1 Disease-associated missense variants in ZBTB18 disrupt DNA 2 binding and impair the development of neurons within the 3 embryonic cerebral cortex

4 Isabel A. Hemming1,2,3,†, Olivier Clément1,2,3,5,†, Ivan E. Gladwyn-Ng1,2, Hayley D. Cullen1,2,3,4, Han 5 Leng Ng5, Heng B. See1,2, Linh Ngo1,2 , Daniela Ulgiati5, Kevin D.G. Pfleger1,2,6, Mark Agostino4,7,8,*, 6 Julian I-T. Heng4,6,7,*

7 1The Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands, Australia 8 2Centre for Medical Research, The University of Western Australia, Crawley, Australia 9 3The University of Western Australia, Medical School, Faculty of Health and Medical Sciences, 10 Crawley, Australia 11 4Curtin Health Innovation Research Institute, Curtin University, Bentley, Australia 12 5The University of Western Australia, School of Biomedical Sciences, Faculty of Health and Medical 13 Sciences, Crawley, Australia 14 6ARC Centre for Personalised Therapeutics Technologies, Australia 15 7School of Pharmacy and Biomedical Sciences, Curtin University, Bentley, Australia 16 8Curtin Institute for Computation, Curtin University, Bentley, Australia

17 * To whom correspondence should be addressed. Tel:(61)-413-509-384, Email: 18 [email protected] 19 Correspondence may also be addressed to Mark Agostino. Tel:(61)-8-9266-9719, Email: 20 [email protected] 21 † The authors wish it to be known that, in their opinion, the first two authors should be regarded as 22 joint First Authors.

23

24 ABSTRACT

25 The activities of DNA-binding transcription factors, such as the multi-zinc finger protein ZBTB18 (also 26 known as RP58, or ZNF238), are essential to coordinate mammalian neurodevelopment, including 27 the birth and radial migration of newborn neurons within the fetal brain. In humans, the majority of 28 disease-associated missense mutations in ZBTB18 lie within the DNA-binding zinc finger domain and 29 are associated with brain developmental disorder, yet the molecular mechanisms explaining their role 30 in disease remain unclear. To address this, we developed in silico models of ZBTB18 bound to DNA, 31 and discovered that half of the missense variants map to residues (Asn461, Arg464, Glu486) 32 predicted to be essential to sequence-specific DNA contact, while others map to residues (Leu434, 33 Tyr447, Arg495) with limited contributions to DNA binding. We studied pathogenic variants to residues 34 with close (N461S) and limited (R495G) DNA contact and found that each bound DNA promiscuously, 35 displayed altered transcriptional regulatory activity in vitro, and influenced the radial migration of 36 newborn neurons in vivo in different ways. Taken together, our results suggest that altered 37 transcriptional regulation could represent an important pathological mechanism for ZBTB18 missense 38 variants in brain developmental disease.

39

1

40 INTRODUCTION

41 The activities of DNA-binding transcription factors are crucial to the formation of the mammalian 42 cerebral cortex, during which, coordinated waves of gene expression orchestrate a step-wise process 43 of neurogenesis, cell migration, and circuit formation within the developing organ (Gupta, Tsai, & 44 Wynshaw-Boris, 2002; Nord, Pattabiraman, Visel, & Rubenstein, 2015). ZBTB18 encodes a 45 transcriptional repressor of the broad complex tramtrack bric-a-brac (BTB) zinc-finger family, 46 comprising an N-terminal BTB domain for protein-protein interaction, as well as four Cys2-His2-like 47 (C2H2) zinc fingers at its C-terminus for DNA binding (Mitchelmore et al., 2002). In mice, Zbtb18 is 48 essential to neurodevelopment (Okado et al., 2009; Xiang et al., 2011), including the radial positioning 49 of newborn postmitotic neurons within the developing cerebral cortex (Heng et al., 2013; Ohtaka- 50 Maruyama et al., 2013). Notably, loss of Zbtb18 results in derepression of downstream target genes, 51 including the cytoskeleton gene Rnd2, which, in turn, leads to defective neuronal migration within the 52 embryonic cerebral cortex (Heng et al., 2013; Ohtaka-Maruyama et al., 2013). We reported that 53 ZBTB18 directly binds enhancer motifs within the Rnd2 gene identified as E1 and E2 (Heng et al., 54 2013), in order to modulate its levels for the control of radial migration by immature neurons. The 55 transcriptional repressor functions for ZBTB18 are essential to its role in the regulation of multiple 56 target genes essential to embryo development (Ohtaka-Maruyama et al., 2013; Xiang et al., 2011). 57 In humans, genetic mutations in ZBTB18 are associated with structural brain abnormalities, 58 neuronal migration disorder and intellectual disability (Cohen et al., 2017; de Munnik et al., 2014; 59 Deciphering Developmental Disorders, 2017; Depienne et al., 2017; Hemming et al., 2016; van der 60 Schoot et al., 2018). More than half of all pathogenic variants identified in ZBTB18 are predicted to be 61 truncating, suggesting that haploinsufficiency/loss-of-function represents a general pathological 62 mechanism for disease (Depienne et al., 2017). However, of the remaining variants that are not 63 predicted to be truncating, it is noteworthy that the majority of these map to the C-terminal zinc finger 64 DNA-binding domain of the polypeptide (Supplementary Figure S1 and Supplementary Table S1). 65 This suggests that missense variants could influence the functions for ZBTB18 in DNA binding and 66 transcriptional regulation which, in turn, leads to brain developmental disease. 67 Here, we have used homology modelling, molecular dynamics (MD) simulations, and 68 molecular mechanics-generalised Born/surface area (MM-GB/SA) calculations to investigate the 69 structural and functional properties of wildtype ZBTB18 and two disease-associated variants – 70 NP_991331.1:p.Arg495Gly (R495G) (Rauch et al., 2012) and NP_991331.1:p.Asn461Ser (N461S) 71 (Farwell et al., 2015; rs797044885) – bound to cognate DNA motifs. Through this approach, we 72 propose key residues within ZBTB18 mediating its contact with DNA, and demonstrate that disease- 73 associated missense variants to contact and non-base-contact residues within ZBTB18 influence its 74 sequence-specific DNA binding, as well as its transcriptional activity in vitro and neuronal functions in 75 vivo. 76

77

2

78 MATERIAL AND METHODS

79 Homology modelling of ZBTB18 zinc finger domain in complex with DNA probe sequences 80 The structures of the wild-type ZBTB18 zinc finger region from residues 373-501 in complex with DNA 81 fragments from the E1 and E2 probe sequences were prepared by homology modelling. These motifs 82 were derived from a previously characterised 3’ regulatory enhancer sequence for Rnd2, a 83 downstream target gene which mediates neuronal migration during brain development (Heng et al., 84 2013). Separate templates for each of the four C2H2 subdomains comprising the ZBTB18 zinc finger 85 domain were identified using BLAST searches of each subdomain against the Protein Data Bank 86 (PDB) using Prime Structure Prediction (Supplementary Table S2). The relevant C2H2 subdomains of 87 the templates were overlaid to the relevant C2H2 subdomains of the designed zinc finger protein Aart 88 (PDB 2I13) (Segal, Crotty, Bhakta, Barbas, & Horton, 2006), which afforded the highest sequence 89 identity to the overall ZBTB18 zinc finger domain. The Composite/Chimera feature of Prime was used 90 to build protein-DNA complexes for the wild-type ZBTB18 zinc finger domain, initially retaining the 91 DNA from PDB 2I13. This DNA was trimmed to a 15 base pair fragment, representing the length of 92 DNA most closely bound by the zinc finger domain. 93 C2H2 zinc finger domain structures in complex with DNA similar in sequence to the E1 94 sequence were then identified; these structures guided the placement of the desired DNA sequences 95 with respect to the protein. The PDB was searched for C2H2 zinc finger domain structures (PFAM 96 PF00096) with DNA bound. Pairwise alignment of the E1 sequence to each DNA sequence from the 97 identified structures was performed using the needleall tool of EMBOSS (Rice, Longden, & Bleasby, 98 2000), this identified the DNA contained in PDB 5KE6 as affording the best alignment (Hashimoto et 99 al., 2017). PDB 5KE6 was then aligned to the initially built DNA-protein complex (i.e., the complex of 100 ZBTB18 with the DNA from PDB 2I13). A sequence alignment between the DNA in PDB 5KE6 and 101 the DNA in PDB 2I13 was generated based on the structural alignment between the two DNA 102 fragments. Combining this structure-based sequence alignment with the sequence alignment of the 103 DNA probe sequences with the template DNA allowed an alignment between the DNA probe 104 sequences and the DNA from PDB 2I13 to be generated (Supplementary Table S3). The DNA from 105 PDB 2I13 was then mutated in UCSF Chimera to the relevant sequences based on this alignment.

106 Following model building and inclusion of the appropriate DNA sequences, the complex was 107 energy minimised using Prime Minimization in a step-wise manner; non-template residues were first 108 minimised, followed by all protein and ion components of the complex (i.e., the entire structure, 109 excluding DNA), and finally, the entire complex. Erroneous cis-amides (i.e., cis-amides not derived 110 from the template or immediately prior to a proline residue) and incorrect stereochemistry generated 111 during the model building process was identified and corrected using Maestro. The structures of the 112 zinc finger domain with each of the E1 and E2 probe sequences were produced in this manner; 113 complexes with mutant proteins were prepared by mutating the relevant residue(s) in these 114 representative structures using the mutation facilities of Maestro and used without further 115 minimisation.

3

116 Molecular dynamics simulations

117 Parameterization of the complexes was performed using AmberTools (Salomon-Ferrer, Case, & 118 Walker, 2013). Simulations were performed using GROMACS 5.0.2 (Abraham et al., 2015). ZBTB18 119 was parameterised with the AMBER ff14SB force field (Maier et al., 2015), while bound DNA was 120 parameterised with the parmbsc0 parameter set (Perez et al., 2007) with ε/ζOL1 (Zgarbova et al., 121 2013) and χOL4 (Krepl et al., 2012) modifications. The Zinc AMBER force field (ZAFF) (Peters et al., 122 2010) was used to parameterise the zinc centres in ZBTB18. The resulting topology was then ported 123 to GROMACS format using acpype (Sousa da Silva & Vranken, 2012) and the remaining system 124 setup completed in GROMACS.

125 Complexes were solvated in TIP3P water (Jorgensen, Chandrasekhar, Madura, Impey, & 126 Klein, 1983) in a dodecahedral box with a minimum of 10 Å distance from the protein to the box edge. 127 The system was charge neutralised with the addition of sodium ions, with further sodium and chloride 128 ions added to a concentration of 0.1 M. Equilibrations of the system in the NVT and NPT ensembles 129 were adapted from previously described procedures (Perez et al., 2007; Shields, Laughton, & Orozco, 130 1997, 1998); briefly, short simulations (0.1 ns) with gradually reducing position restraints on the 131 complex heavy atoms were performed (initially an NVT simulation with 10000 kJ/(mol nm2) applied, 132 followed by NPT simulations with 2000 kJ/(mol nm2) applied, gradually decreasing by 400 kJ/(mol 133 nm2)). Following this, the production MD simulation was performed in the NPT ensemble for 50 ns, 134 with a target temperature of 300K and a target pressure of 1 atm. The modified Berendsen thermostat 135 (V-rescale) was employed for temperature coupling (Bussi, Donadio, & Parrinello, 2007). The 136 Parrinello-Rahman barostat was employed for pressure coupling (Parrinello & Rahman, 1981). The 137 smooth particle mesh Ewald method was employed for long-range electrostatics (Essmann et al., 138 1995). All bonds were constrained using the LINCS algorithm (Hess, Bekker, Berendsen, & Fraaije, 139 1997). Coordinates were saved every 10ps.

140 Five simulations of each complex were performed, starting from new random velocities in 141 each replicate. For binding energy calculations, the final 10 ns of three of these simulations were used, 142 determined by examining the root-mean-squared deviation (RMSD) for heavy atoms over this time 143 range; simulations where the RMSD appeared stable and maintaining under 6.0 Å were selected for 144 binding energy calculations.

145 Binding free energy calculations and decomposition

146 Binding energies were calculated using the (MM-GB/SA) approach, facilitated by the MMPBSA.py tool 147 of AmberTools (Miller et al., 2012). The single-trajectory protocol was utilised (Gohlke, Kiel, & Case, 148 2003), with the following equation:

149 ΔGbind = Gcomplex – Gprotein – Gligand = ΔH + ΔGsolvation – TΔS = ΔGvdw + ΔGele + ΔGGB + ΔGSA – TΔS

4

150 where ΔGvdw is the molecular mechanics van der Waals interaction energy, ΔGele is the molecular

151 mechanics electrostatic interaction energy, ΔGGB is the change in polar desolvation energy upon

152 complex formation and ΔGSA is the change in nonpolar desolvation energy upon complex formation. 153 The entropic term (-TΔS) was not calculated, due to high computational cost and poor accuracy. The 154 modified GB model developed by Onufriev et al. (igb = 5) was used to calculate the polar desolvation 155 energy (Onufriev, Bashford, & Case, 2004); this GB model is suggested as a suitable “general 156 purpose” GB model and has been previously applied to protein-DNA complexes (Ruscio & Onufriev, 157 2006). The non-polar component of the desolvation energy was calculated via solvent accessible 158 surface areas calculated with the LCPO method (Weiser, Shenkin, & Still, 1999). MM-GB/SA energies 159 were also decomposed per-residue (idecomp = 1). The GROMACS tool g_cluster was used to identify 160 the most frequently observed structures from the regions of the simulations submitted for MM-GB/SA 161 analysis. The cutoff for the g_cluster calculation was set to 0.25°nm.

162 Mouse handling and Animal Ethics License.

163 All animal procedures were conducted according to standard operating procedures approved by the 164 Animal Ethics Committees (Perkins Institute: AE021, University of Western Australia: RA/3/100/1361), 165 as well as guidelines provided by the National Health and Medical Research Council of Australia. 166 Unsexed C57BL/6J wild-type mouse embryos were employed for in utero electroporation studies.

167 DNA constructs and cloning

168 A Zbtb18 shRNA vector used in this study has been previously reported (Heng et al., 2013). The 169 cDNA sequence encoding isoform 1 of human ZBTB18 (NM_205768.2) and ZBTB18 (R495G) were 170 commercially synthesised (Integrated DNA Technologies). Both cDNAs were individually sub-cloned 171 into pCIG-FLAG vector in which the GFP cassette was excised by restriction digestion and 172 subsequent cloning (pCIG-F[NG]) (Heng et al., 2013). The N461S variant was generated via site- 173 directed mutagenesis using a GeneArt® Site-Directed Mutagenesis PLUS kit (A14604; Life 174 Technologies, Carlsbad, California, United States) on the pCIG-F[NG] ZBTB18(WT) construct with the 175 following primers: sense, 5’-TCCAGTACTCGCACAGCCTGAGCCGCCATGC-3’; antisense, 5’- 176 GCATGGCGGCTCAGGCTGTGCGAGTACTGGA-3’. All constructs were verified by Sanger 177 sequencing (Australian Genome Research Facility, Perth, Australia).

178 Cell Culture and Transfection

179 Human (HeLa, HEK293T) and mouse (P19 embyrocarcinoma) cell lines were cultured in complete 180 growth media (DMEM (10313021; Gibco by Life Technologies, Carlsbad, California, United States), 2 181 mM Lglutamine (25030081; Gibco by Life Technologies, Carlsbad, California, United States), 10% 182 fetal bovine serum (SFBSF; Bovogen Biological, Keilor East, Victoria, Australia)), in an incubator at

183 37°C supplied with humidified 5% CO2. HeLa cells were seeded in 500 µl of growth media in a 24-well 184 plate, at a density of 50 000 cells per well for localisation experiments. P19 cells were seeded in 500 185 µl of growth media in a 24-well plate, at a density of 25 000 cells per well for luciferase assays. 5

186 HEK293T cells were seeded in 2ml of growth media in a 6-well plate, at a density of 500 000 cells per 187 well for Electrophoretic Mobility Shift Assay (EMSA) experiments and initial western blotting, and in a 188 48-well plate at a density of 25 000 cells per well for experiments with MG132 exposure. 24 hours 189 after seeding, cells were transiently transfected with Lipofectamine 2000 (1166810019; Invitrogen by 190 Life Technologies, Carlsbad, California, United States) as per manufacturer’s instructions. The media 191 was changed 5 hours post-transfection (media without Lipofectamine). For immunostaining 192 experiments, HeLa cells were cultured on uncoated 13 mm round coverslips (CS13100; Grale by 193 Trajan, Ringwood, Victoria, Australia) and fixed onto the coverslips at either 24 or 48 hours post- 194 transfection. P19 cells were collected 48 hours post-transfection for luciferase assays. Nuclear extract 195 from HEK293T cells was collected 48 hours post-transfection for EMSAs. For MG132 experiments 196 HEK293T cells were treated with 10 µM MG132 (C2211, SigmaAldrich, St. Louis, Missouri, United 197 States) at 40 hours post-transfection, and then washed with 1x PBS buffer and lysed using KALB 198 buffer at either 0, 4, or 8, hours post MG132 treatment.

199

200 Luciferase assays

201 Firefly luciferase reporter constructs harbouring a synthetic decamerised binding site (BS10), as well 202 as a construct comprising mouse Rnd2 3’-enhancer sequence (Heng et al., 2008), were used. A 203 Renilla luciferase construct was co-transfected to normalise for transfection efficacy (Promega, 204 Madison, Wisconsin, United States). For competition assays between WT, N461S, and R495G, 250 205 ng of each ZBTB18 construct was added, together with 50 ng of firefly reporter construct, and 25 ng 206 of Renilla construct, per well. DNA was added to 100 µl Opti-MEM I (31985070; Gibco by Life 207 Technologies, Carlsbad, California, United States) per sample. The four constructs were pCIG-F[NG]- 208 empty, pCIG-F[NG]-ZBTB18(WT), pCIG-F[NG]-ZBTB18(N461S), and pCIG-F[NG]-ZBTB18(R495G). 209 48 hours post-transfection, P19 cells were washed with 500 µl 1x PBS and lysed with 150 µl 1x 210 Passive lysis buffer as per manufacturer’s instructions (E1960; Dual Luciferase Assay Kit, Promega, 211 Madison, Wisconsin, United States). The lysate was then centrifuged at 15000 x g for 15 min at 4°C. 212 20 µl of supernatant for each 24-well was added to two 96-wells, and reagent added to each well 213 manually. Firefly and Renilla signals were read in each well of the 96-well plate in order of rows (5 214 second recordings per well), using a VictorLight plate reader (Perkin Elmer, Waltham, Massachusetts, 215 United States) according to manufacturer’s instructions.

216 Western Blotting

217 HEK293T cells were collected 48 hours after transfection and Western blotting. Briefly, cells were 218 washed with 1x PBS buffer and were lysed using KALB buffer (1% Triton X detergent, 150mM NaCl, 219 0.02% sodium azide, 1mM EDTA, 50mM Tris-HCl pH 7.4, and protease inhibitor (4693159001; Roche, 220 Basel, Switzerland)), and protein concentration measured using Bradford reagent (500205; BioRad, 221 Hercules, California, United States). Protein samples were analysed on a 10% SDS gel, transferred to

6

222 nitrocellulose membrane, and incubated overnight with primary antibodies: mouse antiβactin 223 (1:5,000 (A5441; SigmaAldrich, St. Louis, Missouri, United States)), mouse antiFLAG (1:5,000 224 (F1804; SigmaAldrich, St. Louis, Missouri, United States)), mouse antiZBTB18 (1:5,000 (H00010472 225 M04; Abnova, Jhongli, Taiwan)), rabbit antiFLAG (1:5,000 (2368; Cell Signaling, Danvers, 226 Massachusetts, United States)) and rabbit antiZBTB18 (1:5,000 (127141AP; Proteintech, Chicago, 227 Illinois, United States)). Membranes were incubated with anti-rabbit IRDye 680LT or anti-mouse 228 IRDye 800 secondary antibodies (1:10,000 (LI COR Biosciences, Lincoln, Nebraska, United States) 229 for 2 hours before analysis using the Odyssey imaging system (LICORBiosciences, Lincoln, 230 Nebraska, United States).

231 Electrophoretic Mobility Shift Assays (EMSAs)

232 EMSAs were performed as previously described (Cruickshank et al., 2015), using nuclear extracts 233 from HEK293T transfected cells collected using the NE-PER™ Nuclear and Cytoplasmic Extraction 234 Kit according to manufacturer's instructions (78833; Life Technologies Carlsbad, California, United 235 States). In short, 2 µg of crude nuclear protein extract was incubated with 75 fmol of double-stranded 236 oligonucleotides in EMSA reaction buffer (4% Ficoll, 20mM HEPES [pH 7.9], 1mM EDTA 237 [pH8.0],1.5mM DTT, 0.5 µg Poly dI:dC) for 30 minutes, before running on a 6% non-denaturing 238 polyacrylamide gel (6% Bis-acrylamide, 2.5% glycerol, 0.75x TBE buffer) for 90 minutes at 150 V. 239 Commercially sourced, HPLC-purified 5’-biotinylated oligonucleotides (SigmaAldrich, St. Louis, 240 Missouri, United States) comprising sense and anti-sense strands of DNA stretches encompassing 241 the binding sites of E1 (sense 5’-CTCTGCTGTTACTCCTAAATAACAGATGTCTGTCTGCATA-3’; 242 antisense 5’-TATGCAGACAGACATCTGTTATTTAGGAGTAACAGCAGAG-3’); E1mut (sense 5’- 243 CTCTGCTGTTACTCCTAAATAACAGTGATCTGTCTGCATA-3’; antisense 5’- 244 TATGCAGACAGATCACTGTTATTTAGGAGTAACAGCAGAG-3’); E2 (sense 5’- 245 TTTGATCCACCAAAGGGAGGGGCAGATGGGAGTAGGGAAG-3’; antisense 5’- 246 CTTCCCTACTCCCATCTGCCCCTCCCTTTGGTGGATCAAA-3’); E2mut (sense 5’- 247 TTTGATCCACCAAAGGGAGGGGCAGTGAGGAGTAGGGAAG-3’; antisense 5’- 248 CTTCCCTACTCCTCACTGCCCCTCCCTTTGGTGGATCAAA-3’). Strand pairs were annealed by 249 resuspension in 1X TE buffer and heated to 95°C for 10 minutes, then slowly cooled to room 250 temperature. Oligonucleotides were run on a 6% polyacrylamide gel and double-stranded 251 oligonucleotides were extracted from the gel in elution buffer (0.1% sodium dodecyl sulfate (SDS), 0.5 252 M ammonium acetate, 10 mM magnesium acetate) at room temperature for 16 hours. 253 Oligonucleotides were precipitated with 100% ethanol at -80°C for two hours, then pelleted by 254 centrifugation at 16 000g for 30 min at 4°C and washed with 800 µl of 70% ethanol and air dried 255 thoroughly. Oligonucleotides were resuspended in nuclease-free water and diluted to 25 pmol/µL 256 stock just before use. Biotin detection was undertaken with a Chemiluminescent Nucleic Acid 257 Detection Module Kit according to the manufacturer’s instructions (89880; Thermo Fisher, Waltham, 258 Massachusetts, United States).

