Establishment of Bex1 mutant mice
Reagents for genome editing
The Bex1 mutant mouse strains were generated using CRISPR-Cas9 technology as described previously [46]. The gRNA was designed using CRISPRdirect (https://crispr.dbcls.jp/). The sequence of the gRNA is 5′-GTCGCAGGCGGTTCCGGGTTCGG-3′. The Bex2 has the identical gRNA-targeting sequence. We confirmed Bex2 was not mutated in all the strains we analysed. The gRNA sequence was cloned into the gRNA cloning vector (Addgene Plasmid ID #41824).
For gRNA synthesis, the T7 RNA polymerase recognition site was attached to the gRNA sequence via the polymerase chain reaction (PCR). The PCR products were purified and used as the template for in vitro RNA synthesis using the mMESSAGE mMACHINE T7 Transcription Kit (Life Technologies). The synthesized gRNA was purified using MEGAclear (Ambion). Recombinant Cas9 protein was purchased (GeneArt Platinum Cas9 Nuclease, Thermo Scientific, B25642).
Microinjection
Mouse zygotes were obtained via in vitro fertilization (IVF) of WT C57BL6/N gametes. gRNA (100 ng/ml) and Cas9 protein (30 ng/ml, TrueCut Cas9 Protein v2, Invitrogen) were mixed and microinjected into the pro-nuclei and cytoplasm of zygotes. The injected embryos were incubated at 37°C until they were transferred into pseudo-pregnant females at the two-cell stage.
Genotyping and breeding
Genomic DNA was extracted from the tail tips of pups, and the genomic sequence around the gRNA target site was PCR-amplified using the following primers: forward 5′-TGGCAAAAACTGTGCACCTA-3′ and reverse 5′-GGTCCCCATGTCATCTTCAG-3′. The PCR products were treated with ExoSAP-IT (Thermo Fisher Scientific) and sequenced with the forward primer. For the analyses of Bex1 mutant mice, at least 4 different strains were analysed and gave rise to similar observations. In this paper, only the Δ7 strain was used for the analyses. The female at the F1 generation having confirmed mutation was first crossed to C57BL/6N male mice to refresh the Y chromosome. The F2 mutant male mice were then crossed with C57BL/6N female mice to refresh the X chromosome and mitochondrial genome for more than 8 generations. The Bex1 mutant allele of the strain harbouring 7-nucleotide deletion we mainly described in this paper was deposited at Mouse Genome Informatics with the allele number Bex1<em1Mori> MGI:6509427.
Expression analysis of Bex family genes
The expression of Bex family genes was analysed utilizing the datasets we deposited with the accession number DRA007101 in the DDBJ Sequence Read Archive (DRA). Prenatal (embryonic day E18.5) and postnatal (postnatal day P1, P7, P14 and P56) gene expression in C57BL/6N mice (n = 3) were processed using Bio-Linux 8 [65] to examine the expression levels in FPKM values. Heatmaps were generated using MeV (http://mev.tm4.org).
Cell culture
ARPE19 human retinal pigment epithelial cells were cultured in DMEM:F12 medium containing 10% foetal bovine serum (FBS) and penicillin/streptomycin. Neuro2a cells were cultured in Eagle's minimal essential medium supplemented with non-essential amino acids, 10% FBS and penicillin/streptomycin. NIH3T3 cells were cultured in DMEM containing 10% FBS and penicillin/streptomycin. In the knockdown experiments, Lipofectamine RNAiMAX (Invitrogen) was used to transfect the siRNA duplexes at 10 nM: human BEX1 siRNA (Sigma-Aldrich, siRNA IDs: SASI_Hs01_00052058, SASI_Hs01_00052059, SASI_Hs01_00052061), mouse Bex1 siRNA (Bioneer, AccuTarget siRNA ID1330657), mouse Bex2 siRNA (Sigma-Aldrich, siRNA ID SASI_Mm01_00072434) and siRNA negative control (Applied Biosystems, AM4611). The knockdown efficiency was assessed 48 h after transfection. The cellular growth was assessed by counting the number of cells 72 h after transfection. The phase-contrast images of the cells were taken with an EVOS FL microscope (Thermo Fisher Scientific).
