medium (Merck Millipore, Burlington, MA, US) with 20 ng/ml epidermal growth factor (EGF, Peprotech, Rocky Hill, NJ, USA), 20 ng/ml basic fibroblast growth factor (bFGF, Peprotech), and penicillin/streptomycin antibiotics. For the neuronal differentiation, ReNcell CX was differentiated in ReN NSC maintenance medium (SCM005, Merck Millipore) containing penicillin/streptomycin antibiotics without EGF and bFGF for the indicated days.
For transient transfection into HEK293 cells, cells were transfected with indicated plasmid constructs using Transporter™ 5 transfection reagent (Polysciences Inc. Warrington, PA, US). For the electroporation, ReNcell CX cells were harvested, resuspended in BTX electroporation buffer (BTX, Holliston, MA, US), and transfected with ECM830 electroporation system (BTX). Electroporation condition was performed by 1 pulse at 70 V discharge for 30 ms.
Generation of CK2β-Cas9 stable cell lines
The CK2β CRISPR/Cas9 containing cas9, green fluorescent protein (GFP), gRNA expression and CK2β HDR expressing puromycin resistance, red fluorescent protein (RFP) Plasmids were purchased from Santa Cruz. HEK293 cells in a 6-well plate were co-transfected with 2 ug of CK2β CRISPR/Cas9 and 2 ug of CK2β HDR plasmids using Transporter™ 5 transfection reagent. The transfected HEK293 cells with GFP and RFP expression were selected with 5ug/ml puromycin (A1113803, Thermo Fisher Scientific) containing medium for 2 weeks. The puromycin‐resistant cells of CK2β stable knockout were maintained with 1 ug/ml puromycin, and confirmed by qRT-PCR with specific primers.
NSC proliferation and differentiation assay
ReNcell CX cells (5 × 103 cells /well) were seeded onto ultra-low attachment 12-well plates with ReN NSC maintenance medium. The medium was changed every 2 days, and the diameter of neurospheres was observed under a microscope. ReNcell CX cells were cultured in a differentiation medium [10 μM forskolin (F3917, Sigma-Aldrich), 20 ng/ml brain-derived neurotrophic factor (BDNF), 5 μM retinoic acid (R2625, Sigma-Aldrich) in NSC maintenance medium]. After 4 days, cells were fixed with 4% paraformaldehyde solution and immunostained with rabbit anti-MAP2 (Cell signalling Technology, Danvers, MA, US) and mouse anti-GFAP (Cell signalling Technology) antibodies.
Conventional reverse transcription (RT)-PCR and quantitative (q)RT-PCR
Total RNA was isolated using an Rneasy mini kit (QIAGEN, Hilden, Germany). A 1 μg of total RNA was used for cDNA synthesis was performed using Verso cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For conventional RT-PCR, the cDNA was amplified with the following gene-specific primers. The PCR products were loaded on 1% agarose gel and photographed.
For qRT-PCR, qRT-PCR was performed with AriaMx Real-Time PCR System (Agilent, Santa Clara, CA, US). Reactions were prepared in a total volume of 14 µl containing: 2 µl of template, 0.6 µl of each primer (final concentration of 400 nM), 7 µl of 2 × SensiFAST SYBR No-ROX mix (Meridian bioscience Inc., OH, US). The PCR reaction was performed according to the manufacturer’s instructions, in brief, 95 °C for 3 min, followed by 40 cycles of 5 s at 95 °C and 10 s at 60 °C. Melting curve analysis was performed under the following condition: 30 s at 95 °C, 30 s at 65 °C, and 30 s at 95 °C. After normalization against GAPDH mRNA levels, relative quantification of gene expression was performed using the 2-ΔΔCT method. The primer sequences are provided in Additional file 2: Tables S4 and S5.
Cells were fixed with 4% paraformaldehyde for 1 h at room temperature (RT) at around 20–22 ℃, followed by three washes in PBS and permeabilization with 0.1% Triton X-100 in PBS for 15 min at RT. After fixation, blocking was performed with blocking reagent (2% normal horse serum in PBS) for 1 h at RT. Primary antibodies were diluted to the indicated concentrations (Additional file 2: Table S1) in PBST (1 × PBS with 0.05% tween 20), incubated overnight at 4 °C, and washed three times in PBS. The secondary antibodies were incubated in PBST for 1 h at RT and washed three times in PBS. For nuclear counterstaining, DAPI solution (BD Biosciences, Franklin Lakes, NJ, US) was incubated for 5 min at RT in PBST, washed, mounted with vectorMount AQ mounting medium (H-5501, Vector Laboratories, Burlingame, CA, US), observed under a fluorescence microscope (IX71, Olympus, Tokyo, Japan), and verified the co-localization using CellSens software. All images were quantitated by ImageJ software.
For visualizing Endoplasmic reticulum (ER) in living cells, ER staining was performed according to the instructions of the manufacturer of ER-tracker kit (Thermo Fisher Scientific). Briefly, HeLa cells were incubated with 1 uM ER tracker dye in Hank's buffered salt solution (#14025092, HBSS, Thermo Fisher Scientific) for 30 min at 37 °C and the cells were fixed with 4% PFA in PBS for 2 min at 37 °C.