7

259 In utero electroporation and tissue collection for immunostaining

260 In utero electroporation was performed on E14.5 embryos as described previously (Haas et al., 2016). 261 Plasmids were delivered at a final concentration of each species at 1 µg/µl, pre-mixed with Fast 262 Green to visualise site of injection. Three days after electroporation, the pregnant dam was 263 euthanatised by cervical dislocation, embryos (E17.5) were decapitated, and their brains were 264 dissected then fixed overnight at 4°C in 4% paraformaldehyde in PBS. Then, the brains were 265 equilibrated in 30% sucrose in PBS for 3 days at 4°C, before embedding in OCT mounting medium, 266 frozen on dry ice and finally stored at -80°C. Coronal sections 16 µm thick were prepared from each 267 brain using a cryostat (Leica, Biosystems), and recovered on Superfrost-PLUS glass slides (Menzel– 268 Glaser by Trajan, Ringwood, Victoria, Australia). Slides were dried for at least 60 minutes at ambient 269 temperature conditions before storage at -80°C until immunostaining was performed, as follows. 270 Briefly, after a blocking step with 10% of normal goat serum in PBS, brain sections were incubated 271 overnight at 4°C with the primary antibodies: chicken antiGFP (1:700 (Ab13970; Abcam, Cambridge, 272 United Kingdom)), mouse antiFLAG (1:500(F1804; SigmaAldrich, St. Louis, Missouri, United 273 States)). Then sections were incubated with fluorescent secondary antibodies (488Goat antiChicken 274 (1:1,000 (A11039; Invitrogen by Life Technologies, Carlsbad, California, United States)); 568Goat 275 antimouse (1:1,000 (A11031; Invitrogen by Life Technologies, Carlsbad, California, United States))) at 276 room temperature for 2 hr before sections were counterstained with 4′6Diamidino 2Phenylindole 277 (DAPI, 1:10000 (D1306; Invitrogen by Life Technologies, Carlsbad, California, United States)). A 278 coverslip was mounted on each slide using Fluorescent Mounting Medium (DAKO, Berlin, Germany).

279 Microscopy, imaging, quantification studies

280 Images of brain sections were captured on an epifluorescence microscope (Olympus, Shinjuku, 281 Tokyo, Japan) equipped with a CCD camera (SPOT, Sterling Heights, Michigan, United States) for 282 neuronal migration, and on a confocal microscope (Nikon, Minato, Tokyo, Japan) for the analysis of 283 neuronal shape. Subdivisions of the embryonic cortex (VZ/SVZ, IZ and CP) were identified based on 284 cell density as visualised with DAPI (4′6-Diamidino-2-Phenylindole) staining, as described previously 285 (Haas et al., 2016). Cell counting was performed blind to the condition on representative fields of 286 sections of electroporated brains using ImageJ software.

287 Statistics

288 Data from all experiments are expressed as the mean ± standard error of the mean (SEM). A Kruskal 289 Wallis analysis was employed to test multiple conditions, while a Mann-Whitney U test was applied for 290 statistical analyses between two treatments. All statistics were performed through Prism software 291 (GraphPad).

292

293 RESULTS

8

294 Structural prediction and binding energy calculations of ZBTB18 zinc finger domain for 295 wildtype and missense variants, in complex with DNA probe sequences

296 We used homology modelling, molecular dynamics (MD) simulations, and molecular 297 mechanics-generalised Born/surface area (MM-GB/SA) calculations to investigate the structural and 298 functional properties of the C-terminal zinc finger domain of ZBTB18 (residues 373-501) bound to two 299 DNA regulatory motifs, E1 and E2, within the Rnd2 gene (Heng et al., 2008; Heng et al., 2013). To 300 explore sequence-specific binding properties, we also modelled ZBTB18 binding to mutated versions 301 of each motif, named E1mut and E2mut, which we also previously reported as refractory to ZBTB18- 302 mediated transcriptional repression (Heng et al., 2013). The predicted structure of the ZBTB18 zinc 303 finger domain comprises four C2H2-type subdomains, featuring a moderately sized insertion between 304 the first two C2H2 subdomains relative to the template structures (Figure 1, Supplementary Video S1 305 and Supplementary Figures S2A-C). MD simulations indicated stability of the majority of protein-DNA 306 complexes, which were typically reproducible when simulations were commenced from new random 307 velocities (Supplementary Figure S3A,D,G,J). 308 Based on our MM-GB/SA calculations, we found that wildtype ZBTB18 formed energetically 309 more favourable interactions with both E1 and E2, compared to the mutated sequences E1mut and 310 E2mut (Table 1). Per-residue binding energy decomposition identified residues – including Tyr458, 311 Asn461 and His493 – as critical to wildtype ZBTB18 to bind E1 and E2 (Figure 2, Table 2 and 312 Supplementary Table S4). For example, Asn461 contributes as much as 7.2% and 7.6% of the total 313 binding energy of ZBTB18 to E1 and E2 respectively. Importantly, binding energy contributions made 314 by these residues were markedly lower for ZBTB18 modelled with E1mut and E2mut mutants, with the 315 exception of His493, which displayed an increased contribution when complexed with E2mut, 316 altogether suggesting roles for each residue in sequence-specific binding (Figure 2B). Based on our 317 models of ZBTB18 with E1 and E2, we also observed that half of the disease-associated missense 318 variants map to residues (Asn461, Arg464, Glu486) essential to sequence-specific DNA contact, 319 while others map to residues exhibiting comparably reduced (Tyr447, Arg495) or negligible (Leu434) 320 contributions to DNA-binding (Table 2). 321 Next, we focussed our attention on disease-associated ZBTB18 variants which featured 322 residues with close (N461S) or limited (R495G) DNA contact. Separate models were generated for 323 N461S (Figure 3A-B, and Supplementary Figure S2D-F) and R495G (Figure 3C-D, and 324 Supplementary Figure S2G-I) binding E1, E1mut, E2 and E2mut. Binding energy calculations suggested 325 that N461S exhibited more selective binding for E1 over E1mut, and preferential binding for E2mut over 326 E2 (Table 1). On the other hand, R495G exhibited similar binding energies for each of the E1, E1mut 327 and E2 motifs, suggesting non-selective binding for all three sequences. Calculations with E2mut 328 indicated that R495G bound with substantially lower energy compared to E1, E1mut and E2 motifs. 329 We performed binding energy decomposition to understand the interactions taking place in 330 the complexes in finer detail. In the case of a N461S substitution, we observed a reduced contribution 331 to the binding energy of this position between E1 and E2, concomitant with a structural rearrangement 332 of Tyr458 and His493, which augmented their binding with E1mut and E2mut, as well as enhanced the 9

333 contribution to the binding energy of His493 with E2 (Figure 3B and Table 2). In contrast, Arg495 334 weakly contributes to binding of E1 and E2, and substitution to glycine eliminates any contribution to 335 binding energy made by this position (Figure 3D and Table 2). However, with this substitution, we also 336 observed reduced contributions made by Tyr458 and Asn461 when binding to the E1 and E2 motifs, 337 as well as an increase in contribution to E2 binding by His493, altogether suggesting indirect effects 338 of this missense variant on DNA binding. When modelled with E1mut, Tyr458 also shows a five-fold 339 elevation in binding energy contribution in the R495G variant (-3.5 kcal/mol for this residue within 340 R495G complexed with E1mut, versus -0.7 kcal/mol within WT ZBTB18 complexed with E1mut; see 341 Table 2). Therefore, our models suggest that missense variants to residues relevant to close (N461S) 342 or limited (R495G) DNA contact differentially affect the capacity for ZBTB18 to bind DNA, and could 343 lead to spurious binding. 344 345 Missense variants in ZBTB18 disrupt sequence-specific DNA-binding and transcriptional 346 repression

347 To substantiate our molecular predictions, we performed electrophoretic mobility shift assays (EMSAs) 348 with DNA probes comprising each motif. We found that WT ZBTB18 formed distinct molecular 349 complexes with a DNA probe comprising E1 (Figure 4A, arrows), with an additional complex formed 350 by R495G (Figure 4A, arrowhead). In agreement with our modelling results, these complexes were 351 either detected at very low intensity or not detected at all in assays with WT ZBTB18 and E1mut 352 (Figure 4B). In contrast to WT ZBTB18, however, each missense variant formed distinct complexes 353 with the E1mut motif (Figure 4B, arrow and closed arrowhead), with R495G forming a prominent high- 354 molecular weight complex (Figure 4B, open arrowhead). In the case of the E2 motif, ZBTB18 and its 355 missense variants formed molecular complexes (Figure 4C, arrows), with additional complexes 356 formed by R495G (Figure 4C, open and closed arrowheads). However, such protein-DNA complexes 357 were not identified in parallel experiments using a probe comprising the E2mut motif (Figure 4D). 358 Together, these modelling and in vitro data indicate that ZBTB18 binds its DNA motifs E1 and E2 in a 359 sequence-specific manner, while its missense variants bind E1, E2, as well as E1mut, but not E2mut. 360 To understand the impact of each missense variant ZBTB18 on transcriptional regulation in 361 vitro, we performed luciferase reporter assays using a previously validated synthetic promoter 362 construct (termed BS10) (Aoki et al., 1998), as well as a construct harbouring the Rnd2 3’ enhancer 363 (comprising E1 and E2 motifs) (Heng et al., 2013). We found that wildtype ZBTB18 suppressed 364 luciferase reporter activity, while N461S failed to suppress reporter activity mediated by the BS10 365 synthetic enhancer, but stimulated luciferase signal mediated by the Rnd2 3’ enhancer (Figure 4E-F). 366 Unexpectedly, we found that R495G significantly enhanced luciferase signal in both reporter 367 constructs. The capacity for N461S and R495G to stimulate reporter activity was extinguished when 368 equal quantities of the WT construct were co-transfected (Supplementary Figure S4). Next, we 369 explored transcriptional reporter activity of ZBTB18 and each missense ZBTB18 variant using reporter 370 constructs bearing either E1mut, E2mut, or both E1mut and E2mut motifs within the Rnd2 3’ enhancer 371 sequence. For wildtype ZBTB18, transcriptional repression was only abolished in experiments with a 10

372 reporter bearing both E1mut and E2mut motifs, confirming its sequence-specific binding to native E1 373 and E2 motifs (Figure 4G). In contrast, statistically significant transcriptional activation by N461S was 374 observed in constructs bearing E1+E2 or E1mut+E2mut motifs, but not an E1mut+E2 motif or an 375 E1+E2mut motif. When compared to WT ZBTB18, the N461S variant has reduced capacity for 376 repression in the context of E1+E2, E1mut+E2 and E1+E2mut motif pairs, and activation in context of 377 the E1mut+E2mut motif pair (Figure 4H and summarised in Supplementary Figure S5B). These data 378 suggest that N461S variant has reduced repressor activity compared to WT, and may influence 379 transcriptional activation in some contexts. Conversely, R495G robustly stimulated reporter activity in 380 all four reporter constructs, suggesting this variant has strong capacity for activation in all contexts 381 (Figure 4I and Supplementary Figure S5C). 382 Next, we performed transfection experiments utilising FLAG-tagged ZBTB18 constructs to 383 explore the impact of each missense variant on subcellular localisation and expression. Twenty four 384 hours after transfection we observed reduced nuclear localisation of N461S compared to wildtype 385 ZBTB18, concomitant with a corresponding increase in pancellular signal distribution (Supplementary 386 Figure S6A-C). The proportion of cells with a punctate distribution of R495G within the nucleus was 387 also significantly increased compared to wildtype ZBTB18 (Supplementary Figure S6D). These 388 effects were transient and they were not observed 48 hours post-transfection (Supplementary Figure 389 S6E-G). We also routinely observed significantly fewer FLAG-N461S immunopositive cells compared 390 with wildtype and R495G-transfected cells suggesting that N461S might be unstable (Supplementary 391 Figure S7A-B). Furthermore, low steady-state levels of FLAG-N461S protein from lysates of 392 transiently transfected cells were observed (Supplementary Figure S7C-D). To further investigate the 393 hypothesis that the N461S variant might be unstable, we examined FLAG-tagged ZBTB18 expression 394 in transfected cells treated with the proteasome inhibitor MG132 for up to 8 hours followed by 395 Western blotting analysis (Supplementary Figure S7E-F). In this experiment, we reproducibly 396 observed a very low immunoblotted FLAG signal in the lysate of N461S transfected cells compared to 397 WT and R495G conditions at 0 hours of MG132 treatment (Supplementary Figure S7E). However, 398 after 4 hours of MG132 treatment the FLAG signal was significantly increased for N461S, compared 399 to no MG132 treatment. Following 8 hours MG132 exposure, N461S, WT and R495G variant levels 400 were significantly increased (Supplementary Figure S7F). These results indicate that the N461S 401 variant exhibits an exaggerated protein instability compared to WT, and is prone to degradation in 402 cells. 403 404 Disease-associated missense variants to ZBTB18 disrupt radial migration within the 405 embryonic cerebral cortex.

406 The transcriptional regulatory activities of ZBTB18 are essential to mammalian neurodevelopment, 407 and suppression of Zbtb18 impairs radial migration of cortical projection neurons from their birthplace 408 within the germinal ventricular zone (VZ), including their multipolar-to-bipolar transition within the 409 intermediate zone (IZ) as they reach the cortical plate (CP) of the embryonic cerebral cortex (Heng et 410 al., 2013; Ohtaka-Maruyama et al., 2013). Hence, we assessed the capacity for ZBTB18 and its 11

411 missense variants to influence the radial migration of E14.5-born cortical neurons within the E17.5 412 cortex through a series of in utero electroporation experiments. Treatment with Zbtb18 shRNA 413 disrupted the capacity for cells to migrate into the CP; however, this impairment is corrected by co- 414 delivery of wildtype ZBTB18 (Figure 5A-G), consistent with previous findings (Heng et al., 2013; 415 Ohtaka-Maruyama et al., 2013). Strikingly, we found that co-delivery of N461S significantly 416 augmented the radial migration of Zbtb18 shRNA-treated cells into the CP and markedly potentiated 417 their intracortical positioning within the upper CP (Figure 5F-G). We could not detect N461S 418 immunofluorescence in rescued cells (Supplementary Figure S8), consistent with the notion that this 419 protein variant is unstable (see Supplementary Figure S7), and beyond the limit of immunodetection 420 in electroporated cortical cells. In contrast, co-delivery of R495G exacerbated their migration defect 421 (Figure 5F-G). We also performed electroporation experiments on E14.5 cortices collected two days 422 later (ie at E16.5) to observe that the proportion of cells within the CP was not significantly different 423 across all conditions (Supplementary Figure S9A-B). Therefore, our data suggests that the differential 424 effects on radial migration by N461S and R495G variants are relevant to the transition from multipolar 425 to bipolar migration as cells migrate within the IZ and CP. 426 Next, we analysed the cell shapes of IZ and CP neurons across each treatment group and 427 found that knockdown of Zbtb18 led to a significant reduction in the proportion of uni/bipolar-shaped 428 cells in the CP and a concomitant increase in the proportion of multipolar shaped cells, as well as 429 significantly shortened, leading neurites (Figure 5H-O). Co-delivery of WT ZBTB18 resulted in 430 significant restorative effects on cell shape and leading neurite length (Figure 5M-O). In contrast, co- 431 delivery of N461S did not affect IZ cell shape but instead led to an increase in multipolar-shaped cells 432 within the CP with shorter leading neurites (Figure 5O), suggestive of dysregulated locomotion which 433 results in their enhanced localisation within the upper CP. On the other hand, co-delivery of R495G 434 led to an increase in multipolar-shaped cells in the IZ, which we interpret as a disruption of multipolar- 435 to-bipolar transition within the cortical subcompartment. Cell shape profiles and leading neurite 436 lengths of the few cells arriving within the CP were significantly different to wildtype ZBTB18 rescue, 437 consistent with a detrimental impact of R495G expression in CP cells. Taken together, missense 438 variants of ZBTB18 impair radial migration in a cell autonomous fashion, and disrupt neurite 439 outgrowth in immature cortical neurons.

440

441 DISCUSSION

442 In this study, we have applied molecular modelling approaches to investigate the binding of 443 ZBTB18 to two native DNA motifs (E1 and E2) within the 3’ enhancer region for the migration- 444 promoting gene, Rnd2; as well as their mutagenised counterparts (E1mut and E2mut). We demonstrate 445 that MM-GB/SA analysis is effective to model ZBTB18 wildtype and variant complexes with different 446 DNA sequences. This is remarkable considering the difficulties associated with the development of 447 accurate DNA force fields for simulations, and the assumptions required for use of the MM-GB/SA 448 approach (Genheden & Ryde, 2015). A strength to our approach has been to incorporate information 12

449 from multiple templates, as well as using a sequence homology-based approach to identifying 450 relevant DNA structural templates, during the initial homology modelling. As a result, we have 451 developed stable DNA-protein complexes and calculated binding energies for ZBTB18 and its 452 variants, with observations substantiated by biochemical observations in this study. 453 A recent study by van der Shoot and colleagues identified via exome sequencing ZBTB18 454 variants at Arg464 in patients with neurological abnormalities, and used homology modelling against a 455 single template structure and a model DNA fragment to illustrate its potential for direct involvement in 456 DNA binding (van der Schoot et al., 2018). While our study also identified Arg464 as a major 457 contributor to DNA binding by ZBTB18, our rigorous modelling approach utilising multiple templates to 458 develop a model bound to cognate DNA motifs can provide fine detail into the energetic contributions 459 to binding made by each residue, including Asn461 and His493. Crucially, we applied this modelling 460 approach also to enable us to investigate the impact of ZBTB18 missense variants on DNA binding. 461 Indeed, we found that substitution of Asn461 to serine alters the DNA-binding specificity of ZBTB18, 462 and disrupts the capacity for this transcription factor to mediate transcriptional repression and radial 463 migration. Furthermore, our models of ZBTB18 with mutated DNA motifs indicated that residues with 464 very limited DNA contact, such as Arg495, might be relevant to binding of spurious DNA motifs. 465 Indeed, we find that the missense variant R495G dramatically altered the DNA-binding specificity, 466 transcriptional repressor activity and neurobiological functions of ZBTB18. It has recently been 467 established that non-base-contacting residues are crucial to the evolution of C2H2 zinc finger DNA 468 binding (Najafabadi et al., 2017) and our results suggest that molecular modelling approaches are 469 capable of informing the structural and functional consequences of substitution variants and non- 470 base-contacting residues. 471 Our biochemical characterisation of N461S and R495G variants demonstrates that each 472 variant displays distinct effects on its subcellular localisation, as well as its capacity to mediate 473 transcriptional repression in a luciferase reporter assay. The altered capacity for N461S to traffic into 474 the nucleus could arise as a consequence of disruptions to its association with nuclear import proteins, 475 thereby influencing its availability within the nucleus to mediate transcription. Also, we reported low 476 steady-state levels of N461S observed in transfected cells and electroporated neurons, demonstrating 477 that the N461S variant in unstable both in vitro and in vivo. This suggests that the N461S variant has 478 a pathophysiological role with very low expression levels in cells. In the case of the R495G variant, we 479 find that the presence of this variant influences the sub-nuclear localisation of ZBTB18, leading to its 480 accumulation in nuclear puncta, suggestive of protein aggregation. Over time, we find that the 481 distribution patterns of N461S and R495G are not significantly different to wildtype, hence we 482 conclude that each missense variant likely causes a transient delay in the appropriate localisation of 483 ZBTB18 within cells, leading to asynchronisation of its nuclear functions, ultimately disrupting its 484 capacity to orchestrate neurodevelopment. 485 In addition to transient disruptions in subcellular localisation, we find that the transcriptional 486 repressor functions for each missense variant are disrupted in different ways. For example, we find 487 that the N461S variant has lost the capacity for suppressing transcription via the BS10 and Rnd2 3’

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488 enhancer constructs in our luciferase assays, and can potentiate transcriptional activation in certain 489 contexts. We speculate that the impaired capacity for ZBTB18-mediated repression, together with low 490 steady-state levels, explains the pathological impact of the N461S variant, leading to an imbalance in 491 the regulation of downstream target genes. In contrast, we find that the R495G variant appears to 492 have acquired strong transcriptional activation potential in vitro, with attendant consequences on gene 493 regulation in cells. Given that ZBTB18 mediates its transcriptional regulatory functions in part through 494 its recruitment of co-repressors such as Dnmt3a (Fuks et al., 2001), we further speculate that N461S 495 and R495G are altered in their capacity to bind protein partners for transcriptional regulation, and may 496 recruit protein complexes that potentiate gene expression. Our capacity to visualise such changes in 497 protein-DNA complexes in our EMSA experiments was confounded by the presence of endogenous 498 ZBTB18 binding to the E1 motif in the lysates of mock-transfected cells. Also, our experiments with 499 E2mut motifs did not yield specific complexes for ZBTB18 and its missense variants. Nevertheless, we 500 did observe unique protein-DNA complexes formed by N461S on the E1mut motif, as well as by 501 R495G on the E1, E1mut and E2 motifs. Thus, we surmise that luciferase reporter activity mediated by 502 N461S can be explained by its preferential binding to E1, E1mut and E2 motifs, while R495G recruits 503 additional factors to potentiate reporter activity upon binding to these motifs (Supplementary Figure 504 S5). As such, the “loss-of-repression” variant N461S and the “gain-of-transactivation” variant R495G 505 are postulated to influence gene expression during neurodevelopment, likely through dysregulation of 506 downstream genes which influence the radial positioning and differentiation of neurons. 507 Through a series of functional assays, we found that the presence of N461S and R495G 508 affects the development and positioning of newborn neurons within the embryonic cerebral cortex in 509 different ways. Notably, the impact on transcriptional regulation during radial migration is likely to be 510 two-fold. Firstly, the presence of each missense variant could lead to spurious binding of DNA 511 regulatory sequences, resulting in changes in gene expression levels beyond ZBTB18 targets. 512 Secondly, the presence of each missense variant has a unique effect on radially migrating neurons 513 within the embryonic cerebral cortex. Thus, our results support a scenario in which the “loss-of- 514 repression” variant, N461S, has a different impact on radial migration, in contrast to the “gain-of- 515 transactivation” variant, R495G (see Supplementary Figure S10 for an illustrative summary). In such a 516 model, a loss-of-repression owing to the presence of the N461S variant influences gene expression 517 regulation within radially migrating neurons, leading to their exuberant migration and preferential 518 insertion within the Cortical Plate as multipolar-shaped cells, suggestive of disrupted radialglial-guided 519 locomotion. In contrast, the R495G variant potentiates gene expression so as to severely impair their 520 radial migration, culminating in a failure in multipolar-to-bipolar transition of cortical cells, and their 521 accumulation within the IZ. Indeed, we previously reported that too much or too little Rnd2 disrupts 522 radial migration, such that its overexpression in cortical neurons impairs the capacity for cells to adopt 523 appropriate morphologies to undergo movement (Heng et al., 2008; Pacary et al., 2011). Consistent 524 with this interpretation, our analysis of IZ and CP neurons in the rescue assays revealed that the 525 presence of the R495G variant augmented the multipolar phenotype of Zbtb18 shRNA-treated 526 neurons, thereby suggesting an additive effect on cell shape which could underlie its negative impact