Gene expression analysis (qPCR)
The total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) from cultured cells according to the manufacturer’s instruction. The extracted RNA was quantified using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific) and reverse transcribed using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and a PCR Thermal Cycler Dice (Takara) according to the manufacturer’s instructions. qPCR was performed using a LightCycler 480 SYBR Green I Master Kit on a LightCycler 480 instrument (Roche) using the reverse-transcribed cDNA as a template. The specificity and quality of the qPCR amplification was assessed by performing a melting curve analysis. The data were normalized to human TUBB or mouse Tubb5 as indicated in the Figure legends. The sequences of the primers are as follows: mouse Bex1 5′-GGAGCAGGTCTGAGAAGCAG-3′ and 5′-CACGCCTTGATCTTTGGACT-3′; mouse Bex2 5′-TGACTGGAAACCGAGAGTCC-3′ and 5′-CCTCCTTTTCCTGATGGTCA-3′; mouse Tubb5 5′-GATCGGTGCTAAGTTCTGGGA-3′ and 5′-AGGGACATACTTGCCACCTGT-3′; human BEX1 5′-AGAATCGGGAGAAGGAGGAG-3′ and 5′-TTTCTTGGTTGGCATTTTCC-3′; human TUBB 5′-TGGACTCTGTTCGCTCAGGT-3′ and 5′-TGCCTCCTTCCGTACCACAT-3′.
Immunocytochemistry
ARPE19 cells, Neuro2a and NIH3T3 cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT), permeabilized with 0.1% Triton X-100 for 2 min, blocked with 2% FBS at RT and incubated with antibody against Bex1 (ProteinTech, 12390-1-AP, 1:100), α-catenin (CST, #3240, 1:200), β-catenin (CST, #8480, 1:200), Vimentin (Santa Cruz, sc-6260, 1:100), Filamin (Thermo Scientific, #MS-1211, 1:40), Fibrillarin (FBL, 38F3, Novus, #NB300-269, 1:500), Nucleolin (CST, #87792S, 1:400), ARL13B (Abcam, ab1366481:100), acetylated tubulin (Sigma, T6793, 1:500), IFT88 (ProteinTech, 13967-1-AP, 1:100), PCNT (Abcam, ab4448, 1:500), PCNT-Alexa 594 (Novus, NB100-61071AF594, 1:200), LIN28B (CST, #4196S, 1:200), G3BP1 (Bethyl, A302-033A, 1:500) and tubulin (CST, #2148, 1:200) at 4°C overnight. After washing with PBS for 10 min 3 times, cells were incubated with appropriate secondary antibodies anti-rabbit-Alexa Fluor 488, anti-mouse-Alexa Fluor 594 or anti-mouse-Alexa Fluor 647 (1:1,000, Invitrogen) at RT for 1 h. Slides were mounted with Prolong Gold with DAPI (Invitrogen). Images were taken by confocal microscope TCS SP8 (Leica). For the analysis of the primary cilia, the frequency of ciliated cells and the number of cilia per cell were counted. Because a cell density or cell cycle status can influence ciliogenesis, the analysis of cilia was performed at similar cell density both for the normal and the Bex1-depleted conditions. The length of the primary cilia was measured with Leica Application Suite X software. The sphericity of nuclei was calculated with the images of Fibrillarin immunostaining, using the circularity plugin of the NIH ImageJ Fiji [66] using the formula: circularity = 4π(area/perimeter2).
Western blot analysis with mouse tissues
For sampling mouse tissues, dissected tissues were crushed using a homogenizer (μT-12, Taitec) in chilled RIPA buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. The lysates were centrifuged at 20,400×g for 10 min to remove debris. Laemmli sample buffer was added to the lysates, followed by boiling at 95°C for 2 min. The protein samples (10 μg per lane) were separated by SDS-PAGE and transferred to a Hybond-P PVDF membrane (GE Healthcare). Because an antibody reactive to mouse Bex1 protein was not commercially available, we developed a new antibody reactive to mouse Bex1. Polyclonal antisera were generated by immunizing rabbits with the peptide [NH2-C+KKEEKEEKPQDTIR-COOH] derived from the Bex1-specific amino acid sequence. The N terminal cysteine [C+] of the peptide was added for the purpose of conjugating the peptide to carrier protein. As loading control, alpha-tubulin (1:4000, Cell Signaling Technology, #3873) and Gapdh (1:4000, Cell Signaling Technology, #5174) were used. Secondary antibodies conjugated with horseradish peroxidase (Thermo Fisher Scientific, anti-mouse 32430 and anti-rabbit 32460) were used at 1:1000 dilution. The immunoreactive bands were detected using Chemi-Lumi One L or Chemi-Lumi One Ultra (Nacalai Tesque, Kyoto, Japan).