Western blot analysis
Briefly, cells were lysed on ice using RIPA buffer [50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% NP40, 0.1% SDS, 1 mM PMSF, 1 × protease inhibitor cocktail (Roche, Basel, Switzerland)], separated using 12% SDS-PAGE, and transferred to the PVDF membrane (Millipore). Membranes were incubated with specific primary antibodies in PBST overnight at 4 °C (Additional file 2: Table S1). Subsequently, the membrane was incubated with a secondary antibody in PBST containing 0.5% skim milk for 1 h at RT. The proteins were visualized using a chemiluminescence kit (Miracle-Star, Intron Biotech, Seoul, Republic of Korea).
Protein stability analysis
HEK293 cells were transfected with 10 μg each of HA-SPAST-M1 or Flag-FBXL17 plasmids. After 24 h, cells were treated with 50 μg/ml cycloheximide for 0, 2, and 4 h. At the indicated time points, the cells were harvested, and proteins were detected using western blotting. The quantification experiment was performed three times under the same conditions. The signal intensity was determined using ImageJ software.
Separation of cytosolic and nuclear extractions
Cells (3 × 106 cells /100 mm dish) were transfected with 10 μg of plasmids as indicated and harvested 24 h post-transfection. A subcellular fraction from transfected cells was isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Immunoprecipitation assays and pull-down assay
For the immunoprecipitation assay, HEK293 cells were transfected with 10 μg of plasmids as indicated and harvested 24 h after transfection. Cells were then lysed in NET gel buffer [50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, pH 8.0 containing protease inhibitor cocktails] by sonication, cleared by centrifugation at 13,000 rpm for 10 min at 4 °C and supernatants were incubated with bead-conjugated anti-Flag (Sigma-Aldrich) or anti-HA antibody (Thermo Fisher Scientific) for 3 h at 4 °C on a gentle rotator shaker. ReNcell CX (5 × 106 cells) were lysed in NET gel buffer by sonication, centrifuged, and the supernatants were incubated with antibodies against FBXL17 or control IgG overnight at 4 °C. And then, the cell lysate containing antibodies was incubated with Protein A Magnetic Beads (Thermo Fisher Scientific) for 2 h at 4 °C. After incubation, the magnetic beads were placed into the magnet stand, washed three times with 1 ml PBST. Precipitated proteins were separated using SDS-PAGE and analyzed using western blotting.
For pull-down assay, cell lysates and reaction mixtures after in vitro assay were incubated with glutathione Sepharose 4B beads (Sigma-Aldrich, St Louis, MO, USA) or Ni–NTA agarose (QIAGEN) for 3 h at 4 °C with rotation. The beads were precleared three times in 1 ml PBST. After incubation, the bound proteins were washed three times in PBST and analyzed using western blotting with indicated antibodies.
Purification of recombinant proteins expressed in E. coli and in vitro binding assay
E. coli BL21 cells containing the pET28a-His-SPAST plasmid were grown at 37 ℃ until the optical density at 600 nm (OD600) reached 1. Protein expression was induced by incubation with 0.5 mM isopropyl-b-D-thiogalactoside (IPTG, Sigma-Aldrich) and 2% Ethanol at 18 ℃ for 48 h. E. coli. BL21 cells containing pET28a-His-FBXL17-ΔNT1, pGEX4T1-GST-SPAST-M1, pGEX4T1-GST-KLHL, and pGEX4T1-GST plasmids were grown at 37 ℃ to reach an OD 600 nm of 0.8, induced with 1 mM IPTG and 2% ethanol at 20 ℃ for 24 h. The purification of his-tagged recombinant protein from induced E. coli was performed as previously described . Briefly, cells were lysed using lysis buffer [20 mM Tris–HCl, 300 mM NaCl, 10 mM imidazole, and 1 mM Phenylmethanesulfonyl fluoride (PMSF, Roche), pH 7.5], incubated with Ni–NTA agarose for 3 h at 4 °C. The bead-protein complexes were loaded on a column and washed with washing buffer [20 mM Tris–HCl, 300 mM NaCl, and 20 mM imidazole, pH 7.5]. The washed beads were subsequently eluted in elution buffer [20 mM Tris–HCl and 250 mM imidazole, pH 7.5]. The eluted proteins were dialyzed in dialysis buffer [10 mM Tris–HCl, 10% glycerol, and 1 mM PMSF, pH 7.5] at 4 °C overnight. For purification of GST-tagged recombinant protein, cells were lysis with lysis buffer [1 × PBS containing 0.5% Triton X-100 and 1 mM PMSF] incubated with glutathione sepharose beads overnight at 4 °C. The bead-protein complexes were washed three times with washing buffer [1 × PBS containing 1 mM PMSF] and eluted in elution buffer [10 mM Tris–HCl and 150 mM NaCl, 25 mM reduced glutathione, pH 7.5]. The eluted proteins were dialyzed in a dialysis buffer [1 × PBS containing 10% glycerol, 1 mM PMSF].