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527 on radial migration. In contrast, treatment with N461S did not have such an effect in Zbtb18 shRNA- 528 treated cells in the IZ or the CP. In addition, Zbtb18 shRNA-treated CP neurons co-electroporated 529 with R495G extended longer leading neurites while N461S treatment did not have such an effect, 530 further suggesting different cellular mechanisms that underlie the aberrant migration of treated 531 neurons in each respective condition. Based on our results, we surmise that the presence of each 532 substitution variant has a significant effect on the development of neurons within the mammalian brain. 533 Future studies will address how each substitution variant disrupts the long-term positioning, dendritic 534 branching and synaptic connectivity of cortical neurons within the postnatal cortex. 535 It has been reported that more than half of all pathogenic variants identified in ZBTB18 are 536 predicted to be truncating, suggesting that haploinsufficiency/loss-of-function represents a general 537 pathomechanism (Depienne et al., 2017). Yet, the majority of disease-associated missense variants 538 to ZBTB18 lie within the C-terminal zinc finger DNA-binding domain. Therefore, our investigation of 539 the N461S and R495G variants has been crucial to demonstrate that altered transcriptional regulation 540 also represents an important pathological mechanism for ZBTB18 variants in brain developmental 541 disease. Altogether, our in silico models and functional studies demonstrate that missense variants to 542 base-contact and non-base-contact residues influence DNA binding, transcriptional repression and 543 neurodevelopmental signalling. 544 The differences in functions for each pathogenic missense variant draws parallels with the 545 differences in phenotypic presentation of the two human subjects diagnosed with ZBTB18 variants 546 (Cohen et al., 2017; Rauch et al., 2012). Particularly, while intellectual disability was diagnosed in 547 both subjects, the subject harbouring an N461S variant was also diagnosed with microcephaly and 548 hypoplasia of the corpus callosum (Cohen et al., 2017). In contrast, the subject diagnosed with the 549 R495G variant was diagnosed with macrocephaly. We speculate that the presence of missense 550 variants to the zinc finger domain of ZBTB18 influences its capacity to bind DNA in a sequence- 551 specific fashion, affect its transcriptional regulatory functions, and negatively impact on neural 552 development to cause a spectrum of brain disorders in children. Functional studies of common 553 missense variants that lie within the DNA-binding domain of ZBTB18 will also be important to clarify 554 their potential impact on transcriptional regulation in human health and disease. 555

556 DATA AVAILABILITY

557 The datasets analysed during the current study are available from the corresponding author on 558 reasonable request.

559

560 SUPPLEMENTARY DATA

561 Supplementary Data are available at NAR online.

562

15

563 ACKNOWLEDGEMENTS

564 I.A.H. and O.C. performed biochemistry and molecular biology studies with J.I-T.H., H.L.N., H.B.S., 565 D.U. and K.D.G.P.. H.D.C., I.E.G-N. performed in utero electroporation experiments with L.N. and 566 O.C.. M.A. performed structural modelling calculations in consultation with J.I-T.H., I.A.H. and O.C.. 567 J.I-T.H., I.A.H., O.C. and M.A. wrote the manuscript and prepared figures, with all authors providing 568 comment.

569

570 FUNDING

571 Curtin Research Fellowship [CRF130006 to M.A.]; Raine Priming Grant from the Raine Medical 572 Research Foundation [M.A.]; National Health and Medical Research Council of Australia [1011505 to 573 J.I.-T.H.]; Telethon-Perth Children’s Hospital Research Fund [J.I.-T.H.]; NHMRC RD Wright 574 Biomedical Research Fellowship [1085842 to K.D.G.P.]; Work was supported by computational 575 resources provided by the Australian Government through the Pawsey Supercomputing Centre under 576 the National Computational Merit Allocation Scheme [pa6/pawsey0196].

577

578 CONFLICT OF INTEREST

579 None declared.

580

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673 in the developing cortex. Dev Biol, 331(2), 140-151. doi:S0012-1606(09)00277-2 [pii] 674 10.1016/j.ydbio.2009.04.030 675 Onufriev, A., Bashford, D., & Case, D. A. (2004). Exploring protein native states and large-scale 676 conformational changes with a modified generalized born model. Proteins-Structure Function 677 and Bioinformatics, 55(2), 383-394. doi:10.1002/prot.20033 678 Pacary, E., Heng, J., Azzarelli, R., Riou, P., Castro, D., Lebel-Potter, M., . . . Guillemot, F. (2011). 679 Proneural transcription factors regulate different steps of cortical neuron migration through 680 Rnd-mediated inhibition of RhoA signaling. Neuron, 69(6), 1069-1084. doi:S0896- 681 6273(11)00116-4 [pii] 10.1016/j.neuron.2011.02.018 682 Parrinello, M., & Rahman, A. (1981). Polymorphic Transitions in Single-Crystals - a New Molecular- 683 Dynamics Method. Journal of Applied Physics, 52(12), 7182-7190. doi:Doi 10.1063/1.328693 684 Perez, A., Marchan, I., Svozil, D., Sponer, J., Cheatham, T. E., 3rd, Laughton, C. A., & Orozco, M. 685 (2007). Refinement of the AMBER force field for nucleic acids: improving the description of 686 alpha/gamma conformers. Biophys J, 92(11), 3817-3829. doi:10.1529/biophysj.106.097782 687 Peters, M. B., Yang, Y., Wang, B., Fusti-Molnar, L., Weaver, M. N., & Merz, K. M., Jr. (2010). 688 Structural Survey of Zinc Containing Proteins and the Development of the Zinc AMBER Force 689 Field (ZAFF). J Chem Theory Comput, 6(9), 2935-2947. doi:10.1021/ct1002626 690 Rauch, A., Wieczorek, D., Graf, E., Wieland, T., Endele, S., Schwarzmayr, T., . . . Strom, T. M. (2012). 691 Range of genetic mutations associated with severe non-syndromic sporadic intellectual 692 disability: an exome sequencing study. Lancet, 380(9854), 1674-1682. doi:10.1016/S0140- 693 6736(12)61480-9 694 Rice, P., Longden, I., & Bleasby, A. (2000). EMBOSS: the European Molecular Biology Open 695 Software Suite. Trends Genet, 16(6), 276-277. 696 Ruscio, J. Z., & Onufriev, A. (2006). A computational study of nucleosomal DNA flexibility. Biophysical 697 Journal, 91(11), 4121-4132. doi:10.1529/biophysj.106.082099 698 Salomon-Ferrer, R., Case, D. A., & Walker, R. C. (2013). An overview of the Amber biomolecular 699 simulation package. Wiley Interdisciplinary Reviews-Computational Molecular Science, 3(2), 700 198-210. doi:10.1002/wcms.1121 701 Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., 3rd, & Horton, N. C. (2006). Structure of Aart, 702 a designed six-finger zinc finger peptide, bound to DNA. J Mol Biol, 363(2), 405-421. 703 doi:10.1016/j.jmb.2006.08.016 704 Shields, G. C., Laughton, C. A., & Orozco, M. (1997). Molecular dynamics simulations of the d(T·A·T) 705 triple helix. J. Am. Chem. Soc., 119, 7463-7469. 706 Shields, G. C., Laughton, C. A., & Orozco, M. (1998). Molecular dynamics simulation of a 707 PNA.DNA.PNA triple helix in aqueous solution. J. Am. Chem. Soc., 120, 5895-5904. 708 Sousa da Silva, A. W., & Vranken, W. F. (2012). ACPYPE - AnteChamber PYthon Parser interfacE. 709 BMC Res Notes, 5, 367. doi:10.1186/1756-0500-5-367 710 van der Schoot, V., de Munnik, S., Venselaar, H., Elting, M., Mancini, G. M. S., Ravenswaaij-Arts, C. 711 M. A., . . . Stevens, S. J. C. (2018). Toward clinical and molecular understanding of

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712 pathogenic variants in the ZBTB18 gene. Mol Genet Genomic Med, 6(3), 393-400. 713 doi:10.1002/mgg3.387 714 Weiser, J., Shenkin, P. S., & Still, W. C. (1999). Approximate atomic surfaces from linear 715 combinations of pairwise overlaps (LCPO). Journal of Computational Chemistry, 20(2), 217- 716 230. doi:Doi 10.1002/(Sici)1096-987x(19990130)20:2<217::Aid-Jcc4>3.0.Co;2-A 717 Xiang, C., Baubet, V., Pal, S., Holderbaum, L., Tatard, V., Jiang, P., . . . Dahmane, N. (2011). 718 RP58/ZNF238 directly modulates proneurogenic gene levels and is required for neuronal 719 differentiation and brain expansion. Cell Death Differ. doi:cdd2011144 [pii] 720 10.1038/cdd.2011.144 721 Zgarbova, M., Luque, F. J., Sponer, J., Cheatham, T. E., 3rd, Otyepka, M., & Jurecka, P. (2013). 722 Toward Improved Description of DNA Backbone: Revisiting Epsilon and Zeta Torsion Force 723 Field Parameters. J Chem Theory Comput, 9(5), 2339-2354. doi:10.1021/ct400154j 724 725

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726 FIGURES LEGENDS 727 Figure 1. Homology model for ZBTB18 in complex with a 15-residue duplex derived from E1 728 sequence (TAACAGATGTCTGTC), shown in “front” (C2H2 domains 1 and 4 facing viewer) and “rear” 729 (C2H2 domains 2 and 3 facing viewer) views. Zinc Fingers 1-4 (coloured as N- to C-terminal rainbow, 730 blue to red), as well as zinc atoms (grey spheres) and DNA residues (dA – white; dC – pink; dG – 731 pale green; dT – tan) are shown. Residues of ZBTB18 impacted by disease-associated missense 732 variants (Leu434, Tyr447, Asn461, Asn464, Glu486 and Arg495) are shown in the lateral panels, with 733 hydrogen bonds in dashed green lines. Arg464, Gln486 and Asn461 are rotated slightly around the x- 734 axis relative to the full complex views. See Supplementary Figure S1B and Table S1 for degree of 735 amino acid sequence conservation and binding energy decomposition for these six residues, 736 respectively.

737

738 Figure 2. Structural analysis of ZBTB18-DNA complexes. (A) Model of ZBTB18 zinc fingers 1-4 739 bound to the E1 sequence, coloured rainbow (blue to red) from N- to C-terminal, with zinc atoms as 740 grey spheres, and DNA residues coloured as follows: dA – white; dC – pink; dG – pale green; dT – 741 tan. (B) Major interactions of Tyr458, Asn461, and His493 of ZBTB18 with E1, E1mut, E2 and E2mut 742 sequences. Hydrogen bonds (dashed green lines), coordination bonds (dashed purple lines), and 743 residues involved in hydrophobic contacts in at least one panel (transparent spheres) highlighted. 744 Views rotated slightly relative to panel (A) to improve visibility of contacts.

745

746 Figure 3. Structural analysis of DNA complexes with ZBTB18 missense variants. (A) Model of 747 ZBTB18 N461S missense variant zinc fingers 1-4 bound to the E1 sequence. (B) Major interactions of 748 Tyr458, Ser461, and His493 of the N461S variant with E1, E1mut, E2 and E2mut sequences. Hydrogen 749 bonds (dashed green lines), coordination bonds (dashed purple lines), and residues involved in 750 hydrophobic contacts in at least one panel (transparent spheres) highlighted. Views rotated slightly 751 relative to panel (A) to improve visibility of contacts. In all panels, the protein is coloured rainbow (blue 752 to red) from N- to C-terminal, with zinc atoms as grey spheres, and DNA residues coloured as follows: 753 dA – white; dC – pink; dG – pale green; dT – tan. (C) Model of ZBTB18 R495G missense variant zinc 754 fingers 1-4 bound to the E1 sequence. (D) Major interactions of Tyr458, Asn461, and His493 of 755 N461S variant with E1, E1mut, E2 and E2mut sequences. Hydrogen bonds (dashed green lines), 756 coordination bonds (dashed purple lines), and residues involved in hydrophobic contacts in at least 757 one panel (transparent spheres) highlighted. Views rotated slightly relative to panel (C) to improve 758 visibility of contacts.

759

760 Figure 4. Functional analysis of ZBTB18 and its disease-associated variants during DNA-binding and 761 transcriptional regulation, in vitro. (A-D) EMSAs conducted with biotinylated DNA probes in the 21

762 presence of WT, N461S and R495G. Control represents experiments with lysate harvested from 763 mock-transfected cells. Square brackets indicate binding complexes of very high molecular weight 764 detected in EMSAs with all four DNA probes, suggesting these bands to comprise non-specific factors 765 in all cell lysates. (A) WT, N461S and R495G produced complexes with E1 (arrows), with R495G 766 forming higher molecular weight complexes (arrowhead). (B) N461S and R495G formed complexes 767 (closed arrowhead and arrow) with an E1mut probe, with R495G forming an additional, prominent high 768 molecular weight complexes (open arrowhead). A very weak signal representing a complex between 769 WT and E1mut was detected (arrow). (C) WT, N461S and R495G formed complexes with the E2 probe 770 (arrows), with WT and R495G forming additional molecular weight complexes which were common 771 (closed arrowhead) and unique (open arrowhead) to these two species. (D) No specific binding 772 complexes were observed when assessed with E2mut probe. (E) Luciferase assays with a BS10 773 reporter show that WT ZBTB18 suppresses reporter activity while N461S does not. R495G 774 potentiates reporter activity. (F) Luciferase reporter assays with an Rnd2 3’ enhancer show that 775 N461S and R495G potentiate reporter activity. (G) Luciferase reporter assays utilising Rnd2 3’ 776 enhancer constructs harbouring native E1 and E2 motifs (black segments); or mutated E1mut and 777 E2mut motifs (grey segments) confirm sequence-specificity of WT ZBTB18 to repress activity from this 778 reporter through E1 and E2, and not via E1mut and E2mut. (H) With N461S, statistically significant 779 transcriptional activation by N461S was observed in constructs bearing E1+E2 (p<0.05) or 780 E1mut+E2mut (p<0.01) motifs, but not an E1mut+E2 motif or an E1+E2mut motif. When compared to WT 781 ZBTB18, the N461S variant has reduced capacity for repression in the context of E1+E2 (p<0.1), 782 E1mut+E2 (p<0.05) and E1+E2mut (p<0.0001) motif pairs; as well as apparent activation of the 783 E1mut+E2mut (p<0.01) motif pair. (I) R495G augments luciferase reporter activity across all constructs. 784 Values represent fold-change of signal relative to control condition ± SEM. For all graphs Kruskal– 785 Wallis performed, followed by a two-way Mann–Whitney U test; *P<0.05, **P<0.01, ***P<0.001, 786 ****P<0.0001 compared to control; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 compared to WT 787 ZBTB18.

788

789 Figure 5. The effects of ZBTB18 and its disease-associated variants on radial migration of E14.5- 790 born cortical cells within the embryonic E17.5 mouse cerebral cortex. (A-E) In utero electroporation of 791 E14.5 mouse cortex with either; control shRNA and an empty vector (A), Zbtb18 shRNA and an 792 empty vector (B), Zbtb18 shRNA and WT (C), Zbtb18 shRNA and N461S (D), or Zbtb18 shRNA and 793 R495G (E). Brains were collected three days later (E17.5), white bars indicate the cortical plate (CP), 794 intermediate zone (IZ), and ventricular zone/subventricular zone (VS/SVZ). (F) Quantification of 795 distribution of GFP-labelled cells within the CP, IZ, and VS/SVZ. (G) Quantification of GFP-labelled 796 cells within the upper, middle, and lower CP (uCP, mCP, and lCP, respectively). (H–L) Representative 797 images of neurons within either the cortical plate (CP) or the intermediate zone (IZ) of in utero 798 electroporation brains (E14.5, collected E17.5) for either; control shRNA and an empty vector (H), 799 Zbtb18 shRNA and an empty vector (I), Zbtb18 shRNA and WT (J), Zbtb18 shRNA vector and N461S

22

800 (K), or Zbtb18 shRNA and R495G (L). White arrowheads indicate round cells, unfilled arrowheads 801 indicate multipolar cells, and indented arrowheads indicate bipolar cells. (M-N) Proportions of cells 802 exhibiting different morphologies (round, uni/bipolar, and multipolar) in each brain region. (O) Leading 803 processes of GFP+ cells within the CP were analyzed. FLAG-tagged WT and R495G was 804 immunodetectable in cortical cells, but not in N461S treatment (Supplementary Figure S8). For all 805 graphs KruskalWallis performed, followed by a two-way MannWhitney U test (Mean SEM n 806 6 (161 processes per brain for (o)); *P<0.05, **P<0.01 compared to control; #P<0.05, ##P<0.01 807 compared to Zbtb18 shRNA. Scale bar in (e) represents 200 µm. Scale bar in (l) represents 25 µm.

808

809 TABLES

810 Table 1. Binding energies for ZBTB18-DNA complexes.

ΔGbind (kcal/mol) DNA motifs ZBTB18 variant E1 E1mut E2 E2mut WT -119.8 ± 0.3 -117.1 ± 0.3 -125.2 ± 0.3 -94.0 ± 0.3 N461S -129.6 ± 0.3 -119.2 ± 0.4 -108.1 ± 0.3 -124.5 ± 0.4 R495G -126.4 ± 0.3 -126.0 ± 0.3 -126.9 ± 0.4 -102.0 ± 0.3 811

812

813 Table 2. Binding energy decompositions for selected residues in ZBTB18-DNA complexes. Three 814 residues (Tyr458, Asn461 and His493) with significant contributions to DNA-binding are indicated in 815 grey. Binding energy calculations for additional residues (Leu434, Tyr447, Arg464, Glu486, Arg495) 816 impacted by pathogenic missense variants are also listed. A comprehensive list describing binding 817 energy decomposition values for all residues making a major contribution to the binding energy in at 818 least one complex is provided as Supplementary Table S4.

ΔGbind (kcal/mol) Protein WT N461S R495G DNA E1 E1mut E2 E2mut E1 E1mut E2 E2mut E1 E1mut E2 E2mut Leu434 -0.1 +0.1 -0.0 -0.1 -0.1 -0.1 -0.1 +0.0 -0.0 -0.1 +0.1 -0.1 Tyr447 -2.1 -0.5 +0.0 -0.1 -1.6 -2.9 +0.1 -1.7 -0.9 -3.4 +0.1 +0.0 Tyr458 -3.2 -0.7 -3.4 -0.7 -2.2 -2.5 -3.1 -4.3 -1.9 -3.5 -1.1 -4.2 Asn/Ser461 -8.6 -2.3 -9.6 -1.4 -2.5 -1.5 -2.3 -3.3 -7.7 -3.9 -3.6 -5.5 Arg464 -4.0 -3.1 -4.9 -3.8 -3.9 -2.7 -4.0 -4.3 -4.0 -5.5 -4.5 -3.1 Glu486 -3.1 -2.7 -4.3 -2.5 -2.8 -2.9 -2.9 -3.4 -3.6 -2.4 -3.5 -2.8 His493 -5.9 -0.3 -0.7 -2.9 -5.3 -3.9 -5.8 -4.2 -2.3 -1.3 -4.9 -4.2 23

Arg/Gly495 -0.7 -2.5 -0.3 -2.7 -1.2 -1.4 -0.6 -1.0 +0.0 +0.0 +0.0 -0.0 819

24

Figure 1 Figure 2 A Figure 3 E1 E1mut E2 E2mut A

1

E1 E1mut E2 E2mut

1

E19 sequences in the wildtype, while in the N461S and R495G variants, this preference is reversed. The corresponding pair to this residue, dG 4’, is also a major contributor to the binding energy, particularly in complexes involving E18 sequences. dG 6 generally makes major contributions to the binding energy in the binding of the original sequences, while its contribution is generally diminished in the binding of the mutant sequences, despite this residue being the same in both the original and mutant sequences. A clear preference for dG at position 9 is observed, with mutation to dA at this position generally poorly tolerated, and in some complexes, contributing unfavourably to the binding energy.

DISCUSSION

…

AcknowledgementsTable SXX. Templates for each of the C2H2 subdomains of the ZBTB18 zinc finger domain.

M.A.Region is supportedTemplate by a Curtin (PDB ID) ResearchSequence alignment/region Fellowship, and a recipient of a Raine Priming Grant (Raine modelleda MedicalC2H2 1 Research3UK3 Foundation). ThisFMCPLCNKVFPSPHILQIHLSTH work was supported by computational resources provided by the Australian Government throughRECSYCGKFFRSNYYLNIHLRTH the Pawsey Supercomputing Centre under the National ComputationalC2H2 2 1MEY Merit(1) Allocation Scheme.PTCSLCGKTFSCMYTLKRHERTH YKCPECGKSFSQSSNLQKHQRTH C2H2 3 1MEY (1) YTCTQCGKSFQYSHNLSRHAVVH YKCPECGKSFSQSSDLQKHQRTH i C2H2 4 5K5H (2) HACKWCERRFTQSGDLYRHIRKFH YECYICHARFTQSGTMKMHILQKH aIn this column, the first row indicates the ZBTB18 sequence; the second row indicates the template sequence. Regions not shown in this table were modelled de novo by Prime during model building. A E1 motif B E1mutmotif C E2 motif D Emut motif Table SYY. Templates for the E18 and E19 probe sequences.