Mouse histological analysis
The ocular histology was analysed by the ophthalmologists. Following the euthanization of the mice, the eyes were enucleated and were fixed with 4% PFA at RT for 30 min. For haematoxylin and eosin (HE) staining, samples were paraffin-embedded and sectioned at 3 μm thickness. The images were taken by Nikon ECLIPSE 90i (Nikon). For immunohistochemistry, after removal of the optic nerve, cornea and lens, the retina was cut into quadrants, making angles at 90 degrees to the optic nerve head at the centre. The flat-mounted retina was analysed by immunohistochemistry as described below.
For the histological analysis in brains and kidneys, the mice were systemically fixed with 4% PFA followed by dissection of the organs. The dissected organs were post-fixed with 4% PFA for 3 h followed by sectioning at 3 μm thickness. The tissue sections were stained with HE. The images were taken by NanoZoomer S210 slide scanner (Hamamatsu) and analysed with NanoZoomer U12388-01 software (Hamamatsu). Histological diagnosis was made by the nephrologist.
For the immunohistochemistry with the retina and striatum, the flat-mounted retina or the PFA-fixed brain was permeabilized with 0.1% Triton X-100 for 2 min and blocked with 2% FBS at RT for 60 min. The tissues were incubated with the primary antibody against acetylated tubulin (Sigma, T6793, 1:500) at 4°C overnight. After washing with phosphate buffer saline (PBS), the tissues were incubated with the secondary antibody anti-mouse-Alexa Fluor 594 (1:1,000, Invitrogen). After washing with PBS, the tissues were mounted with Prolong Gold with DAPI (Invitrogen). Images were taken by confocal microscope TCS SP8 (Leica). For the analysis of the primary cilia, the frequency of ciliated cells was counted. The length of the primary cilia was measured with Leica Application Suite X software.
Protein expression and purification
The expression plasmid for mouse Bex1 were constructed with fusion proteins tagged with hexahistidine (His) at the N-terminus by inserting Bex1 into the pETDuet-1 vector. The pETDuet-1-Bex1 vector was introduced into the E. coli strain Rosetta-gami 2(DE3)pLysS. Transformed bacteria were grown in Luria broth medium containing ampicillin. Uniformly 15N-labelled proteins were grown in M9 minimal medium with 0.5 g/L 15NH4Cl. Bacterial cells were grown to an OD600 of ~ 0.6 at 37°C, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the medium. Cells were further incubated at 37°C for 5 h and were then harvested by centrifugation at 5000×g for 10 min at 4°C. The collected bacterial cells were suspended in the buffer containing 20 mM sodium phosphate (pH 8.0), 1 M NaCl, 0.2 mg/mL lysozyme, DNase I and 1 mM phenylmethanesulfonyl fluoride (PMSF) and were rotated for 1 h at 4°C. The cells were sonicated three cycles with intermittent pulses for 1 min (pulse of 0.7 s, interval of 0.3 s, output level of 3) using an ultrasonic disruptor equipped with a standard flat tip (Sonifier 450 Advance, Branson Ultrasonics, Connecticut). After removal of cell debris by centrifugation at 12,000×g for 45 min at 4°C, the cell extract was loaded onto a Ni-NTA resin (QIAGEN). The column was washed with a solution containing 20 mM sodium phosphate (pH 8.0), 300 mM NaCl and 20 mM imidazole, followed by the elution with the buffer containing 20 mM sodium phosphate (pH 8.0), 300 mM NaCl and 500 mM imidazole. The purity of the protein was analysed by SDS-PAGE. The purified protein was collected, dialyzed against suitable solution for each experiment and concentrated with a powder of absorbent gel (Spectra/Gel® Absorbent, Spectrum-Labs, Rancho Dominguez, CA, USA) at 4 °C. The concentration of Bex1 protein was calculated from the spectra measured by NanoDrop One (Thermo Scientific), using an absorption coefficient of 7000.
CD spectroscopy
Circular dichroism CD spectra between 195 and 250 nm were collected on a J-805 spectropolarimeter (JASCO, Tokyo, Japan) at 10, 25, and 37 °C. We measured 200 μL solution of 5-10 uM BEX1 in 20 mM sodium phosphate buffer, 140 mM NaCl, pH7.3 in a quartz cuvette with a 1 mm light path length. The results were expressed as apparent ellipticity with the unit of ellipticity.
Nuclear magnetic resonance (NMR) experiments
The two-dimensional 1H-15N HSQC spectrum was measured with a Bruker Avance III 900-MHz spectrometer equipped with a cryomagnetic probe. NMR experiments were performed at the protein concentration of 28 μM. The solvent conditions were 20 mM sodium phosphate buffer, 140 mM NaCl, 5% D2O, pH7.3. The chemical shift values were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS).