For in vitro binding assay, purified recombinant proteins from E. coli, each 0.5 μg protein used per binding reaction was incubated in 200 μl binding buffer [1 × PBS, 0.1% NP-40 containing 1 mM PMSF and protease inhibitor cocktails], pulled-down using glutathione sepharose beads and analyzed using western blotting.
In vivo and in vitro ubiquitination assay
In vivo and in vitro ubiquitination was performed as previously described [1, 2]. Briefly, for in vivo ubiquitination assay, HEK293 cells were transfected with 10 μg Flag-tagged SPAST-M1, Flag-SPAST-M87, the lysine to arginine mutants of SPAST-M1, and 5 μg HA-tagged ubiquitin plasmid using Transporter™ 5 transfection reagent. At 24 h after transfection, cells were treated 10 μM MG132 for 16 h, lysed in NET gel buffer, and the cell lysates were incubated using an anti-Flag magnetic bead overnight at 4 °C. The immunoprecipitated proteins were washed three times in PBST and analyzed using western blot using indicated antibodies. For in vitro ubiquitination assay, the reaction mixtures were incubated with 0.5 μg His-E1, 0.5 μg His-UbcH10b, 0.5 μg His-FBXL17-ΔNT1, 100 μg/reaction HeLa cell S100 extract, 5 μg GST-SPAST-M1, GST-KLHL or 5 μg GST, and 25 μg/mL Flag-ubiquitin (Boston Biochem, Cambridge, MA, USA) in reaction buffer [25 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 2.5 mM DTT, 5 mM adenosine triphosphate containing the ATP-regeneration system [1 mM creatine phosphate, 1 mM creatine kinase, 0.5 μg/mL ubiquitin aldehyde] at 37 °C for 1.5 h, followed by pull-down using glutathione sepharose beads and analyzed using western blotting.
In vitro casein kinase II (CK2) kinase assay
GST-tagged SPAST, KLHL, and GST alone or His-tagged FBXL17-ΔNT1 proteins were purified from the E. coli expression system. The recombinant proteins (each 0.5 μg/reaction) were incubated in kinase reaction buffer [50 mM Tris–Cl. pH7.5, 200 mM NaCl, 10 mM MgCl2, 2 mM EDTA, 1 mM DTT, 200 μM ATP] with 500 U casein kinase II (P6010, New England Biolabs, Ipswich, MA, US), composed of two α-subunits and two β-subunits in the presence or absence of 10 μCi of [γ-32P]ATP for 30 min at 30 ℃. The reaction was terminated by adding SDS sample loading buffer [50 mM Tris–Cl. pH 6.8, 2% SDS, 10% glycerol, 0.2% bromophenol blue dye, 4% β-mercaptoethanol]. After resolution using SDS-PAGE, the gel was transferred into the PVDF membrane and visualized by direct exposure of the membrane to X-ray film (AGFA, Belgium).
Animal experiments and dissection of the forebrain and spinal cord from mouse embryos
Mice were housed in a specific-pathogen-free animal laboratory (humidity 60–65%; temperature 22 °C) in 12 h light–dark cycles. All animal housing and experiments conducted were in accordance with the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal Care and Use Committee Guidelines (KRIBB-AEC-18090).
The mouse embryonic tissues were harvested for the day of vaginal plug was dated as embryonic day (E) 0.5. The dissection of spinal cords from embryos was performed as previously described [3, 4]. Briefly, the pregnant mouse (E 10.5–18.5) was anesthetized using carbon dioxide (CO2) gas as a euthanasia agent, dissected the lower abdomen, and the uteri containing embryos were placed in a 100-mm Petri dish containing ice-cold sterile HBSS. The extraembryonic membranes overlying the embryos were removed from cranial to caudal and separated from the forebrain to the spinal cord using a dissection microscope (Nikon C-PSN, Tokyo, Japan). The isolated tissues were washed in ice-cold PBS and analyzed using RT-PCR or western blotting.
Mouse embryo tissue preparation and immunofluorescence assay
The pregnant mouse (E 14.5) was sacrificed by cervical dislocation and carefully removed the embryos from the uterus. Embryos were removed extraembryonic membranes and fixed overnight in 4% paraformaldehyde (PFA) in PBS at 4 °C following immersed in 30% sucrose in PBS for 48 h at 4 °C. For the frozen section, fixed embryos embedded in OCT compound (Tissue-Tek, #4583, Sakura Finetek USA, Torrance, CA, US), and sectioned at 10 μm thickness using a cryostat (CM1520, Leica, Wetzlar, Germany). The sections were treated with 0.5% Triton X-100 in PBS for 10 min and blocked with 2.5% normal horse serum in PBS for 1 h. After the blocking, sections were incubated with anti-SPAST antibody and anti-FBXL17 antibody for 24 h at 4 °C in PBS. After washing, the sections were proceeded according to the immunofluorescence assay method.
The student’s t-test was used to determine statistical significance. Differences were considered statistically significant at P < 0.05.