DNA probe Template (PDB ID) Sequence alignmentsa E18 5KE6 (3) TAACAGATGTCTGTC GAGGTGTGGC AGGGAAAAGCCCGGG E19 5KL2 (4) GGGCAGATGGGAGTA GCGTGGGAGT AGGGAAAAGCCCGGG aIn each row of this column, the first line indicates the DNA probe sequences to be modelled, the second line indicates the DNA sequence contained in the PDB template and the third line indicates ce from PDB 2I13. Figure captions

Fig XX. Predicted structural basis of ZBTB18 recognition of . A. 3D structure (prepared using PyMOL) of representative complex from MD simulations. ZBTB18 depicted as cartoon with N- to C-terminal colouring from blue to red. Approximate domain colourings: C2H2 1 – blue-cyan; C2H2 2 – green-yellow; C2H2 3 – yellow-orange; C2H2 4 – orange-red. Zinc centres shown as grey spheres, protein residues listed in Table VV shown as sticks. depicted WT WT WT WT as mixed cartoon-sticks with white backbone and nucleosides coloured by type: deoxyadenosine – N461S N461S N461S N461S R495G R495G R495G R495G control control control control white; deoxyguanosine – pale green; deoxythymidine – pale yellow; deoxycytidine – pale pink. B. 2D o t o t o t o t representation (prepared using NUCPLOT) of hydrogen bonds in the representative complex.

REFERENCES

1. Segal, D.J., Crotty, J.W., Bhakta, M.S., Barbas, C.F. and Horton, N.C. (2006) Structure of Aart,

13 11

10

9

8 a designed six-finger zinc finger peptide, bound to DNA. J. Mol. Biol., 363, 405-421. G control BS10 enhancer 7 6

5

4

3 WT E BS10 3 3

13

12 13 11 10 2 9 Table SXX. Binding energy decomposition for all residues making a major contribution to the binding 8 #### 7

fo a 6

5 energy in at least one complex. 4 **** 1 3 3 3 ** wt N461S R495G Residue E18 E18mut**** E19 E19mut**** E18 E18mut E19 E19mut E18 E18mut E19 E19mut

0 rela;ve 0 control huRP58(wild-type) control huRP58(wild-type) control huRP58(wild-type) Lys386 -2.9 -4.4 -1.2 -2.3 -2.7 -3.4 -1.4 -1.9 -2.2 -4.0 -1.3 -4.5

2 Ser390 -1.2 -0.9 -3.3 -2.1 -2.1 -0.4 -1.8 -0.4 -0.6 -1.8 -3.6 -1.9 #### His392 -2.1 -0.8 -1.7 -5.0 -3.8 -1.5 -3.1 -3.4 -2.0 -1.1 -1.8 -4.2 fo His397 -3.4 -4.2 -1.5 -1.4 -3.7 -4.6 -0.3 -2.2 -4.6 -3.6 -0.3 -2.0 H Thr400 -3.2 -2.6 -1.3 -0.6 -4.4 -3.8 -0.4 -1.9 -4.8 -3.6 -0.7 -0.3 1 Arg403 -1.5 -3.3 -2.2 -1.9 -4.9 -5.2 -1.8 -3.5 -5.0 -2.1 -0.9 -0.4

13 13 12 control 11

10

9 8 N461S rela;ve 7 ## 6

**** 5

4

3 3 00 3 ** oto ## #

2 fo *

F 1 ####

Rnd2 3’enhancer rela;ve

0 0 control huRP58(N461S) control huRP58(N461S) control huRP58(N461S) control huRP58(N461S)

13

12 13 11 10 ## 9

8

7 6 ** 5

4

3 3 I 3 ## control 13

13 12 R495G 11 ## 10 9 ## 2 #### 8 ** 7 #### fo 6

5 * 4 ** 3 **** 3 3 **

1

fo 2

rela;ve **

1

0 control 0 rela;ve oto 0 0 control huRP58(R495G) control huRP58(R495G) control huRP58(R495G) control huRP58(R495G) mCP uCP lCP 0

Intermediate zone 10 Cortical plate IntermediateIntermediate zone zone Intermediate zone CorticalCortical plate plate Cortical plate 20 Intermediate GFP+ cells % zone Cortical plate IntermediateIntermediate zone zone CorticalCortical plate plate control 30 Intermediate zone controlcontrol Cortical plate control B A Intermediate zone IntermediateCortical plate zone 40 Cortical plate control control e C

I E14.5 àE17.5 P Z A

H CP SVZ

controlIZ Embryonic subcompartments VS/

50 SVZ VZ CP IZ

Control controlRP58 shRNA 02 04 06 080 70 60 50 40 30 20 10 0 SVZ CP IZ control c PCIGF - scr PCIGF - sh3 sh3 - hu(WT) sh3 - hu (NS) hu(RG) - sh3

Control 0 control RP58RP58 shRNA shRNA RP58 shRNAcontrol 10 I RP58 shRNA Zbtb18 ** ** SVZ

CP RP58 shRNA IZ # 20 0 # F SVZ

VZ/ RP58 shRNA shRNA CP

IZ RP58 shRNA * SVZ/VZ 0 0 0 40 50 60 70 0 10 20 30 percentage of GFP posi;ve cells CP ecnaeo F+cells GFP+ Percentage of IZ 10 30

0 GFP+ cells % * p*=0.078 RP58 shRNA B **

RP58 shRNA ## RP58 shRNA **

** RP58 shRNA+ RP58 WT J #

10 RP58 shRNA 20 40 RP58 shRNA RP58 ## Zbtb18 Zbtb18 * ## # # + RP58+ RP58 WT WT *

20 +WT + RP58 WT RP58 shRNA 30 50 GFP+ cells % p*=0.078 ## shRNA RP58 shRNA ** # shRNA * ## shRNA

30

** RP58 shRNA * 40 60

## + RP58 WT ##

** + RP58 WT

## RP58 shRNA # ## K

40 RP58 shRNA ** ## 50 70 + RP58 WT # Zbtb18 RP58RP58 shRNA shRNA RP58 shRNA +N461S ## RP58 shRNA+ RP58 N461S 50 RP58** shRNA

** + RP58 WT shRNA 60 ## ## * **

+ RP58 N461SC

## + RP58 N461S 60

## + RP58+ N461SRP58 WT

+ RP58** WT RP58 shRNA Zbtb18 70

## RP58 shRNA RP58 + RP58 WT L 70 RP58 shRNA Zbtb18 RP58 shRNA+ RP58 N461S +R495G + RP58 N461S G shRNA+WT shRNA RP58 shRNA

C shRNA mCP Z V S Z/ +RP58 RP58 shRNA N461S uCP RP58 shRNA l mCP

uCP RP58 shRNARP58 shRNA Z + RP58 R495G CP lCP

EmbryonicRP58 subcompartments shRNA 0

+ RP58 N461S mcp ucp lcp

0

0 mCP uCP M lCP N + RP58 R495G percentage of GFP posi;ve cells + RP58 R495G ** 100 **

IZ GFP+ Cell (%) CP GFP+ Cell (%) 5 + RP58+ R495GRP58 N461S * 20 40 60 80 20 40 60 80 ## * ## 0

0 0

## 20 40 60 80 100

p#=0.078 RP58 shRNA 0 20 40 60 80

## 0 **

#

** + # p*=0.055 RP58 N461S 10 RP58 shRNA 10

10 round mCP round uCP

lCP D

round round

* ## **

0 RP58 shRNA

Zbtb18 15 * + RP58 R495G * + RP58N461S ##

+ RP58 R495G RP58 20

** ##

# **

5

20 n/ioa mul;polar uni/bipolar 20 n/ioa mul;polar uni/bipolar

* RP58 shRNA

## * * ##

10 GFP+ cells %

p#=0.078 **

* # p*=0.055

+ RP58 R495G

uni/bipolar # uni/bipolar 10

* * 25 RP58 shRNA ##

shRNA+N461S Percenetage of GFP+ cells # * 10 20 30 40 50 60 70 80 90 30 # shRNA ## # ##

RP58 shRNA 0 + RP58 R495G

15 30

Percenetage of GFP+ cells 30 Percenetage of GFP+ cells *

* Percenetage of GFP+ cells 10 20 30 40 50 60 70 80

10 20 30 40 50 60 70 80 90 + RP58 R495G

10 20 30 40 50 60 70 80 90 Percenetage of GFP+ cells

* *

0 20

0 40 10 20 30 40 50 60 70 80 90 ecnaeGP cells Percentage GFP+

35 0 multipolar

# multipolar *

* GFP+ cells %

0

20 Percenetage of GFP+ cells + RP58 R495G* * Percenetage of GFP+ cells * ## 10 20 30 40 50 60 70 80

40 **

10 20 30 40 50 60 70 80 Percenetage of GFP+ cells # # Dendritc length (µm) 25 40 0 10 20 30 40 50 60 70 80 #

10 12 14 16 18 20 ## 0 0 2 4 6 8 0 O 30

45 Percenetage of GFP+ cells

Dendritc length (µm) CP Leading Process Length (μm)

20- 40- 60- Dendritc length (µm) 50 10 20 30 40 50 60 70 80 90 10 12 14 16 18 20 0- 10 12 14 16 18 20 # Percenetage of GFP+Dendri cellstc length (µm)

50 0 2 4 6 8

0 10 12 14 16 18 20 10 20 30 40 50 60 70 80 90 E 0 2 4 6 8 round 35 50 0 2 4 6 8

Percenetage of GFP+ cells0

Zbtb18 30 70 80 Percenetage of GFP+ cells 10 20 30 40 50 60 Percenetage of GFP+ cells + RP58R495G ** control RP58 shRNA RP58 shRNA+WT RP58 shRNA+N461S RP58 shRNA+R495G 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 40 RP58 round ## round round 0 Z Control Zbtb18 Zbtb18 Zbtb18 0 round btb18 Percenetage of GFP+ cells ** Percenetage of GFP+ cells

Dendritc length (µm) 10 20 30 40 50 60 70 80 90 45 0-20µm 10 20 30 40 50 60 70 80 10 12 14 16 18 20 round 0 round Dendritc length (µm) 0 shRNA+R495G

0 2 4 6 8 Percenetage of GFP+ cells round Percenetage of10 GFP+12 14 cells16 18 20 10 20 30 40 50 60 70 80 90

shRNA 50 0 2 4 6 8 ## shRNA shRNA 10 20 30 40 50 60 70 80 shRNA shRNA 0 0-20µm **

0-20µm Percenetage of GFP+ cells 0 PercenetageDendri of GFP+tc length cells (µm) 40 0-20µm 70 80 90 10 20 30 40 50 60 RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control 10 12 14 16 18 20 10 20 30 40 50 60 70 80 round 0 ## 0 2 4 6 8 0 ** Percenetage of GFP+ cells round Dendritc length (µm)

+ + + 10 12 14 16 18 20 10 20 30 40 50 60 70 80 RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control

0 2 4 6 8 RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control WT N G round 0

Dendritc length (µm) RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control round 10 12 14 16 18 20 round ** ##

0 2 4 6 8 20-40µm

Dendritc length (µm) 0-20µm

10 12 14 16 18 20 50 round

0-20µm

round

0 2 4 6 8

uni/bipolar

20-40µm

20-40µm round

20-40µm round 0-20µm RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control uni/bipolar uni/bipolar round RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control uni/bipolar round uni/bipolar 0-20µm uni/bipolar round uni/bipolar 0-20µm RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control 1 uni/bipolar 20-40µm 40-60µm RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control 0-20µm 20-40µm 1 1 RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control 1 uni/bipolar 40-60µm 40-60µm uni/bipolar 20-40µm 40-60µm RP58 shRNA+R495G RP58 shRNA+N461S RP58 shRNA+WT RP58 shRNA control uni/bipolar 20-40µm uni/bipolar uni/bipolar 20-40µm uni/bipolar 1 uni/bipolar 1 uni/bipolar 20-40µm >60µm 40-60µm uni/bipolar mul 40-60µm 1 uni/bipolar uni/bipolar >60µm >60µm mul t mul mul >60µm polar 40-60µm 1 mul uni/bipolar t 1 mul t 40-60µm mul t polar polar mul t polar 40-60µm polar t 1 t polar t polar polar 40-60µm >60µm mul >60µm mul mul t polar >60µm mul t mul polar t >60µm mul polar t mul t >60µm polar mul polar mul t polar t >60µm mul t polar mul polar t polar t mul t polar polar t polar Supplementary Figure 1

N461S R464H R464C Y447H Q486E H15R C54R A186P R495G L434P

B ZF1 ZF2 Humans HSTVKESVSTNNRV------QYEPAHLAPLREDSVLRELDREDKASDDE--MMTPESERVQVEGGMESSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGIRSKPAADVNVPTCSLCGKTFSCMYTLKRHERTHSG Chimps HSTVKESVSTNNRV------QYEPAHLAPLREDSVLRELDREDKASDDE--MMTPESERVQVEGGMESSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGIRSKPAADVNVPTCSLCGKTFSCMYTLKRHERTHSG Rhesus(M) HSTVKESVSTNNRV------QYEPAHLAPLREDSVLRELDREDKASDDE--MMTPESERVQVEGGMESSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGIRSKPAADVNVPTCSLCGKTFSCMYTLKRHERTHSG Mice HSTVKESVSTNNRV------QYEPAHLAPLREDSVLRELDREDKASDDE--MMTPESERVQVEGGMENSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGIRSKPAADVNVPTCSLCGKTFSCMYTLKRHERTHSG Rats HSTVKESVSANNRV------QYEPAHLAPLREDSVLRELDREDKASDDE--MMTPESERVQVEGGMESSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGIRSKPAADVNVPTCSLCGKTFSCMYTLKRHERTHSG Cows HSTVKESVSTNNRV------QYEPAHLAPLREDSVLRELEREDKASDDE--MMTPESERVQVEGGMESSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGLRSKPAADVNVPTCSLCGKTFSCMYTLKRHERTHSG Chickens HSTVKESVSTNNRV------PYEPAHLAPMREDSVLRELDREDKASDDE--MITPENERVQVEGNMDSSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGIRSKPATDVNVPTCSLCGKTFSCMYTLKRHERTHSG Dogs HSAVKESVSTNARV------QYEPAHLAPLREDSVLRELERDDKASDDE--MMTPDAERGPVEGGMESSLLPYVSNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQDGLRSKPAADVNVPTCSLCGKTFSCMYTLKRHERTHSG Frogs RNTVKESSSSNIRA------PYEPVHLAPIREDSVLRELDHDDKASDDD---ITPENERVQMETNMDSSLLPYVPNILS-PAGQIFMCPLCNKVFPSPHILQIHLSTHFREQEGIRSKPANDVHVPTCSLCGKTFSCMYTLKRHERTHSG Zebrafish HSGVKEPPSTNGTHLSLVAQRRLGLEAHLSALREASLASELERDDKGGDDDTDVLGGDSDRVQEAAGVESSLLPYVSSMLGAPHTQIFMCPLCNKVFPSPHILQIHLSTHFREQEGVRAKPAGDVNVPTCSICGKTFSCMYTLKRHERTHSG :. *** *:* .**: :** *: **:::**..**: : : :* .::.****** .:*. * *****************************:*:*:*** **:*****:********************

ZF3 ZF4 L434 Humans EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPTVRDWTLEDSSQELWK Chimps EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPTVRDWTLEDSSQELWK Rhesus(M) EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPTVRDWTLEDSSQELWK Mice EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPTVRDWTLEDSSQELWK Rats EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPTVRDWTLEDSSQELWK Cows EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPAVRDWTLEDSSQELWK Chickens EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPTVRDWTLEDSSQELWK Dogs EKPYTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALSLPTVRDWTLEDSSQELWK Frogs EKPFTCTQCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSETLGLPAVRDWTLEDSSQELWK Zebrafish EKPYTCTTCGKSFQYSHNLSRHAVVHTREKPHACKWCERRFTQSGDLYRHIRKFHCELVNSLSVKSEALNLPTVRDWALEDSSQELWK ***:*** ***********************************************************:*.**:****:**********

Y447 N461 R464 Q486 R495 Supplementary Figure

WT N461S R495G E1 complexes complexes mut E1 E2 complexes complexes mut E2 Supplementary Figure

B 4 4 4 4 3 3

2 2

1 1 Relative fold-change Relative Relative fold-change Relative

0 0 0 0 WT - + -+ WT - + -+ N461S -- ++ R495G -- ++ Supplementary Figure 5

B

? ? ? ? ? ?

WT N461S R495G

? unknown Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 5 Pancellular Pancellular Exclusively Nuclear Exclusively Nuclear SupplementaryA Figure 5 SupplementarySupplementary Figure Figure55 Uniform Punctate Pancellular Uniform Exclusively Nuclear Punctate Uniform Punctate Pancellular Exclusively Nuclear Pancellular Pancellular Exclusively Nuclear Exclusively Nuclear Uniform Punctate Uniform Punctate FLAG / DAPI Uniform Punctate a’ a’’ a’’’ FLAG / DAPI a’ a’’ a’’’ 24h post transfection 24h post transfection

FLAG / DAPI

1.2 100 FLAG / DAPI FLAG / DAPI

100 FLAG / DAPI 24h post transfection *** 24h post transfection a’ a’’ a’’ a’’’ a’’ a’ a’’ a’ a’ ’ a’a’’ 1.0 ’ a’’’ ’ 80 100

1.2 100 100 *** 0.8

1.0 60 24h post transfection 24h24h80 24h post post post transfection transfection transfection 100 24h post transfection B C 0.6 D 24h post transfection 24h post transfection 0.8 40 100 24h post transfection 1.2 1.2 100 100 1.2 100 *** 100100 60 100 0.4 *** ****** 0.6

1.0 1.0 1.2 1.0 20 100 80 80 80 100 % distribution 100 80 40 0.2 100 100 *** 0.4

0.8 0.8 *** % exclusively nuclear 0.8

0 60 0.0 60 HUMAN N461S R495G 60 WT NS RG 60 20 0

0 1.0 % distribution 0.2

80 0.6 100 0.6 WT N461S R495G WT N461S R495G 0.6 % exclusively nuclear

40 4040

40 0 0.0 HUMAN N461S R495G *** *** 0 WT NS RG 00.4 *** 0.4 0.4 48h post transfection WT N461S R495G 0.8 WT N461S R495G % pancellular 48h post transfection 20 60 2020

% distribution 20 % distribution

0.2 distribution% 0.2 0.2

1.2 100 100

0.6 % exclusively nuclear % exclusively nuclear % exclusively nuclear

0 0 0.0 0 0.0 HUMAN N461S R495G HUMAN N461S R495G 0 48h WTpost transfectionNS RG WT NS RG 48h0.0 post transfection HUMAN N461S R495G 0 0 WT NS RG 0 0 0 1.0 0 80 100 40 1.2 WT N461S R495G *** 100 100 WTWTWT N461SN461SN461SR495GR495GR495G WTWT N461SN461S R495GR495G 0.4 0.8

60 1.0 80 100

48h post transfection 48h48h post post transfection transfection0.6 48h post transfection 20 48h post transfection 48h post transfection % distribution 0.8 E F 0.2 G 40 1.2 100 1.2 100 100

1.2 100 100100 60 100 0.4

0.6 % exclusively nuclear 1.0 1.0

1.0 20 80 100 8080 100 0 80 40 0.2 100 0.0 HUMAN N461S R495G WT NS RG % distribution 0 0 0.4

0.8 0.8

% exclusively nuclear 0.8

0 0.0 60 60 HUMAN N461S R495G 60 0 WT NS RG WT0 N461S R495G60 20 WT N461S R495G

0.2

0.6 % distribution 0.6 WT N461S R495G WT N461S R495G 0.6 % exclusively nuclear

40 4040

40 0 0.0 HUMAN N461S R495G 0 0 WT NS RG 0.4 0.4

0.4 WT N461S R495G WT N461S R495G % pancellular

20 2020 48h post transfection20 0.2 48h post transfection0.2 0.2 % distribution % distribution distribution% % exclusively nuclear % exclusively nuclear

% exclusively nuclear 1.2 100

0 00 0.0 0.0 HUMAN N461S R495G HUMAN N461S R495G 0 WT NS RG WT NS RG 0 00.0 HUMAN N461S R495G 0 100 0 0 0 WT NS RG WT N461S R495G WT N461S R495G WTWT N461SN461SR495GR495G WTWT N461SN461S R495GR495G

1.0 80 100

0.8

60

0.6

40

0.4

20

0.2 % distribution % exclusively nuclear

0 0.0 HUMAN N461S R495G 0 0 WT NS RG WT N461S R495G WT N461S R495G SupplementarySupplementarySupplementarySupplementarySupplementary FigureSupplementary FigureSupplementary FigureSupplementary FigureSupplementary Supplementary Figure FigureSupplementary FigureSupplementary FigureSupplementary FigureSupplementary FigureSupplementary Supplementary FigureSupplementary FigureSupplementarySupplementary FigureSupplementarySupplementary FigureSupplementary Figure FigureSupplementary FigureSupplementary Figure FigureSupplementary Figure FigureSupplementary FigureSupplementary Supplementary Figure FigureSupplementary FigureSupplementary FigureSupplementary FigureSupplementary FigureSupplementary Supplementary Figure FigureSupplementary FigureSupplementary Figure Figure Figure Figure Figure

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0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 SupplementarySupplementarySupplemenSupplementary FigurFiguretarye 7 FigureSupplementarySupplementary Figure0.6 7 0.6 0.6 FigurFigure0.6 e0.6 7 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.70 0.4 0.4 0.4 0.4 0.4 0.4 0.70 0.70 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.8 0.8 0.8 # # # # # # # # # # B B# # # # # # 0.8B# # B# # # # # # 0.8B# # B 0.8 # # 0.6 0.6 0.6 # # # # # # # # 0.60 0.60 0.60 0.2 0.2 0.6 0.2 0.2 0.2 0.6 0.2 0.2 0.2 0.6 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.70 0.2 0.2 0.2 0.2 0.2 0.2 0.70 0.70 GFP+/DAPI+ GFP+/DAPI+

0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+

0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+

0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ Proportion of 0.2 0.2 GFP+/DAPI+ Proportion of 0.2 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ Proportion of GFP+/DAPI+ Proportion of 0.2 Proportion of GFP+/DAPI+ GFP+/DAPI+ Proportion of Proportion of