In the NMR titration experiment with Bex1 and GTP, GTP was added to 28 μM Bex1 at concentrations of 28 μM, 56 μM, 112 μM and 280 μM, and each 1H-15N HSQC spectrum was measured for 2.5 h at 25 °C. Chemical shift perturbation (Δδ) was calculated by the following equation (1);
$$\Delta \updelta =\sqrt{{\left({\Delta \updelta}_{\mathrm{H}}\right)}^2+{\left(\Delta {\updelta}_{\mathrm{N}}/5\right)}^2}$$
(1)
where ΔδH and ΔδN are the chemical shift differences in the presence and absence of the GTP, respectively.
The concentration of GTP was plotted on the horizontal axis against Δδ on the vertical axis and fitted to obtain binding constant (Kd) by the following eq. (2);
$${\Delta \updelta}_{\mathrm{H},\mathrm{N}}={\Delta \updelta}_{\mathrm{max}}\times \frac{\left(\left[\mathrm{Bex}1\right]+\left[\mathrm{GTP}\right]+{K}_{\mathrm{d}}\right)-\sqrt{{\left(\left[\mathrm{Bex}1\right]+\left[\mathrm{GTP}\right]+{K}_{\mathrm{d}}\right)}^2-4\left[\mathrm{Bex}1\right]\times \left[\mathrm{GTP}\right]\ }}{2\times \left[\mathrm{Bex}1\right]}$$
(2)
where Δδmax is ΔδH,N in case of moving to the maximum, and Δδmax and Kd were calculated by fitting Eq. (2) using Igor application.
Structural modelling
The tertiary structures of Bex family proteins were assessed by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). The property of Bex family proteins as intrinsically disordered proteins (IDPs) was assessed by PrDOS (http://prdos.hgc.jp/) and DISOPRED3 (http://bioinf.cs.ucl.ac.uk/psipred/).
In vitro reconstitution assays
The Bex1 condensate formation was tested by the method described previously [32]. The recombinant Bex1 protein (10 μM) was labelled with DyLight 594 NHS ester according to the manufacturer’s instruction (ThermoFisher Scientific). To remove unbound dye, dialysis was performed with the dialysis buffer containing 25 mM HEPES and 150 mM KCl using BioDesignDialysis Tubing (molecular weight cut off = 8000 Da, BioDesign Inc. of New York). The labelled Bex1 protein (final concentration = 25 nM) was diluted in the buffer containing 25 mM HEPES, 150 mM KCl, 0.5 mM dithiothreitol (DTT) in the presence (9%) or absence (0%) of polyethylene glycol (PEG) 4000 and incubated for 5 min at 25°C. Images of the Bex1 condensates were taken by IX83 inverted microscope (Olympus). Pseudo-coloured images were generated to show Bex1 signals in green.
The potency of the Bex1 condensates to promote tubulin polymerization was assessed by incubating the Bex1 condensates with tubulin protein. For the tubulin, Alexa 488-labelled (catalogue number: 048805, PurSolutions, Tennessee) or unlabelled (catalogue number: 032005) tubulin was purchased and mixed at 1:4. The Bex1 condensates were incubated with the mixed tubulin at 2.5 μM in the buffer containing 25 mM HEPES,150 mM KCl, 0.5 mM DTT, 9% PEG and 2 mM GTP and incubated for 10 min at 25°C. Images of polymerized tubulin were obtained by IX83 inverted microscope (Olympus) and confocal microscope TCS SP8 (Leica). Pseudo-coloured images were generated to show Bex1 signals in green and tubulin signals in magenta.
Fluorescent recovery after photo-bleaching (FRAP)
For the in vivo experiments, FRAP of EGFP-Bex1 in ARPE19 cells was performed on Fluoview FV3000 confocal microscope system (Olympus). Using a 60× oil immersion objective, a whole EGFP-Bex1 granule was bleached using a laser intensity of 20% at 488 nm. Recovery was recorded for every second for a total of 300 s after bleaching. Analysis of the recovery curves was carried out with cellSens software (Olympus).
For the in vitro experiments, FRAP was carried out with samples in glass bottom 8-well chamber slides using a Fluoview FV3000 confocal microscope equipped with 60× oil immersion objectives, as above. Condensates were bleached using a laser intensity of 30% at 561 nm. Recovery was recorded for every second for a total of 180 s after bleaching. Analysis of the recovery curves was carried out with cellSens software (Olympus).
Statistical analysis
For all quantified data, mean ± standard deviation (SD) was presented. Statistical significance between two experimental groups was indicated by an asterisk and comparisons were made using the Student’s t test. P values less than 0.05 were considered significant.