0.2 GFP+/DAPI+ Proportion of GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ Proportion of Proportion of GFP+/DAPI+ 0.2 0.2 Proportion of

0.2 GFP+/DAPI+ GFP+/DAPI+ Proportion of 0.2 GFP+/DAPI+

0.2 Proportion of 0.2 Proportion of GFP+/DAPI+ GFP+/DAPI+ Proportion of Proportion of

Proportion of 0.2 GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ Proportion of Proportion of Proportion of Proportion of 0.2 0.2 Proportion of Proportion of Proportion of Proportion of 0.2 0.2 Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of

0.50 Proportion of Proportion of Proportion of Proportion of Proportion of 0.50 0.50 Proportion of Proportion of 0.6 Proportion of 0.60 0.6 0.6 0.6 0.60 0.60

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Proportion of 3.0 FLAG 3.0

Proportion of FLAG Proportion of Proportion of FLAG FLAG Proportion of Proportion of 3.0 FLAG 3.0 FLAG 3.0 Proportion of 3.0 Proportion of Proportion of FLAG FLAG Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 3 3 Proportion3 of 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 # # 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 # # 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.00.0 0.0 0.0 0.0 0.0 0.0 1.0 # # 0.0 # # 0.0 0.0 0.0 # # # # 1.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.4 0.4 0.4 0.4 0.0 0.0 0.0 0.0 0.0 3 0.0 0.0 0.0 1.0 # # # # 0.0 1.0 0.00.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 # # # # 1.0 0.0 0.0 # # 0.0 # # 0.0 0.4 0.4 0.4 0.4 0.40.0 0.00.4 0.0 0.40.0 0.0 30.0 0.0 0.01.0 0.0 0.0 0.030.0 0.0 1.0 0.00.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.00.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0

FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.01.0 0.0 0.0 0.6 0.0 1.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0

FLAG+GFP+/GFP+ 1.0 FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 Supplementary Figure 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0

FLAG+GFP+/GFP+ 1.0 0.4 0.4 0.4 0.4 FLAG+GFP+/GFP+ 0.0 0.00.0 0.0 0.0 0.60.0 0.0 0.0 1.0 0.0 0.00.0 0.6 0.0 0.0 0.0 0.0 0.0

Supplementary Figure 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 1.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Supplementary Figure FLAG+GFP+/GFP+ 1.0 Supplementary FigureSupplementary Figure 0.4 0.4 0.4 0.4 0.0 0.4 0.0 0.4 0.0 0.0 0.40.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0

FLAG+GFP+/GFP+ 1.0 Supplementary FigureSupplementarySupplementary Figure Figure 0.2 FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0

FLAG+GFP+/GFP+ 1.0 Supplementary FigureSupplementary FigureSupplementary Figure 0.2 0.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 0.0 0.00.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ SupplementarySupplementary Figure FigureSupplementary Figure0.2 0.2 0.2 0.2 FLAG+GFP+/GFP+ Supplementary Figure0.6Supplementary Figure 0.2 0.2 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ T T T T SupplementaryFLAG+GFP+/GFP+ FigureSupplementary Figure 0.2 0.2 0.2 0.2 Supplementary Figure S S S S FLAG+GFP+/GFP+ T T T T T T T FLAG+GFP+/GFP+ T 0.6 0.6 Supplementary Figure S S S S S FLAG+GFP+/GFP+ S S S FLAG+GFP+/GFP+ T T T T T T T T FLAG+GFP+/GFP+ T T T T T T T T Supplementary Figure S S S S S FLAG+GFP+/GFP+ S S S Supplementary FigureSupplementary Figure S S S S S S S S T T T T T T T T T T T T T T T T T T T T

S FLAG+GFP+/GFP+ S S S FLAG+GFP+/GFP+ Supplementary FigureSupplementary Figure S S S S S S S S S S S S S FLAG+GFP+/GFP+ S S S Supplementary Figure T T T T T T T S T T T 0.2ST T S S T T T T T T T Signal intensity (arbitrary units) FLAG+GFP+/GFP+ Supplementary Figure S S S S S S S S S S S S S FLAG+GFP+/GFP+ S S S Supplementary Figure T T T T Supplementary Figure T 0.2 T T TS T T T ST T S T S S T T S S S Signal intensity (arbitrary units) FLAG+GFP+/GFP+ T 0.2 0.2S TS ST TS T S S TS S S0.2T T FLAG+GFP+/GFP+ TS S T T S T S T T TS T S S

Supplementary Figure Signal intensity (arbitrary units) S S S S S 0.2S S S

Supplementary Figure Signal intensity (arbitrary units)

Supplementary Figure Signal intensity (arbitrary units) 0.2 T T S T S T ST 0.2TST S T T T SS S S T S T TS ST T TS S T S S S T TS T S S Supplementary FigureSupplementary Signal intensity (arbitrary units) Figure T Signal intensity (arbitrary units) T T T T T T T T T T T T T T T T T T T Supplementary Figure Signal intensity (arbitrary units) 0.2 0.2S S S 0.2S 0.2S S S S S S S S S S S S SupplementarySignal intensity (arbitrary units) Figure S S S S Signal intensity (arbitrary units) T T T T T T T T T S T ST T ST S S T TS T S S Signal intensity (arbitrary units) S S S S S S S S

T T T T T T T T Signal intensity (arbitrary units) Signal intensity (arbitrary units) S S T S S TS ST T S T T S S T T T ST S T T S SS S S Signal intensity (arbitrary units)

T T T T Signal intensity (arbitrary units) T S T S ST TST S T SS TS S S T T S ST T TS ST S S S T TS T S S Signal intensity (arbitrary units)

T T T T Signal intensity (arbitrary units) T T T T T T S T T S S S T T T T Signal intensity (arbitrary units) Signal intensity (arbitrary units) S S S S S S * * * S T S TS T S T T S S T S T S T S S T T TS T T S S T S S T T S S S Signal intensity (arbitrary units) T S TS ST TS T S S TS S ST T TS S T T S T S T T TS T S S Signal intensity (arbitrary units) S S S S GFP+/DAPI+ S S S S Signal intensity (arbitrary units) Signal intensity (arbitrary units) * * * T T T T T * * * T T T T T T T T T T T T T T T S S S S S SS S S S S S GFP+/DAPI+ S S S S S S S S Signal intensity (arbitrary units) T Signal intensity (arbitrary units) T T T T T T T T T T T T T T T

Signal intensity (arbitrary units) S S S S S S S S GFP+/DAPI+ GFP+/DAPI+ S S S S S S S S Signal intensity (arbitrary units) Signal intensity (arbitrary units) 0.2 GFP+/DAPI+ Mouse T T T T T GFP+/DAPI+ T T T 0.2 Signal intensity (arbitrary units) S S S S S S S S GFP+/DAPI+ α Mouse GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) 0.2 0.2 Signal intensity (arbitrary units) GFP+/DAPI+ GFP+/DAPI+ Mouse Mouse α Mouse 0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 Signal intensity (arbitrary units) 0.2 0.2 Signal intensity (arbitrary units) α α Mouse Mouse 2.5 α Signal intensity (arbitrary units) Mouse α Mouse Mouse α α Mouse 0.2 0.2 Signal intensity (arbitrary units) 0.2 0.2 α α 2.5 2.5

Mouse α GFP+/DAPI+

Mouse Mouse Proportion of

α GFP+/DAPI+ α Mouse α Proportion of Mouse

GFP+/DAPI+ GFP+/DAPI+ 0.2 GFP+/DAPI+

Mouse Proportion of Proportion of α Mouse α GFP+/DAPI+ Proportion of Mouse 0.2 Proportion of GFP+/DAPI+ α α GFP+/DAPI+ Proportion of α Mouse 0.2 0.2 Proportion of GFP+/DAPI+ GFP+/DAPI+ 0.4 0.4 0.4 0.4 0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 Proportion of Proportion of Mouse Mouse α Proportion of Proportion of Mouse 2.5 0.2 0.2 0.0 α Mouse α Mouse 0.0 α Mouse α Mouse 0.0 Mouse α 2.5 0.4 0.4 0.4 0.4 0.4 0.40.2 0.2 0.4 0.2 0.2 0.0 α Mouse 2.5 2.5

α 0.0 Proportion of 0.0 0.0 0.0 α Mouse 0.0 α 0.0 2.5 2.5 Mouse Mouse Proportion of 0.0 0.0 α Proportion of 0.0 0.0 α Mouse 0.0 Proportion of Mouse Proportion of Proportion of α

0.0 0.0 Proportion of 0.0 0.0 0.0 Proportion of Mouse α Mouse Proportion of Proportion of 0.0 0.4 α Proportion of Proportion of

0.0 Proportion of α 0.0 Proportion of

α Proportion of 0.0 Proportion of 0.0 Proportion of Proportion of 0.0 0.0 0.0 Proportion of Proportion of Proportion of Proportion of 0.0 WT N461SWT R495G0.0N461SWTWT0.4WTN461SR495GN461SN461SWTR495GR495GN461SR495GWT0.0 WTN461SR495GWT 0.4N461SN461SR495GWTR495GR495G0.0 N461SWTWTR495GWTN461SR495GN461SN461SWTR495G0.4R495GR495GN461SR495GWTR495GWTN461SR495GR495G0.40.0 N461SR495G R495G0.4 0.0 0.4 0.40.0 0.4 0.0 0.00.4 0.4 0.0 0.4 0.00.4 Proportion of Proportion of 0.0 WT N461SWT R495GN461SWTWT WTN461SR495GN461SN461SWTR495G0.5 R495GN461SR495GWT WTN461SR495GWTWTN461SN461SR495GN461SWTWTR495GR495GN461SWTN461SWTWT WTN461SWTN461SR495GN461SN461SWTN461SR495GR495GN461SWTR495GWTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWT WTN461SR495GN461SR495G R495G 0.0 0.0 0.0 Proportion of 0.0 0.0 WT0.0 N461SWT0.5 R495GN461SWT WTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWTWT WTN461SR495GN461SWTN461SR495GR495GN461SWTR495GWTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWT WTN461SR495GN461SR495G R495G Proportion of 0.0 0.0 R495G R495G R495G R495G 0.0 0.5 0.0WT N461SWT R495GN461SWT WTR495GN461SR495GN461SR495GR495GR495GR495GWTR495GN461SWT N461SWT WTN461S N461S 0.5 R495G R495G R495G R495G WT N461SWT R495GN461SWT WTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWTWT WTN461SR495GN461SWTN461SR495G0.5 N461SWTR495GWTN461SWTN461SN461SWT N461SWT WTN461S N461S Proportion of Proportion of 0.0 Proportion of 0.0 0.0 0.0 0.0 WT N461S0.0WT R495GN461SWT WTN461SR495GN461SR495G R495G WT Proportion of N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G 0.5 0.5 0.0

0.4 Proportion of 0.0 Proportion of R495G R495G R495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G0.5 R495G WT N461SWT N461SWT WTN461S N461SR495G R495G R495G R495G 0.5 R495G R495G R495G R495G WT N461SWT N461SWT WTN461S N461S R495G R495G R495G R495G WT 0.5 N461SWT N461SWT0.0 WTN461SWTN461SN461SWT R495GN461SWT WTN461SR495GN461SR495G R495G WT N461SWT N461SWT WTN461S N461S

Proportion of Proportion of 0.0 0.0 Proportion of Proportion of 0.0 0.0 WT0.0 N461SWT0.5 R495GN461SWT WTN461SR495GWTN461SR495GN461SWTR495G0.0 R495GN461SWTWT WTN461SR495GN461SWTN461SR495GR495GN461SWTR495G0.0 WTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWT WTN461SR495GN461SR495G R495G 0.4 0.4 0.0 0.5 0.0 R495G R495G R495G R495G 0.5 R495G R495G R495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G 0.0 * * * WT N461SWT R495GN461SWT 0.0 WTN461SR495GWTN461SR495GN461SWTR495G0.0 R495GN461SWTWT WTN461SR495GN461SWTN461SR495G0.5 N461SWTR495G0.0 WTN461SWTN461SN461SWT N461SWT WTN461S N461S WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G 0.5 0.5 WT N461SWT R495GN461SWTWT WTN461SR495GN461SN461SWTR495G0.5 R495GN461SR495GWT WTN461SR495GWTWTN461SN461SR495GN461SWTWTR495GR495GR495GN461SWTN461SWTWT WTN461SR495GWTN461SR495GN461SN461SWTN461SR495GR495GR495GN461SR495GWTR495GWTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWT WTN461SR495G* * * N461SR495G R495G0.5 * * * 0.5 T WT N461SWT0.5 R495GN461SWT WTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWTWT WTN461SR495GN461SWTN461SR495GR495GN461SWTR495GWTN461SR495GWTN461SR495GN461SR495GWTR495GR495GN461SWTR495GWTN461SR495GR495GN461SR495GR495GR495G S 0.5 WT N461SWT R495GN461SWT WTR495GN461SR495GN461SR495GR495GR495GR495GWTR495GN461SWT N461SWT WTN461S N461S T S WT T N461SWTS R495GN461SWT WTN461SR495GWTN461SR495G0.5 N461SWTR495GR495GN461SWTWT WTN461SR495GN461SWTN461SWTR495G0.5 N461SN461SWTR495GWTWTN461SR495GWTN461SWTN461SN461SWTN461SR495GWT N461SR495GR495GN461SWT R495GWTN461SR495GN461SWTR495GN461SR495GWT R495GN461SWT WTN461SR495GN461SR495G R495G T T 0.5 0.5 S T T S WT N461SWT R495GN461SWT WTN461SR495GN461SR495G0.5 R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495GR495GR495G R495G R495G R495G 0.5 R495G R495G R495G R495G WT N461SWT N461SWT WTN461S N461S R495G R495G R495G R495G S T S T WT 0.5 N461SWT N461SWT WTN461SWTN461SN461SWT R495GN461SWT WTN461SR495GN461SR495G R495G WT N461SWT N461SWT WTN461S N461S T S S T T S S T T S WT N461SWT0.5 R495GN461SWT WTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWTWT WTN461SR495GN461SWTN461SR495GR495GN461SWTR495GWTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWT WTN461SR495GN461SR495G R495G T T S T 0.5 R495G R495G R495G R495G S S T T S S T WT T N461SWTS R495GN461SS WT WTN461SR495GWTN461SR495G0.5 N461SWTR495GR495GN461SWTWT WTN461SR495GN461SWTN461SR495G0.5 N461SWTR495GWTN461SWTN461SN461SWT R495GN461SWT WTN461SR495GN461SR495G R495G T S S T S T T S 0.5 0.5 T S TS S T T S S TS S T T T 0.5 S S T T S T 0.5 S T S β-ACTIN T S T T S TS S T T T S T S S TS T S T T S TS S S T T T S S T T S S S SupplementaryT FLAG+GFP+/GFP+ SupplementarySupplementary FigureSupplementary FigureT Figure Figure β-ACTIN T S S S S T # # # SupplementaryFLAG+GFP+/GFP+ SupplementarySupplementary FigureSupplementaryT Figure Figure Figure T β-ACTIN S S T S S β-ACTIN β-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ # # # T T T T FLAG+GFP+/GFP+

SupplementarySupplementarySupplementarySupplementarySupplementarySupplementary FigureSupplementary FigureSupplementary Figure Figure Figure Figure Figure Figure FLAG+GFP+/GFP+ SupplementarySupplementarySupplementary FigureSupplementarySupplementaryS FigureSupplementary Figure FigureSupplementary FigureSupplementary Figure Figure Figure β-ACTIN β-ACTIN # # # T S T S S T S T FLAG+GFP+/GFP+ # # # SupplementarySupplementarySupplementary FigureSupplementary# # # Supplementary FigureFLAG+GFP+/GFP+ Supplementary Figure FigureSupplementary FigureSupplementary Figure Figure Figure β-ACTIN β-ACTIN β-ACTIN S S T S T S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN oc β-ACTIN β-ACTINβ-ACTINβ-ACTINSupplementarySupplementaryβ-ACTIN# # # Supplementaryβ-ACTINSupplementarySupplementaryβ-ACTINSupplementary Figure# # # Supplementary FigureSupplementarySupplementarySupplementary Figureβ-ACTIN Figure# # # FLAG+GFP+/GFP+ Supplementary FigureFLAG+GFP+/GFP+ Supplementary Figure# # # FigureSupplementary FigureSupplementary Figure# # # Supplementary Figure# # # Supplementary Figure FigureS# # # Figure Figure# # # Figure Figure S# # # # # # # # # β-ACTINWT R495G N461S SupplementaryFLAG+GFP+/GFP+ SupplementarySupplementary FigureSupplementary Figure Figure Figure # # # # # # β-ACTIN oc oc SupplementaryFLAG+GFP+/GFP+ SupplementarySupplementary FigureSupplementary Figure Figure Figure β-ACTIN β-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTINWT R495G β-ACTINN461SWTSupplementarySupplementaryR495G SupplementarySupplementarySupplementarySupplementaryN461S FigureSupplementary FigureSupplementary Figure FigureSupplementary Figure FigureSupplementary Figure Figure Supplementary FigureSupplementarySupplementary FigureFLAG+GFP+/GFP+ Supplementary Figure FigureSupplementary FigureSupplementary Figure Figure Figure FLAG+GFP+/GFP+ β-ACTIN β-ACTIN β-ACTIN SupplementarySupplementarySupplementary FigureSupplementarySupplementary FigureFLAG+GFP+/GFP+ Supplementary Figure FigureSupplementary FigureSupplementary Figure Figure Figure FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ oc WT R495G oc N461S ocβ-ACTIN0.4 β-ACTINβ-ACTIN β-ACTIN0.4 SupplementarySupplementaryβ-ACTINSupplementaryβ-ACTINSupplementarySupplementaryβ-ACTINSupplementary0.4 FigureSupplementary FigureSupplementarySupplementarySupplementary Figureβ-ACTIN FigureFLAG+GFP+/GFP+ Supplementary FigureFLAG+GFP+/GFP+ Supplementary Figure FigureSupplementary FigureSupplementary FigureSupplementary FigureSupplementary Figure Figure Figure Figure Figure Figure WT R495G 2.0 N461S WT R495G N461S WT N461SWT N461SR495G WTR495G N461SWT N461SR495G R495GWTN461SWTWT N461SR495GN461S WTR495GR495GN461SWT0.4 N461SR495GR495G WT2.0 0.4 N461S R495G 2.0 0.4 WT N461S R495G WT 2.0 N461S R495GWT N461S R495G WT N461S R495G WT N461S R495G WT N461SWT R495GN461SWT2.0 R495GN461SWT R495GN461S R495GWT N461SWT R495GN461S R495G WT N461S R495G 0.8 0.8 WT0.2 N461S0.8WT N461S0.8R495G0.20.8WTR495G N461SWT0.2 N461S0.8R495G0.20.8R495G0.2WT0.8WT N461SWTN461SWT0.80.2 N461SR495GN461S0.8R495G0.2WTR495GR495G0.2N461SWTWT0.8 N461SR495GN461S0.2 R495GR495G0.2WT N461SN461SWT 2.02.0 N461SR495GR495G WTR495GWTN461SWT N461SN461SN461SWTR495G2.02.0 R495GN461SR495GR495GWTWTR495GN461SWTN461SN461SR495GR495GWTR495GN461SWT N461SWTR495GN461SR495G WTR495GN461S R495G WT 0.0 N461S R495G WT0.8 N461SWT R495GN461SWT 0.8R495GN461S R495G WT 0.8 N461SWT R495GN461S R495G 0.0 WT N461S 0.8R495G 0.8 WT N461S R495G 0.0 R495G 0.8 0.0 WT N461S 0.8R495G0.0 WT N461S R495G 0.8 WTWT N461SN461S R495GWT N461S R495G WT N461S R495G 0.2 0.2 0.2 0.2 0.0 0.0 WT N461S R495G WT N461S R495GWT N461S R495G WT N461S R495G 0.8 0.0 0.0 0.0 0.0 WT N461S R495G 0.0WT N461S R495GWT N461S R495G WT N461S R495G B 0.8 0.8 0.8 0.0 0.0 0.0 B 0.2 0.2 0.2 0.2 0.0 0.2 0.2 0.2 0.2 0.0 B 0.8 0.8 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 B 0.8 B 0.8 0.8 0.0 0.0 0.0 0.0 0.0 0.0 B B 0.0 0.0 B B 0.0 B 0.8 0.8 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 B 0.8 0.8 0.0 B 0.0 B 0.8 0.0 B 0.8 0.0 B 0.0 B 0.0 B 0.0 0.0 B 0.0 0.0 B Supplementary0.0 Figure 0.0 0.0 B 0.0 Supplementary Figure 0.0 0.0 0.0 0.0 B GFP+/DAPI+ 0.0 0.0GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ 0.0 0.0 0.0 0.0 0.6 B B SupplementarySupplementary Figure Figure Supplementary0.0 FigureSupplementary 0.0 Figure 0.0 0.0 0.0 0.0 B B Supplementary FigureSupplementary Figure0.0 0.0 0.0 0.0 0.0 0.0

0.6 0.2 0.2 GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ 0.6 0.6 0.6 B 0 B B B 0 B SupplementarySupplementary0.0 Figure0.0 FigureSupplementarySupplementary GFP+/DAPI+ 0.0 Figure0.0GFP+/DAPI+ Figure GFP+/DAPI+ GFP+/DAPI+ 0.6 0.6 0.6 0.6 0.6 0.6 0 0 B B 0 0 B0.2 B B 00.2 B0.2 0.2 0.2 0.2 0.2 0.6 0.6 0.6 0.6 Supplementary0 Supplementary0.6 Figure Figure0 0 0 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Proportion of SupplementarySupplementaryProportion of Figure FigureProportion of Proportion of 0 0.6 0.6 T Supplementary FigureSupplementary 0 Figure 0 0.6 0.6 T SupplementaryS FigureSupplementary T Figure 0 0

0.2 0.2 GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ S

S GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Proportion of Proportion of Proportion of Proportion of T T SupplementarySupplementary Figure FigureProportion of SupplementarySupplementary ProportionT of Figure0 FigureProportion of Proportion of 0 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.60 0 0 0.60 0 0 S S 0 0 0.2 T 0 T T 0 TS 0.2S S S T 0.2T0 S T S0.2T S T TS 0.2S S S T 0.2T T TS S0.2S S T T T TS S S S Mouse Proportion of Proportion of Proportion of Proportion of 0.6 0.6 α 0.6 Proportion of 0.6 Proportion of T T Proportion of S Proportion of 0 0 T 0 0 0 Proportion of Proportion of Proportion of Proportion of Mouse Proportion of Proportion of Proportion of Proportion of S S

Mouse T T Proportion of Proportion of Proportion of Proportion of T 0 α α S S T T T TS SProportion of S S T T Proportion of S T S T S T TS SProportion of S S ProportionT of T T TS S 0S S T T T TS S S S Mouse α Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Mouse Mouse Proportion of Proportion of Proportion of Proportion of 0.2 α α 1.5 1.5 1.5 0.2 3.0 Mock 0.2WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 Mock 3.0 WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 3.0 Mock 3.0 WT3.0 3.0 R495G3.0 3.0 3.0 1.5 3.0 3.0 3.0 3.0 3.0 Mock3.0 3.0WT 3.0 R495G 1.5 Mock WT R495G Mock WT R495G 3.0 Mock WT3.0 R495G3.0 3.0 0.2 1.5 3.0 Mock WT3.0 1.5 R495G3.0 3.0 Mock 3.0 WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 1.5 1.5 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 0.2 3.0 Mock WT0.23.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 Mock 3.0 WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 3.0 Mock 3.0 WT3.0 3.0 R495G3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Mock3.0 3.0WT 3.0 R495G Mock WTWT R495GN461S R495GMock WT R495G WT3.0 MockN461SWT3.0 R495GR495G3.0 3.0 WT N461S R495G 3.0 Mock WTWT3.0 R495GN461S3.0 3.0 Mock 3.0 R495GWT3.0 R495G3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3 3 3 3 3.0 3.0 3.0 3.0 R495GR495G WTWT N461SN461S R495GR495G 3.0 3.0 3.0 3.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ WTWT N461SN461S WT N461S R495G R495GWTMockN461SWT R495GR495G WTMockN461SWT R495GR495G 3 3 3 3 WT N461S3.0 3.0 3.0 3.0 3 3 3 3 3 3 3 3 3 3 3 WT 3 N461S R495G WT N461SMock R495GWTWT R495GN461S R495G3.0 Mock WTWT3.0 R495GN461S3.0 3.0 R495G GFP+/DAPI+ R495GR495G 0.8 0.8 0.8R495GR495GWT0.8 WT N461SN461S R495GR495G WTWT N461SN461S R495GR495G FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ WTWT N461SN461S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 3 FLAG+GFP+/GFP+ 3 3 FLAG+GFP+/GFP+ 3 33 3 3 33 3 3 3 330.8 0.83 0.8 33 0.8WT0.83WT0.8 0.8N461S0.8N461S0.80.8 0.8 0.8 0.2 GFP+/DAPI+ 3 3 3 3 WTWT0.2WTN461SWTN461SN461SN461SR495GR495GR495GR495G GFP+/DAPI+ 30.2 3 3 WT3WTR495GWT3 N461SWT33N461SN461S3 N461SR495G33R495GR495G0.8 R495G330.83 0.83 0.833R495G0.8 30.8 WT0.833 0.80.830.8N461S0.8330.80.80.80.80.8R495G330.830.80.83 0.8330.8 0.8 WT0.833 0.80.8 0.8N461S0.830.80.80.8 0.8R495G3 0.8 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 3 3 3 3 3 33 33 33 3 3 3 3 WTWT3 N461SN461S R495G0.8 WT0.8WT N461S0.8N461S 0.8R495GWT N461S R495G WT N461S R495G GFP+/DAPI+ R495GR495G WTWT N461SN461S R495GR495G R495GR495G Proportion of WTWT N461SN461S WTWT N461SN461S R495GR495G WTWT N461SN461S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 3 FLAG+GFP+/GFP+ 3 3 3FLAG+GFP+/GFP+ 33 3 3 33 3 B3 3 330.8 B0.83 0.8 33B0.8 0.83 0.8B 0.8 0.8 0.80.8 0.8 0.8 0.8 0.8 0.8 0.8 0.2 GFP+/DAPI+ 0.8 0.8 0.8 0.8 GFP+/DAPI+ 3 3 3 3 Proportion of Proportion of BBB 3 BBB3BBBB3B3B3 B 3 0.8B0.83 0.8 3B0.8 30.8 0.8B 0.8B0.8 0.830.8 0.8 0.8B3 0.8 3B 0.8B 0.8 0.8 0.6FLAG 0.6 FLAG FLAG 0.2 0.2 BBB3BBB 3BB3 BB3BBBB33B 3 B3 BB0.8 33B0.8 B3 BB30.8B0.8B 33 0.8 0.8 30.8 0.8 3 Proportion of 0.6 0.6 0.6 0.6 0.6 Proportion of

Proportion of B B B B 0.6 Proportion of 0.6 0.6 Proportion of 0.6 BProportion of BB BBB BBBBBB FLAG 0.6 0.6 0.6 0.6 0.6 0.6 0.60.60.60.6 0.60.6 0.6 BBBBBBBB BBBBBBBBBB B BBB B BBBBBB B 0.6 0.6 FLAG FLAG 0.6 0.6 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Proportion of 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Proportion of 0.6 Proportion of 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.60.60.6 0.6 0.60.6 0.6 3.0 0.6 0.6 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 3.0 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 3.0 3.0 3.0 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 1.0 0.0 0.0 0.0 0.0 3.0 3.0 3.0 3.0 0.0 0.0 0.0 0.0 3.0 3.0 1.0 0.0 0.0 1.0 0.0 0.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 2.5 2.5 0.0 2.5 2.5 0.0 0.0 0.0 3.0 2.5 2.5 2.5 2.5 3.0 3.0 2.5 2.5 2.5 2.5 2.5 3.0 2.5 2.5 2.5 2.5 3.0 2.5 3.0 2.5 2.5 3.0 2.5 2.5 2.5 2.5 2.5 2.5 3.0 2.5 2.5 2.5 2.5 2.5 3.0 2.5 3.0 3 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3.0 2.5 2.5 2.5 2.5 3.0 3 1.01.0 3.0 0.0 0.0 0.0 0.0 2.5 2.5 2.5 2.5 3.0 0.0 0.0 0.0 0.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 FLAG+GFP+/GFP+ 3 3 3 2.5 2.5 2.5 2.5 0.8 2.5 2.5 2.5 2.5 3 1.01.0 1.01.0 3 2.5 2.5 2.5 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 2.5 2.5 2.5 0.0 2.5 0.0 2.5 0.02.5 2.5 0.0 0.0 0.0 0.0 3 0.8 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 3 3 Supplementary Figure 2.5 2.5 2.5 2.5 0.80.8 3 0.8 0.8 2.5 2.5 2.5 2.5 0.8 2.5 2.5 2.5 2.5 3 2.5 2.5 2.5 2.5 3 0.80.8 3 0.8 SupplementarySupplementary Figure Figure 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3 0.8 0.8 3 2.5 2.5 2.5 2.5 3 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3 3 3 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3 2.5 2.5 2.5 2.5 FLAG+GFP+/GFP+ 3 3 2.5 2.5 2.5 2.5 0.4 3 0.8 2.5 2.5 2.5 2.5 3 2.5 2.5 2.5 2.5 0.4 E 3 3 0.8 2.5 2.5 2.5 2.5

FLAG+GFP+/GFP+ 3 FLAG+GFP+/GFP+ B E T T T T 0.8 0.8 3 0.8 0.8 Supplementary Figure0.4 0.4 0.4 S S S S B E T T T 3T 0.8 0.8 0.4 E 0.4 E S SBB S S 3 B 3 T T T T 0.8 0.8 B 0.4 0.4 E E T T T T 3 0.8 0.8 3 SupplementarySupplementary Figure Figure0.4 S S SS S S S S B E T T T T B E 0.4 E T 3 T T 3T 0.4 0.4 0.4 E S SBB SS S SBB S S 0.4 0.4 E Signal intensity (arbitrary units) 0.4 E 0.4 E E E

0.4 Signal intensity (arbitrary units) 0.4 E E E Signal intensity B(arbitrary units) E T T T T 0.4 0.4 0.4 S S S S B 0.4 E E E T T T T 0.4 S BSB S S B B 0.4 0.4 E T T E TT T 0.6 T T T 0.4 S E S SS S S S S B B E 0.4 E 0.6 T T TT T T T T 0.4 0.4 0.4 0.6 0.6E S BSB SS S BSB S S 0.4 0.4 E Signal intensity (arbitrary units) 0.4 E 0.4 0.6 0.6E 0.6 0.6 E 0.6 0.6E 0.6 0.6 Signal intensity Signal (arbitrary units) E E Signal intensity (arbitrary units) Mouse α Mouse α Mouse α 0.6 0.6 0.6 0.6 0.6 0.6 Mouse 0.0ocα oc ocMouse oc α Mouse α 0.0 0.6 0.6 0.6 0.6 0.6 0.6 0.60.6 0.60.6 0.60.6 0.6 0.60.6 0.6 0.60.60.60.60.60.60.60.60.6 0.6 0.6 0.6 0.6 0.60.6 0.6 0.60.60.60.60.60.60.60.60.6 0.6 0.6 0.6 0.6 0.60.6 0.6 0.60.60.60.6 0.6 0.6 WT R495G WTocN461SWTR495G ocWTR495G N461SocR495G 2.5 N461SocN461S 0.0 0.0 oc oc oc0.0ocWToc R495G ocWT ocN461SWTR495G ocWT0.0ocR495G N461SR495G 2.5 N461SocN461Soc oc WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S WT R495G WTocN461SWTR495G ocWTR495G N461SWTocR495G 2.5 N461SR495G ocWTocN461SN461SWTR495G ocWTR495G N461SocR495G 2.5 N461SocN461S 2.5 0.6 0.6 0.6 0.6 oc oc ocWTococR495G WTococN461SWTR495G ocWTocR495G N461SWTocR495G ocN461SR495G ocWTN461SN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S 2.5 0.6 0.6 0.6 0.6 WT R495G WT N461SWTR495G WTR495G N461SWT R495G N461SR495G WTN461SN461SWTR495G ocWTR495G N461SocR495G 2.5 N461SocN461Soc oc oc ococWToc R495G ocWT ocN461SWTR495G ocWTocR495G N461SR495G 2.5 N461S0.6oc0.6 N461Soc0.6 0.6oc0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S WT N461SWT R495GN461SWT WTN461SR495GN461S R495G R495G WT N461SWT WTR495GN461SWTR495G WTocWTN461SR495GN461SWTR495G ocN461SWTR495G N461SR495GWTocR495G 2.5 N461SR495G ocR495GWTocN461SN461SWTR495G ocWTR495G N461SocR495G 0.62.5 N461SocN461S0.6 0.6 0.6 0.6 0.6 0.6 0.6 oc0.0 oc oc oc 0.0 oc oc ocWTococR495G WT ocN461SWTR495G ocWTR495G N461SWTocR495G 2.5 N461SR495G 0.6 0.6WTN461SN461SWTR495G 0.6 0.6 WTR495G 0.6 N461S0.6 R495G 0.6 2.5 0.6N461S0.6 0.6 N461S0.6 0.6 0.6 0.6 0.6 0.6 WT R495G WT N461SWTR495G WTR495G N461SWT R495G 2.5 N461SR495G WTN461SN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S oc oc WToc ocN461SWT R495GN461SWT 0.5 WTN461SR495GWTN461SR495GN461SWTR495GWTN461SR495GWTN461SR495GN461S R495GWT R495GN461SWT WTN461SR495GWTN461SR495GN461SWTR495GWTN461SR495GN461SR495G R495G 0.4 oc oc ococ0.0WToc R495G ocWT ocN461SWTR495G ocWTocR495G 0.0N461SR495G 2.5 N461SocN461Soc oc 0.0 0.0 2.0 2.0 2.0 2.0 WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S 2.5 0.4 WT R495G WT N461SWTR495G WTR495G N461SR495G 0.5N461SN461S oc oc oc ococ oc oc oc 0.5 2.0 2.0 2.0 2.0 oc oc oc2.5 oc 0.4 0.4 oc 0.4 oc ocWTococR495G WT ocN461SWTR495G ocWTR495G N461SWTocR495G N461SR495G WTN461SN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S 2.0 2.0 2.0 2.0 2.0 WT2.0 R495G 2.0 WT2.0 N461S2.0 WTR495G WTR495G N461S2.0 R495G 2.5 N461S2.0 N461S2.0 2.5 oc oc oc oc 0.4 WT R495G WT N461SWTR495G 0.4 WTR495G N461SWT R495G N461SR495G WTN461SN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S 2.0 WT2.0 R495G 2.0 WT2.0 N461S2.0 WTR495G WTR495G N461S2.0 R495G 2.5 N461S2.0 N461S2.0 0.4 0.4 oc oc ocococ oc oc ococ 3.0 oc oc oc 0.4 2.0 2.0 2.0 2.0 2.0 WT2.0 R495G 2.0 WT2.0 N461S2.0 WTR495G WTR495G N461S2.0 R495G 2.5 N461S2.0 N461S2.0 2.5 0.4 WT R495G WTocN461SWTR495G ocWT3.0 R495G N461SWTocR495G N461SR495G ocWTocN461SN461SWTR495G ocWTR495G N461SocR495G 2.5 N461SocN461S WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461S2.0 R495G2.0 R495G2.0 2.0 0.4 3.0 WT3.0 R495G WT N461SWTR495G WTR495G N461SWT R495G 2.5 N461SR495G WTN461SN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S oc oc oc oc 3.0 0.4 0.4 oc oc oc oc 3.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 WT R495G WT N461SWTR495G WTR495G N461SWT R495G 2.5 N461SR495G WTN461SN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S 3.0 0.4 0.4 3.0 WT N461SWT R495GN461SWT 0.50.5 WTN461SR495GWTN461SR495GN461SWTR495GWTN461SR495GWTN461SR495GN461S R495GWT R495GN461SWT WTN461SR495GWTN461S2.0 R495GN461SWT2.0 R495GWTN461S2.0 R495G2.0 N461S2.0 R495G2.0 R495G2.0 2.0 0.4 0.4 3.0 3.0 3.0 3.0 0.4 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 T 0.4 S 0.50.5 0.5 2.0 2.0 2.0 2.0 0.5 0.4 0.4 T S 0.4 2.0 2.0 2.0 2.0 T 0.4 S T 0.4 S 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.4 0.4 2.0 T 2.0 2.0 2.0 2.0 T 2.0 3.02.0 2.0 0.4 S 2.0 2.0 2.0 2.0 S 2.0 0.4 2.0 2.0 2.0 2.0 2.0 2.0 2.0 3.0 0.4 3.0 3.0 2.0 2.0 2.0 2.0 0.4 3.0 3.0 0.4 2.0 2.0 2.0 2.0 0.4 0.4 2.0 2.0 2.0 3.0 2.0 2.0 2.0 2.0 3.0 2.0 0.4 0.4 3.0 3.0 2.0 2.0 2.0 3.0 3.0 2.0 2.0 2.0 2.0 2.0 β-ACTIN T S T S T S T S T S 3 3 3 3 T S 2.0 3 3 3 3 2.0 2.0 2.0 2.0 3 3 3 3 2.0 2.0 2.0 β-ACTIN β-ACTIN β-ACTIN 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.40.4 0.4 0.40.40.40.4 0.4 0.40.4 # # # 0.4 0.4 0.4 3 0.43 0.43 30.4 0.40.4 # # # 0.4 0.4 0.4 3 0.43 30.4 30.4 0.4 # # # 3 3 Time (hrs) 0 4 8 oc Time (WT hrsR495G ) 0 4 8 0 4 8ocβ-ACTIN N461S 0 4 8 0 4 80 4 8 β-ACTIN0 4 8 0 4 8 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.40.4 0.4 0.40.40.40.40.40.4 0.4 0.40.4 0.40.40.40.6 0.60.40.60.40.40.60.4 0.4 0.40.40.4 0.40.4 0.4 0.40.4 0.40.40.40.6 0.60.40.60.40.40.60.4 0.4 0.40.4 0.4 0.4 0.4 0.6 0.6 0.6 30.6 3 Time (Mouse Time (hrsTime (hrs) 0 4 8 Mouse ) 0 4 8 ochrsoc) 0 4 8 Mouse ocWTTime (WTTime (WTMouse hrsTime (R495G hrs) 0 4 8 Time (N461S 0 4 8R495G ) 0 4 8 ochrs 0 4 8ochrs) 0 4 8 0 4 8N461SWT) 0 4 8 ocN461SocWTR495GTime (WTR495G 0 4 8R495G hrsTime (WT0 4 8 0 4 8R495G ) 0 4 8 0 4 8Time (N461S0 4 8ochrsTime ( 0 4 8N461SR495G ) 0 4 8 hrs 0 4 8N461Sochrs) 0 4 8 0 4 8 WT) 0 4 8 Time (oc0 4 8 N461Soc0 4 8R495GTime (0 4 8 WT0 4 8hrsR495G Time ( 0 4 8) 0 4 8 0 4 8hrsWT0 4 8ochrsR495G ) 0 4 8 0 4 8N461S) 0 4 8 oc0 4 8 0 4 8ocR495G Time (0 4 8 WT 0 4 8N461SWT0 4 8 WT0 4 8hrsTime (0 4 8 WTN461SR495G ) 0 4 8 R495G 0 4 80 4 8ochrsR495G N461STime ( 0 4 8R495G ) 0 4 8 0 4 8 0 4 8N461S0 4 8ochrs0 4 8 WTN461S) 0 4 8 N461SR495GocTime (0 4 8 WT0 4 8R495G WT 0 4 80 4 8 0 4 8hrsTime (0 4 8 WT0 4 8R495G ) 0 4 8 0 4 8N461SochrsN461STime (0.4R495G ) 0 4 8 0 4 8 0 4 8N461S0.4ochrs0 4 8 WT0 4 8) 0 4 8 0 4 8 N461S0.4R495GocTime (0.4WT0 4 8R495G 0.6 WT 0 4 80.60.4hrs0.6WT0 4 80.4R495G ) 0 4 8 0.60 4 8 0 4 8N461S0.4ocN461S0.40.4R495G 0 4 8 0 4 8N461S0.4 WT0 4 80 4 8 N461S0.4R495G0.40 4 8R495G 0.6 0 4 80.60.40.60 4 80.40.60 4 8 N461S0.4 0.4 0 4 8 0 4 80 4 8 0.6 0.6 0.6 0.60 4 8 α α Mouse α WT αMouse R495G Mouse N461SMouse WTTime (WT hrs2.0 R495G ) 0 4 8 N461SocN461S WTTime (WT 0 4 8hrsR495G ) 0 4 8 N461SocN461S0 4 8 0 4 80 4 8 0 4 8 0 4 8 Mouse Mouse Mouse Mouse α Mouse Time (Mouse αhrs) 0 4 8 Mouse ocα Mouse α Mouse Time ( 0 4 8Mouse hrsWT) 0 4 8 Time (Mouse oc2.0 hrsR495G Mouse ) 0 4 8 0 4 8ocN461S 0 4 8WT0 4 8 0 4 8R495G 0 4 8N461S0 4 80 4 8 0 4 8 α α α α 2.0 2.0 α α Mouse α WT αMouse αR495G Mouse Mouse N461SMouse α Mouse αMouse WT αMouse Mouse R495G Time (WTMouse Mouse hrsN461SR495G Mouse ) 0 4 8 ocN461S2.0 0 4 8 0 4 8 0 4 8 Mouse Mouse Mouse α Mouse Mouse α Mouse α Time (αMouse hrsα Mouse ) 0 4 8 ocα Time (α2.0 hrs) 0 4 8 α 0 4 8ocWT R495G Time (0 4 8 0 4 8N461S2.0 hrs) 0 4 8 oc0 4 8 0 4 8 0 4 80 4 8 0 4 8 0 4 8 α α Mouse α αMouse Mouse Mouse 2.0 0.8 α α α α α α α2.0 α WT R495G N461S2.0 Time (WT hrsR495G ) 0 4 8 ocN461S Time (WT 0 4 8hrsR495G ) 0 4 8 ocN461S0 4 8 0 4 80 4 8 0 4 8 0 4 8 Mouse Mouse Mouse Mouse α Mouse Time (Mouse αhrs) 0 4 8 Mouse ocα Mouse α Mouse Time ( 0 4 8Mouse hrsWT) 0 4 8 Mouse oc2.0 R495G Mouse 0 4 8N461S 0 4 8WT0 4 8 R495G 0 4 8N461S 0 4 8 α α α α 2.0 2.0 WT 0.8 N461S R495G 0.8 WT αN461S α R495GMouse α WT αMouse αR495G Mouse N461SMouse α Mouse α WT αMouse R495G Mouse N461SMouse 2.0 Mouse Mouse Mouse Mouse Mouse Mouse Mouse α Mouse Mouse α Mouse α αMouse α Mouse * * * α α2.0 α 2.0 α α α α α R495Gα α2.0 * * * α 2.0 α α Mouse α αMouse Mouse Mouse WT 2.0 N461S R495GWT N461S R495G WT N461S WT * * * * * * N461S R495G * * * Mouse Mouse Mouse Mouse α Mouse Mouse α Mouse α Mouse α Mouse Mouse Mouse 2.0 Mouse * * * 0.0 1.5 1.5 1.5 1.5 * * * 2.0 * * * α α α α 2.0 α α Mouse α αMouse α Mouse Mouse α Mouse α αMouse Mouse Mouse 2.0 Mouse 1.5 Mouse 1.5 Mouse 1.5 Mouse 1.5 * * * * * * * * * * * * Mouse Mouse Mouse α Mouse Mouse α Mouse α αMouse α Mouse α α2.0 α 2.0 1.5 1.5 1.5 1.5 1.5 Mouse 1.5 1.5 Mouse 1.5 Mouse 0.0 Mouse 2.0 0.0 α α α α 1.5 1.5 1.5 1.5 0.8 α α α α α α α2.0 α 2.0 Mouse Mouse Mouse Mouse 1.5 1.5 1.5 1.5 2.0 Mouse α Mouse α Mouse α Mouse α Mouse 1.5 Mouse 1.5 Mouse 1.5 Mouse 1.5 1.5 1.5 1.5 2.5 1.5 α 1.5 α 1.5 α 1.5 α 1.5 1.5 1.5 2.0 1.5 1.5 2.0 0.2 α α Mouse α αMouse α Mouse Mouse α Mouse α αMouse Mouse Mouse 2.0 B 0.8 0.8 2.5 1.5 1.5 1.5 1.5 0.2 Mouse Mouse Mouse α Mouse Mouse α Mouse α αMouse α Mouse α α2.0 α 2.0 0.2 0.2 0.2 α α α 1.5 α 2.5 α2.5 1.5 1.5 α 1.5 1.5 α2.0 1.5 α 1.5 2.5 1.5 1.5 2.0 1.5 1.5 2.5 1.5 B 1.5 1.5 1.5 1.5 B 2.5 2.5 1.5 1.5 1.5 1.5 0.2 0.2 1.5 1.5 1.5 1.5 0.2 0.2 0.2 1.5 1.5 1.5 1.5 0.00.0 2.5 2.5 1.5 2.5 2.5 1.5 1.5 1.5 0.2 1.5 1.5 1.5 1.5 0.2 1.5 1.5 1.5 1.5 0.0 0.2 0.2 1.5 1.5 1.5 1.5 1.5 0.0 1.5 1.5 1.5 0.00.0 0.4 0.4 0.4 0.4 0.2 0.2 1.5 1.5 1.5 1.5 1.5 0.41.5 1.5 0.4 1.5 0.4 0.4 2.5 0.2 0.2 0.2 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 0.41.5 0.4 1.5 1.5 0.4 0.41.5 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 2.5 0.4 0.4 0.4 0.4 B 1.5 1.5 1.5 1.5 0.2 0.4 0.4 0.4 0.4 2.5 2.5 0.4 0.4 0.4 0.4 0.2 0.2 0.2 1.5 1.5 1.5 1.5 1.5 0.41.5 0.4 1.5 2.50.4 0.41.5 1.5 0.4 0.4 0.4 0.40.41.5 0.4 1.5 2.50.4 0.41.5 0.4 0.4 0.4 0.4 B B 2.5 1.5 1.5 1.5 1.5 2.5 1.5 1.5 1.5 1.5 0.6 0.2 0.2 2.5 2.5 0.6 0.6 0.2 0.2 0.2 0.2 0 0.4 0.4 1.5 1.5 1.5 1.5 1.5 2.5 2.5 1.5 1.5 1.5 0.2 0.2 0.2 0.2 0.4 0.4 0.2 0 0.4 0.4 0 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.2 0.2 0.4 0.4 0.40.4 0.4 0.4 0.4 0.4 0.4 0.4 0.40.4 0.4 0.4 0.4 0.4 0.4 0.4 0.40.4 0.4 0.40.40.40.40.40.40.40.40.4 0.4 0.4 0.4 0.4 0.40.4 0.4 0.40.40.40.4 0.4 0.4 0.6 GFP+/DAPI+ GFP+/DAPI+ 0.4 0.2 GFP+/DAPI+ GFP+/DAPI+ 0.6 0.2 GFP+/DAPI+ 0 0.4 0.6 GFP+/DAPI+ GFP+/DAPI+ 0.4 0.4 0.2 GFP+/DAPI+ 0.4 0.4 GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ * * * 0.2 0.2 GFP+/DAPI+ 0 0.4 0 0.4 * * * 0.2 GFP+/DAPI+ 0.4 0.4 GFP+/DAPI+ 0.4 0.4 0.2 0.2 0.2 GFP+/DAPI+ * * * * * * * * * * * * GFP+/DAPI+ 0.2 GFP+/DAPI+ * * * * * * GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ Proportion of 0.2 0.2 * * * * * * * * * * * * GFP+/DAPI+ FLAG FLAGProportion of FLAG FLAG 0.2 0.2 GFP+/DAPI+

Proportion of 0.2 GFP+/DAPI+ GFP+/DAPI+

FLAGProportion of FLAG FLAG FLAG 0.2 Proportion of GFP+/DAPI+ GFP+/DAPI+

FLAG FLAG FLAG FLAG Proportion of FLAG FLAG FLAG FLAGFLAG FLAGProportion of FLAG FLAG GFP+/DAPI+

0.2 Proportion of GFP+/DAPI+ GFP+/DAPI+

Proportion of 0.2 FLAG Proportion of 0.2FLAG FLAG FLAGFLAG FLAG FLAG FLAG GFP+/DAPI+ Mouse FLAG FLAG 0.2FLAG FLAGFLAG FLAGFLAG 0.2FLAGFLAGFLAGProportion of FLAG FLAG GFP+/DAPI+

Proportion of 0.2 GFP+/DAPI+ GFP+/DAPI+

0.2 FLAGProportion of FLAG FLAG FLAG α Mouse 0.2 0.2 Proportion of GFP+/DAPI+ GFP+/DAPI+

FLAG FLAG FLAG FLAG Proportion of Mouse FLAG FLAG FLAG FLAGFLAG FLAGProportion of FLAG FLAG Proportion of Mouse α Mouse 0.2 GFP+/DAPI+ GFP+/DAPI+

Proportion of 0.2 FLAG Proportion of 0.2FLAG FLAG FLAGFLAG FLAG FLAG FLAG Proportion of Proportion of α α Mouse α Mouse Mouse FLAG FLAG 0.2 FLAG FLAGFLAG FLAG 0.2 FLAG FLAG Proportion of Proportion of FLAG FLAG FLAGMouse FLAG Mouse Proportion of α Proportion of α Mouse 0.2 0.2 Proportion of α

FLAGProportion of FLAG FLAG FLAG Proportion of Proportion of α Proportion of α Mouse Proportion of Mouse α FLAG FLAG FLAG FLAG Proportion of Mouse Proportion of Proportion of Proportion of FLAG FLAG FLAG FLAGFLAG FLAG FLAG FLAG Proportion of Proportion of α Mouse Proportion of Mouse FLAG Proportion of FLAG FLAG FLAGFLAG FLAG αFLAG FLAG α Proportion of Proportion of FLAG FLAG FLAGMouse FLAG Mouse Proportion of α Mouse FLAG FLAG FLAG FLAGFLAG Proportion of FLAG FLAG FLAG α Proportion of

FLAGProportion of FLAG FLAG FLAG Proportion of Proportion of α α Mouse Proportion of α

FLAG FLAG FLAG FLAG Proportion of Proportion of

Proportion of 3.0 3.0 3.0 3.0 Mouse Proportion of FLAG FLAG FLAG FLAGFLAG FLAG FLAG FLAG Proportion of Mouse α Mouse Mock WTProportion of R495G 1.5 FLAG Proportion of FLAG FLAG FLAGFLAG FLAG FLAG FLAG Proportion of α α Mouse α Mouse 1.5 FLAG FLAG FLAG FLAGFLAG FLAG FLAG FLAG Mouse 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Proportion of Mouse Mouse Proportion of α Proportion of α 1.5 Mock WT R495GMock 1.5 WT R495G α Mouse 1.5

Proportion of α Proportion of α Mouse 1.5 oc 1.5 Proportion of Mouse α Mouse Proportion of Proportion of WT R495G 1.5 N461S 1.5 oc 1.5 Proportion of α α Mouse α Mouse ococ WT R495G N461S 1.5 Proportion of Mouse ocMouse Proportion of α oc Proportion of α WTWT R495G R495G N461SN461S 1.5 1.5 oc WT R495G N461S WT R495G 1.5 N461S Proportion of α oc Proportion of α ococ WT R495G N461S WT R495G 1.5 N461S 1.5 Proportion of ococ Proportion of 3.0 3.0 3.0 WT3.0WT R495G R495G N461SN461S 1.5 WTWT R495G R495G N461SN461S 1.5 1.5 Mock WT R495G 1.5 3.0 3.0 3.0 3.0 1.0 1.5 1.0 1.0 1.0 Mock 3.0 3.0 3.0 3.0 1.5 1.5 WT 0.4 R495GMock WT R495G 1.0 1.0 1.0 1.0 1.5 oc 1.5 0.4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 WT R495G 1.0 1.5 1.0 N461S1.0 1.5 oc 1.5 0.4 0.4 0.4 1.5 0.4 ococ1.0 WT 1.0 R495G 1.0 N461S1.0 1.0oc 1.0 1.0 1.0 oc 1.0 1.0 1.0 2.0 1.0 0.4 1.5 0.4 1.0 1.0 WT1.0 WT R495G 1.0 R495G 1.0 N461SN461S1.0 1.0 1.0 3 3 3 3 oc WT R495G 1.5 ocN461S WT R495G 1.5 N461S 2.0 1.0 1.0 0.4 1.0 0.4 1.0 0.4 0.4 ococ WT R495G oc ocN461S WT R495G 1.5 N461S 1.5 3 3 WT1.0 WT 2.0 3R495G 2.0 R495G 1.0 3N461S1.0 N461S1.0 31.0 1.5 WTWT1.0 3R495G R495G 1.0 2.0 3N461S1.0 N461S1.0 1.5 1.0 1.0 2.0 1.0 1.0 1.0 1.0 1.0 1.0 2.0 2.0 1.02.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.4 1.0 2.0 2.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.4 0.4 0.4 0.4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

FLAG+GFP+/GFP+ 2.0 1.0 1.0 1.0 1.0 0.4 0.4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

FLAG+GFP+/GFP+ 3 3 3 3 2.0 1.0 1.0 0.4 1.0 0.4 1.0 0.4 0.4

FLAG+GFP+/GFP+ 1.0 2.0 2.0 1.0 1.0 1.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 1.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0 2.0 1.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 3 3 3 3 2.0 3 3 3 1.0 3 1.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 1.0 2.0 2.0 1.0 1.0 1.0 1.0 2.0 2.0 1.0 1.0 1.0

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

FLAG+GFP+/GFP+ 0.6 FLAG+GFP+/GFP+ FLAG FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

FLAG FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 FLAG+GFP+/GFP+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG+GFP+/GFP+ 0.6 0.6

FLAG FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2

FLAG FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG FLAG FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 FLAG FLAG FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG+GFP+/GFP+ 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAGSignal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) 0.6 FLAG FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG 0.6 Signal intensity (arbitrary units) 0.6 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

0.2 0.2 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Mouse Mouse Mouse Mouse FLAG GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+

FLAG FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

0.2 0.2 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ α α Mouse α αMouse Mouse Mouse GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+

FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) 3.0 FLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

0.2 0.2 0.2 0.2 0.2 0.2 0.2 GFP+/DAPI+ 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Mouse Mouse Mouse Mouse α Mouse Mouse α Mouse α Mouse α Mouse Mouse Mouse Mouse FLAG FLAG 0.2 0.2 0.2GFP+/DAPI+ GFP+/DAPI+ 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ 0.2GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ 0.2 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) α α α α α α Mouse α αMouse Mouse Mouse Mouse Mouse Mouse Mouse 0.2 0.2 0.2 0.2 0.2 Signal intensity (arbitrary units) 0.2 Signal intensity (arbitrary units) 0.2 Signal intensity (arbitrary units) 0.2 Signal intensity (arbitrary units)

α α α α Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Mouse α Mouse αMouse α Mouse α Mouse αα α Mouse Mouse α ααMouse Mouse α Mouse α αMouse Mouse ααMouse α ααMouse α Mouse 3.0 Mouse 3.0 0.2 0.2 0.2 0.2 0.2 0.2Signal intensity (arbitrary units) 0.2 0.20.2 0.2Signal intensity (arbitrary units) 0.2Signal intensity 0.2(arbitrary units) Signal intensity 0.2(arbitrary units) 0.2 0.2 0.2

Mouse Mouse Mouse α Mouse α α α GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Mouse Mouse Mouse Mouse Mouse Mouse Mouse Proportion of Mouse Proportion of Proportion of Proportion of

α α α α GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ α α Mouse α αMouse α Mouse Mouse αProportion of Mouse α Proportion of αMouse Proportion of Mouse Proportion of Mouse GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+

0.2 0.2 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Mouse Mouse Mouse Mouse Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of α Mouse α Mouse α αMouse Mouse GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+

α α α Proportion of α Proportion of Proportion of Proportion of Mouse Mouse Mouse Mouse Proportion of Proportion of Proportion of Proportion of

0.2 0.2 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ α α α α GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+

α α α α Proportion of Proportion of Proportion of Proportion of α α Mouse α αMouse Mouse Mouse Proportion of Proportion of Proportion of Proportion of 3.0

0.2 0.2 0.2 0.2 0.2 0.2 0.2 GFP+/DAPI+ 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ 0.2 0.2 0.2GFP+/DAPI+ GFP+/DAPI+ 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 0.2 0.2 0.2 0.2 Mouse Mouse Mouse Mouse α Mouse Mouse α Mouse α Mouse α Mouse Mouse Mouse Mouse 0.2 0.2 Proportion of Proportion of 0.2 0.2Proportion of Proportion0.2 of Proportion of Proportion of 0.2Proportion of Proportion of 0.2 0.2 α α α α α α Mouse 1.0 α αMouse α Mouse Mouse α Mouse α αMouse Mouse Mouse 3.0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 3.0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Mouse Mouse Mouse α Mouse Mouse α 1.0 Mouse Mouse α αMouse Mouse α Mouse 2.5 Mouse αMouse α2.5 α 2.5 2.5 1.0 α α Mouse α αMouse Mouse Mouse 1.0 1.0 α α α α α Mouse α αMouse Mouse α Mouse Proportion of Mouse Proportion of Proportion of Proportion of 2.5 2.5 2.5 2.5

α α α α Proportion of Proportion of Proportion of Proportion of 0.0 2.5 2.5 2.5 2.5 1.0 Mouse Mouse Mouse Mouse 1.0 Mouse Mouse Mouse Mouse 0.0 Proportion of Proportion of Proportion of Proportion of Mouse 1.0

0.0 α α α α Proportion of Proportion of Proportion of Proportion of 1.0 α Mouse Mouse Mouse Mouse 1.0 Mouse Mouse Mouse Mouse 0.0 Proportion of Proportion of Proportion of Proportion of αProportion of Proportion of Proportion of Proportionα of α α α α α α Proportion of Proportion of Proportion of Proportion of Mouse Mouse 1.0 Proportion of Proportion of Proportion of Proportion of 0.0 α Mouse Mouse Mouse Mouse Mouse Mouse Proportion of Proportion of Mouse Proportion of Mouse Proportion of Mouse Proportion of Proportion of Proportion of Proportion of

0.0 α α Mouse α α Proportion of Proportion of Proportion of Proportion of 0.0 0.0 α α α α Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 0.0 0.0 Proportion of Proportion of Proportion of Proportion of 3 1.0 α α Mouse 1.0 1.0 0.0 α αα α α Mouse Proportion of Proportion of Proportion of Proportion of 0.0 α αα α α Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 0.0 0.0 0.0 Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Mouse Mouse 1.0 1.0 0.0 α Mouse Mouse Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 0.0 0.0 0.0 0.0 0.0 α Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 3 1.0 0.0 α α 3 α α 1.0 0.0 1.0 0.0 0.0 0.0 0.0 1.0 0.0 2.5 2.5 0.0 2.5 2.5 0.5 0.5 0.5 0.5 0.0 1.0 0.0 0.0 1.0 Proportion of 0.0 1.0 Proportion of Proportion of Proportion of 0.0 0.0 0.5 0.00.5 Mouse 0.5 0.5 0.0 1.0 0.0 Proportion of Proportion of Proportion of 2.5 Proportion of 1.0 2.5 2.52.5 2.5 2.5 2.5 2.5 0.5 0.5 0.5 0.5 1.0 0.5 Mouse 0.5 0.5 0.5 0.0 α 0.5 0.5 0.5 0.5 1.0

Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 1.0 Proportion of Proportion of

0.0 Proportion of Proportion of Proportion of Proportion of Mouse Mouse 0.5 0.5 0.5 0.5 1.0 Proportion of Proportion of Proportion of Proportion of 0.0 0.0 α Mouse 0.5 0.5 Mouse 0.5 0.5

0.0 0.0 Proportion of Proportion of Proportion of Proportion of 0.0 0.0 0.5 0.5 0.5 1.5 0.5 Proportion of Proportion of Proportion of Proportion of 3 0.5 0.5 0.5 0.5 0.5 0.5 1.0 0.5 0.5 α α 1.0 0.0 Mouse α Mouse α 1.0 0.0 Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of Proportion of 0.0 0.0 0.0 1.5 0.5 Proportion of Proportion of 0.5 Proportion of Proportion of 0.5 Proportion of Proportion of 0.5 Proportion of Proportion of 0.0 0.0 0.0 Mouse Mouse α Mouse Mouse α 1.0 1.0 0.0 0.0 0.2 0.5 1.5 1.5 0.5 0.5 0.5 0.0 0.5 0.5 0.5 1.5 0.5 0.5 0.5 0.5 1.5 0.5 0.0 0.2 3 α α 3 α α 1.0 1.0 0.4 0.0 0.0 0.0 0.0 0.2 0.2 0.0 0.0 1.5 0.5 0.5 0.5 0.5 1.5 0.5 0.5 0.5 0.5 0.2 0.5 1.5 1.5 0.5 0.5 0.5 E 0.0 0.2 0.5 1.5 1.5 0.5 0.5 0.5 0.4 0.5 0.5 0.00.5 0.5 0.0 0.0 0.2 0.20.0 0.4 E 0.0 0.5 0.00.5 0.5 0.5 0.2 0.2 0.0 0.2 0.2 E 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 T 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1.5 0.5 S T T 1.50.5 0.5 0.5 0.5 S S 0.2 0.5 1.5 1.5 0.5 0.5 0.5 1.5 T S T T T S Signal intensity (arbitrary units) 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1.5 0.5 1.5

S Signal intensity (arbitrary units) 0.5 0.5 0.5 0.5 0.4 T T S S T T T 0.2 0.2 1.5 0.5 0.5 0.5 0.5 1.5 1.5

Signal intensity (arbitrary units) 0.2 S S 0.5 0.5 0.5 0.5 S S 0.2 0.5 1.5 1.5 0.5 0.5 0.5

T Signal intensity (arbitrary units) E Signal intensity (arbitrary units) T S T S S T S T T S 0.2 0.2

T Signal intensity (arbitrary units) 0.4 0.4 T S T S Signal intensity (arbitrary units) S S T Signal intensity (arbitrary units) 0.2 0.2 0.2 0.2

T Signal intensity (arbitrary units) E T E T Signal intensity (arbitrary units) S S S T T S S T T T Signal intensity (arbitrary units)

S S S Signal intensity (arbitrary units) S Signal intensity (arbitrary units) T T S T S T S T S Signal intensity (arbitrary units)

Signal intensity (arbitrary units) S T T T S T S Signal intensity (arbitrary units)

Signal intensity (arbitrary units) S S Signal intensity (arbitrary units) T β-ACTIN β-ACTINβ-ACTINβ-ACTIN T Signal intensity (arbitrary units) S T T S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ S T β-ACTINS β-ACTINβ-ACTINβ-ACTIN Signal intensity (arbitrary units) T S T T T FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

S S S Signal intensity (arbitrary units) S GFP+/DAPI+

T Signal intensity (arbitrary units)

β-ACTIN β-ACTINβ-ACTINβ-ACTIN Signal intensity (arbitrary units) T β-ACTINT β-ACTINβ-ACTINβ-ACTINβ-ACTINT β-ACTINβ-ACTINβ-ACTIN T FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ S S S S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ T GFP+/DAPI+

T Signal intensity (arbitrary units) T β-ACTIN β-ACTINβ-ACTINβ-ACTINT β-ACTIN β-ACTINβ-ACTINβ-ACTIN Signal intensity (arbitrary units) S S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

S S Signal intensity (arbitrary units) T GFP+/DAPI+ GFP+/DAPI+ T β-ACTIN β-ACTINSignal intensity β-ACTIN(arbitrary units) β-ACTIN Signal intensity (arbitrary units)

0.2 GFP+/DAPI+ β-ACTIN β-ACTINβ-ACTINβ-ACTINβ-ACTINT β-ACTINβ-ACTINβ-ACTINT GFP+/DAPI+ Mouse S S FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ S T β-ACTINS β-ACTINβ-ACTINβ-ACTIN Signal intensity (arbitrary units) T T 0.2 GFP+/DAPI+ α Mouse S T T GFP+/DAPI+ S S S Signal intensity (arbitrary units) S Signal intensity (arbitrary units)

β-ACTIN β-ACTINβ-ACTINβ-ACTIN Signal intensity (arbitrary units) T T 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ Mouse Mouse α Mouse S β-ACTINT S β-ACTINβ-ACTINβ-ACTINβ-ACTIN β-ACTINS β-ACTINβ-ACTIN T S 0.2 GFP+/DAPI+ GFP+/DAPI+ 0.2 Signal intensity (arbitrary units) α α Mouse Mouse T β-ACTINS β-ACTINβ-ACTINβ-ACTINT β-ACTINS β-ACTINβ-ACTIN0.2β-ACTIN 0.2 Signal intensity (arbitrary units)

α Signal intensity (arbitrary units) β-ACTIN β-ACTINβ-ACTINβ-ACTINMouse Mouse β-ACTIN β-ACTINβ-ACTINβ-ACTINβ-ACTIN β-ACTINβ-ACTINβ-ACTIN 0.2 0.2 Signal intensity (arbitrary units) 0.2 0.2 β-ACTIN β-ACTINβ-ACTINβ-ACTINα Mouse α α Mouse FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ α α Mouse FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ Mouse α GFP+/DAPI+ β-ACTIN β-ACTINβ-ACTINβ-ACTIN Mouse Proportion of β-ACTIN β-ACTINβ-ACTINβ-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN β-ACTINβ-ACTINβ-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

α Mouse GFP+/DAPI+ β-ACTIN β-ACTINβ-ACTINβ-ACTIN α α Proportion of Mouse β-ACTIN β-ACTINβ-ACTINβ-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

Mouse GFP+/DAPI+ GFP+/DAPI+

β-ACTIN β-ACTINβ-ACTINβ-ACTIN 0.2 GFP+/DAPI+ Mouse Proportion of Proportion of α α GFP+/DAPI+ β-ACTIN β-ACTINβ-ACTINβ-ACTIN Proportion of FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN β-ACTINβ-ACTINβ-ACTIN Proportion of

Mouse FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

β-ACTIN β-ACTINβ-ACTINβ-ACTIN 0.2 GFP+/DAPI+ α α GFP+/DAPI+ Proportion of α Mouse Proportion of

β-ACTIN β-ACTINFLAG β-ACTINβ-ACTIN 0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ β-ACTIN β-ACTINβ-ACTINβ-ACTIN 0.2 GFP+/DAPI+ GFP+/DAPI+ β-ACTIN β-ACTINβ-ACTINβ-ACTINProportion of Proportion of 0.2 Mouse α ocMouse α Mouse αoc ocMouse Mouse ocα Mouse α Mouse Mouse α FLAGFLAGMouse FLAG β-ACTIN FLAGβ-ACTINFLAGβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINFLAGβ-ACTINβ-ACTINFLAGβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTIN0.2 0.2 0.2β-ACTIN Proportion of Proportion of 0.2 0.2 0.2 WT R495G WT N461SWTR495G WTR495G N461Sα R495G 2.5 N461SN461S α α Mouse Proportion of α FLAGFLAG FLAGFLAG Mouse Mouse Proportion of oc oc oc ocα ocProportion of oc oc

Mouse Proportion of 2.5 Mouse α 2.5 WT R495G WT N461SWTR495G Proportion of Proportion of WTR495G N461SWTR495G N461SWTR495G N461SWTR495G N461Sα R495G N461SN461S Proportion of Proportion of

Mouse α Mouse Proportion of Proportion of

α Proportion of Proportion of Proportion of α Proportion of

α Proportion of Proportion of Proportion of Proportion of FLAG Proportion of Proportion of Proportion of Proportion of oc oc oc oc FLAGFLAGFLAG FLAG FLAG Proportion of Proportion of 0.5 FLAG FLAG 2.5 R495G WT N461S R495G WT R495G WT N461SWTR495G WTR495G N461SR495G N461SProportion of N461S WT N461S 0.5 FLAGFLAG FLAGFLAG

R495G WT N461S R495G Proportion of WT N461S 0.4 oc0.5 oc oc ococ oc oc 0.5 2.0 2.0 0.5 2.0 2.0

Proportion of Proportion of 2.5 2.5 R495G Proportion of WT N461S R495G R495G WT N461S WT N461S R495G WT R495GR495G WT N461SWTR495G WTR495G N461SWTR495G N461SWTR495G N461S WTR495G N461SR495G N461SProportion of N461S WT N461S R495G WT N461S 0.5 WT N461S 0.5 Proportion of R495G WT N461S R495G R495G 0.5 Proportion of WT N461S 0.4 R495G WT 0.5 N461S WT N461S 0.4 0.5 2.0 2.0 2.02.0 2.0 2.0 2.0 2.0

R495G Proportion of Proportion of

R495G WT N461S Proportion of Proportion of WT N461S R495G WT N461SWT R495GN461SWT N461S R495G R495GWT N461S R495G WT N461S R495G 0.5 WT N461S 0.5 WT N461S R495G R495G WT N461S R495GWT N461S R495G R495G0.5 0.5 WT N461S R495G WT N461S 0.5 WT N461SR495G WT N461S R495G R495G 0.5 WT N461S R495G WT 0.5 N461S WT N461S 0.5 R495G WT N461S R495G 0.5 WT N461S R495G WT N461S R495GWT N461S R495G WT N461S R495G WT N461S 0.5 R495G WT N461S R495G 0.0 0.0 0.0 0.0 WT N461S 0.4 0.5 0.5 2.0 2.0 0.5 2.0 2.0 WT N461S R495G R495G WT N461S R495G WT N461S R495G 0.0 R495G 0.0 0.0 0.0 WT N461S R495G WT N461S 0.5 WT N461S 0.5 R495G WT 0.0 N461S R495G0.0 0.0 0.0 0.0 0.0 R495G0.0 0.0 0.0 0.0 0.5 0.0 0.0 WT N461S R495G WT 0.5 N461S 0.4 WT N461S 0.4 0.5 2.0 2.0 2.0 2.0 R495G WT N461S R495G 2.0 2.0 2.0 2.0 WT N461S 0.0 0.0 0.0 0.0 0.5 WT N461S R495G WT N461S R495GWT N461S R495G WT N461S R495G 0.0 0.0 0.0 0.0 R495G WT N461S R495G 0.0 0.0 0.0 1.0 0.0 WT N461S 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.5 WT N461S R495G R495G WT N461S R495G WT N461S R495G 1.0 0.0 0.0 R495G 0.0 0.0 0.0 0.0 0.0 0.0 WT N461S R495G WT N461S 0.5 WT N461S0.0 0.0 0.0 0.0 0.5 0.0 1.0 1.0 R495G0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 R495G WT N461S 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 R495G 1.0 WT N461S R495G WT 0.5 N461S 0.0 0.0 0.0 0.0 WT 0.0 0.0N461S0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 R495G 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 WT N461S R495G WT N461S R495GWT N461S 0.0 0.0 WT0.0 N461S0.0 0.0 R495G 0.0 0.00.0 0.0 0.0 0.0 0.01.0 0.0 0.0 0.0 0.0 0.0 1.0 0.01.0 0.00.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.01.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 β-ACTIN 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 β-ACTIN FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 β-ACTIN FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.00.00.01.0 0.0 0.0 0.0 0.00.00.0 0.0 0.0 0.00.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 1.0 0.0 0.0 0.0 0.0 0.0 β-ACTIN β-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 1.0 0.0 0.0 0.0 0.0 Supplementaryβ-ACTIN Figure β-ACTIN 0.0 0.0 0.0 FLAG+GFP+/GFP+ 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.01.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 1.0 1.0 0.0 0.0 0.0 0.0 β-ACTINSupplementary Figureβ-ACTIN β-ACTIN 0.0 0.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ 0.0 0.00.0 0.0 0.0 0.0 0.00.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.01.0 0.01.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN SupplementarySupplementary Figure FigureSupplementary0 SupplementarySupplementarySupplementary0 0 β-ACTIN00 Figure0 Figure00 FigureSupplementary0 FigureSupplementaryβ-ACTIN0Supplementary0Supplementary β-ACTIN00 β-ACTIN00 Figure0 Figure0 Figure0 Figureβ-ACTIN0 β-ACTIN00 β-ACTIN00 0 0 β-ACTIN0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 0 0 β-ACTIN 0 0 FLAG+GFP+/GFP+ 0 0 0 00 0 0 0 0 0 β-ACTIN FLAG+GFP+/GFP+ 0 0 0 0 β-ACTIN β-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ 0 0 0 0 Supplementaryβ-ACTIN Figure β-ACTIN T S T ST T S S 0 FLAG+GFP+/GFP+ 0 0 0 0 0 0 0 FLAG+GFP+/GFP+ β-ACTINSupplementary Figureβ-ACTIN β-ACTIN T S T ST T S S FLAG+GFP+/GFP+ T S T TS 0 T S 0 S 0 0 0 0 0 0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ SupplementarySupplementary Figure Figure0 0 0 0 β-ACTIN T T S S T T ST ST T S T S S S T T FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ T T T0S T T T0S T T 0S T 0S T0 0 0 0 Supplementary FigureSupplementary Figure S S S S S S S S Supplementaryβ-ACTIN0 FigureSupplementary0 β-ACTIN0 0 Figure β-ACTIN T S T T T ST S T STS TT S TS STS TT ST S ST S S T T S S S T TS T S T S S T TS T S S 0 0 0 0 β-ACTIN T T T T T T T T T T T T T T TT T T T T T T T T SupplementarySupplementary Figure FigureSupplementary0 Supplementary 0 0 Figure Figure0 0 0 0 S 0 S Signal intensity β-ACTIN(arbitrary units) S S S S S S S S S S S S SS S S S S S S S S β-ACTIN T T T T T T T T T T T T T T T T 0 0 0 0β-ACTIN0 0 Signal0 intensity (arbitrary units) 0 0 00 0 S S S S S S S S S S S S S S S S

0 0 0 0 0 0 Signal intensity (arbitrary units) Signal0 intensity (arbitrary units) 0 Signal intensity (arbitrary units) Time (hrs) 0 4 8 oc 0 4 8 0 4 8 0 4 8 0 0 0 0 Signal intensity (arbitrary units) Signal intensity (arbitrary units) WT R495G N461S T T T T0 0 0 0 0 0 0 0 0 Signal0 intensity (arbitrary units) 0 0 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Time (hrs) 0 4 8 ocTime (hrs) 0 4 8 oc 0 4 8 0 4 8 0 4 8T 0 4 8 0 4 8ST T S T S S0 4 8 T T T T Signal intensity (arbitrary units) Signal intensity (arbitrary units) WT R495G N461SWT R495G N461ST T S S T S T ST ST S T S T TS ST S T S T S T S T ST TT T T T ST T ST T S T T STST T0STTS ST T S TS TS STS T T0SSTT SS0TT STT 0SST STT SSTS T 0S0T SS T0 S ST 0S T S 0T0 TS 0 T 0T T S T 00 S ST 0 T 0T TS T T S S S S Signal intensity (arbitrary units) S S S S S S S S S T S T S S T SST S T T S T T T TS S T ST T T S T T S S

Signal intensity (arbitrary units) S S S S S S S S S S S S S S S S Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Time (hrs) 0 4 8 oc 0 4 8 0 4 8 0 4 8 Signal intensity (arbitrary units) Signal intensity (arbitrary units) WT R495G N461S Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Mouse Mouse Time (Mouse hrsMouse ) 0 4 8 ocTime (hrs) 0 4 8 oc 0 4 8Mouse 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8 Signal intensity (arbitrary units) Signal intensity (arbitrary units) α αMouse α Mouse α Mouse WTMouse R495G Mouse Mouse 2.0 Mouse N461SMouse WTMouse Mouse α αR495G N461SMouse Mouse α α Mock α WT MockαR495GMockWT α MockR495GWT α α WT αR495G R495G α 2.0 Mouse Mouse Mouse α α α α 2.0 Mouse Mouse Mouse α α α α 0.0 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 Mouse Mouse Mouse Mouse Mock 0.0 WT MockR495GMockWT Mock MockR495GWTMouse Mock WT R495G R495G Mock Mock 4 WT R495GWT R495GWTMock WT R495GWT Mock R495G R495G MockWT MockR495GWT WT R495G R495G 0.0 2.0 Mouse 0.0 α Mock Mock Mock Mock 4 α α α α WT R495GWT R495GWT WT R495G R495G 0.0 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 4 Mouse Mouse Mock WT MockR495GMockWT MockR495GWT WT R495G R495G Mouse Mouse Mouse Mouse Mockα 0.0 WT MockR495GMockWT MockR495GWT WT R495G R495G Mouse Mouse Mouse Mouse Mouse Mock 0.0 WT MockR495GMockWT MockR495GWTMouse WT R495G R495G 4 4 1.5 1.5 1.5 0.0 1.5 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 0.0 α α 2.0 α α α α 0.0 Mouse α 2.0 0.0Mouse 4 α 4 α α 0.0α α Mock 0.0 WT MockR495GMockWT Mock MockR495GWT Mock WT R495G R495G Mock Mock 4 0.0 Mouse Mouse α Mouse 0.0 Mouse α WT R495GWT R495GWTMock WT R495GWT Mock R495G R495G MockWT MockR495GWT WT R495G R495G 1.5 0.0 1.5 1.51.5 0.0 1.5 1.5 1.5 1.5 Mock Mock Mock Mock 4 WT R495GWT R495GWT WT R495G R495G 0.0 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 α α 0.0 0.0 0.0 0.0 0.04 α α 0.0 0.0 0.0 Mock 0.0 WT MockR495GMockWT MockR495GWT WT R495G R495G Mock 0.0 WT MockR495GMockWT MockR495GWT WT R495G R495G Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 0.0 4 0.0 0.0 0.0 0.2 0.0 Mock WT MockR495GMockWT 4 MockR495GWT WT R495G R495G 4 0.0 0.0 0.0 0.0 0.0 0.0 4 0.0 0.0 0.0 0.0 Mock 0.0 WT MockR495GMockWT Mock MockR495GWT Mock WT R495G R495G Mock Mock 4 WT R495GWT R495GWTMock WT R495G Mock R495G Mock Mock 2.5 WT R495GWT R495GWT WT R495G R495G 0.0 0.0 Mock Mock Mock Mock 0.2 4 WT R495GWT R495GWT WT R495G R495G 0.0 Mock Mock Mock Mock 0.2 2.5 WT R495GWT R495GWT WT R495G R495G 4 4 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G Mock 0.0 WT MockR495GMockWT MockR495GWT WT R495G R495G Mock 0.0 WT MockR495GMockWT MockR495GWT WT R495G R495G 2.5 2.5 4 2.5 4 R495G R495G R495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G 1.5 WT1.5 N461SWT 1.5 N461SWT0.0 1.5 WTN461S N461S Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 0.0 0.0 0.0 4 2.5 4 2.5 0.0 R495G R495G R495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495G WT N461SWT N461SWT WTN461S N461S Mock 0.0 WT MockR495GMockWT Mock MockR495GWT Mock WT R495G R495G Mock Mock 4 WT R495GWT R495GWTMock WT R495G Mock R495G Mock Mock 2.5 WT R495GWT R495GWT WT R495G R495G 2.5 2.5 0.0 0.0 R495GR495G R495GR495GR495GR495GR495GR495G R495GR495G R495GR495GR495GR495GR495GR495GWT0.5 WT N461SN461SWTWT N461SWTN461SWT WTN461SWTN461SN461SN461S WTWT N461SN461SWTWT N461SWTN461SWT WTN461SWTN461SN461SN461S0.0 R495G R495G R495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495GR495G R495G R495G R495G 0.0 Mock Mock Mock Mock 4 1.5 WT1.5 N461SWT 1.5 N461SWT 1.5 WTN461S N461S R495G R495GWT R495GR495GR495GWT R495GWTWT WT N461SR495GWT R495G N461S WT WTN461S N461S 1.5 WT1.5 N461SWT 1.5 N461SWT0.0 1.5 WTN461S N461S Mock Mock Mock Mock 2.5 0.0 0.0 WT R495GWT R495GWT WT R495G R495G 0.0 0.0 4 4 0.0 0.0 0.0 0.5 R495G 0.0R495G R495G R495G WT N461SWT R495GN461SWT WTN461SR495GN461SR495G R495GR495G R495G R495G R495G WT N461SWT N461SWT WTN461S N461S Mock R495G0.0 WT MockR495GR495GMockWT R495GMockR495GWT R495G WT R495GWT R495G N461S WT N461SWT WTN461S N461S WT N461SWT N461SWT WTN461S N461S Mock 0.0 WT MockR495GMockWT MockR495GWT WT R495G R495G 2.5 2.5 4 2.5 4 0.0 0.0 0.0 0.5 0.5 R495GR495G R495GR495GR495GR495GR495GR495G R495GR495G R495G0.0R495GR495GR495GR495GR495GWT0.5 WT N461SN461SWTWT N461SWTN461SWT WTN461SWTN461SN461SN461S R495GR495G R495GR495GR495GR495GR495GR495G WTWT N461SN461SWTWT N461SWTN461SWT WTN461SWTN461SN461SN461S0.0 R495GR495G R495GR495GR495GR495GR495GR495GWT0.5 WT N461SN461SWTWT N461SWTN461SWT WTN461SWTN461SN461SN461S WTWT N461SN461SWTWT N461SWTN461SWT WTN461SWTN461SN461SN461S0.0 0.2 4 0.02.5 0.0 4 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0 2.5 2.5 0.5 0.2 0.2 0 0 0 0 0 0 WTWT0 N461SN461SWT0WTR495GN461S0R495GWTN461SWT WTN461SR495GWTN461SR495GN461SR495GN461SR495GR495GR495GWTWT N461SN461SWT0.5 WT0.5R495GN461SR495GWTN461SWT WTN461SR495GWTN461SR495GN461SR495GN461SR495GR495G0.5 R495G0.5 WTWT N461SN461SWTWTR495GN461SR495GWTN461SWT WTN461SR495GWTN461SR495GN461SR495GN461SWTR495G0R495GR495GN461SWTWT0.5 WTR495GN461SWTN461SN461SWTWTN461SWTR495GR495GWTN461SR495GWTN461SN461SWTR495GN461SWTN461SR495GWTR495GN461SR495GN461SR495GN461SR495GWTWTR495GR495GWTN461SR495GN461SWTN461SR495GR495GN461SWTR495GWTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWT WTN461SR495GN461SR495G R495G T 0 WT N461SWT0.50 R495GN461SWT WTN461SR495GWTN461SR495GN461SWTR495G0 R495GN461SWTWT WTN461SR495GN461SWTN461SR495GR495GN461SWTR495GWTN461SR495GWTN461SR495GN461SWTR495GR495GN461SWT WTN461SR495GN461SR495G R495G 0.5 0.5 R495GR495G R495GR495GR495GR495GR495GR495G S 0.5 2.5 T WTWT N461SN461SWTWTR495GN461SR495GWTTN461SWT WTN461SR495GWTSN461SR495GWTWTN461SR495GN461SN461SR495GN461S0WTR495GWTR495GR495GN461SR495GWTWTN461SWTWTWTN461SR495GN461SWTN461SR495GN461SWTN461SWTR495GN461SR495GN461SWT0N461SR495GWTR495GWTN461SWTN461SWT0.5 WTN461SN461SN461SN461SWTWTR495GN461SR495GWTN461SWT WTN461SR495GWTN461SR495GN461SR495GN461SR495GR495GR495G S 0.5 2.5 0 T T T T 0 T 0 0.5 S S S 0.5 0.5 2.5 2.5 2.5 0 T S S T T S S T S S T S 0.5 0.5 T S GFP+/DAPI+ 2.5 2.5 0 2.5 2.5 2.5 0 0 0 T T 0 S S T T S S T T T S S S TT T S S S

0.2 GFP+/DAPI+ 2.5 0.2 GFP+/DAPI+ 0.2 2.5 0 2.5 2.5 T S0 2.5 0 2.5 0 0 2.5 0 0 T T T S S S T T T 2.5 T S S T T T TS S 0S T S T S S 0 T T 0 S 0 T S GFP+/DAPI+ T T T Proportion of FLAG FLAG FLAG FLAG β-ACTINβ-ACTIN S S S S T # # # T S S S T T S S S

0.2 GFP+/DAPI+ GFP+/DAPI+ Proportion of Mouse F α F FLAG0.2 FLAGProportion of FLAG0.2 FLAGFLAGβ-ACTINβ-ACTINFLAG FLAG β-ACTINβ-ACTINβ-ACTINβ-ACTIN β-ACTINβ-ACTINβ-ACTINβ-ACTIN # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # #

Proportion of F Mouse F Mouse F Proportion of FLAG FLAGα FLAGF FLAG α F β-ACTINβ-ACTIN

Proportion of F Proportion of F F

Proportion of β-ACTINβ-ACTIN FLAG FLAGMockProportion of MockWTFLAGR495GFLAG F Mock FLAGWT F R495GFLAG F FLAG β-ACTINβ-ACTINF β-ACTINβ-ACTINF β-ACTINβ-ACTIN 3.0 WT R495G Mock WT R495G F Mock WT R495G β-ACTINβ-ACTINF Mouse F 3.0 Mock WT R495G MockF MockWT WTR495G R495G 3.0 Mock 3.0 WT R495G Mock α F 3.0 WT R495G Mock WT R495G 3.0 3.0 Proportion of F Mouse F Mouse F Mock 3.0 1.5 WT R495G Mock WT R495G 3.0 Mock WT R495G α F 3.0 α F F 3.0 Mock WT R495G Mock WT R495G Proportion of Proportion of F F 3.0 Mock 3.0 1.5 WT R495G Mock Mock WT R495G F 3.0 1.5 WT R495G F Mock WT R495G F 3.0 F 3.0 Mock 3.0 F 3.0 3.0 WT R495G Mock WT R495G F Mock WT R495G F 3.0 Mock WT R495G MockF MockWT WTR495G R495G 3.0 Mock 3.0 WT R495G Mock 3.0 WT N461S R495G WT R495G WTMockN461SWT R495GR495G 3.0 3.0 R495G Mock WT N461S R495G 3.0 WT N461S WT R495G 1.5 Mock WT R495G 3.0 Mock WT R495G 3.0 R495GR495G WTWT N461SN461S R495GR495G WT3.0 WT N461SN461S WT N461S R495G WT N461S R495GWTMockN461SWT R495GR495G WTMockN461SWT R495GR495G 3.0 1.0 1.0 1.0 1.0 R495G Mock WT N461S R495G 3.0 WT N461S R495GWT R495G Mock WT N461S R495G 3.0 WT R495G 1.5 WT N461S R495GR495G1.5 WTWT N461SN461S R495GR495G 3 WT3.0 WT N461SN461S R495GR495G WTWT N461SN461S R495GR495G WT3.0 WT N461SN461S 3 3.0 3 3 3 3 3 3 3 3 3 0.8 0.8 0.83 0.8 3 WTWTWTN461SWTN461SN461S0.8N461SR495G1.00.8R495G0.83R495G0.8R495G3 1.0 WT 3.0 0.8N461S1.01.00.8 0.83WTR495G0.8WTR495GWT1.0 N461SWTN461SN461S1.0 N461SR495GWTR495GR495GR495GN461S1.0 R495GWT1.0 WT N461SN461S R495GR495G WT N461S R495GR495G 1.0 1.0 1.0 1.0 WT N461S WT N461S R495G WT N461S 3 3 WTWT3N461SN461S R495GR495G0.0 WTWT N461SN461S R495GR495GWTWT N461SN461S R495GR495G WTWT N461SN461S R495GR495G 3 0.0 3 3 3 1.0 1.0 0.0 0.0 1.01.0 1.0 1.0 0.0 1.0 1.0 0.0 0.8 0.0 0.0 FLAG+GFP+/GFP+ 3 3 0.8 0.0 0.0 3 3 3 B 0.8 0.8 3 0.8 0.8 0.0 0.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ BBB 3 3 B B 0.8 0.8 0.83 3 B B 0.8 0.8 0.83 BB 3 BB 3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ B 0.0 0.0 0.0 0.0 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ B FLAG 0.6 * * * * BB B B Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity B(arbitrary units) Signal intensity (arbitrary units) B FLAG 0.6 0.6 0.6 FLAG 0.6 * * * * * * * * 0.6 * * * * BB * * * * BB 0 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAG 0.6 0.6 0.6 0.6* * * 0.6 0.6 0.6* * * * * * * * * * * * * Signal intensity * * * * 0 (arbitrary units) 0 0* * * * * * Signal* * * * intensity * * * * (arbitrary units) Signal intensity 0 (arbitrary units) 0* * * * 0 Signal intensity 0 * * * * (arbitrary units) * * * * 0 0* * * * 0 0 * * * * * * * * * * * * FLAG 0.6 0.6 0.6 FLAG 0.6 Signal intensity (arbitrary units) Signal intensity 0.6 (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) 0* * * * Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Mouse α Mouse αMouse Mouse α Mouse α αMouse Mouse α Mouse 0.6 α0.6 0.6* αMouse * Mouse α * 0.6 α0.6 0.6* * * * * Signal0 intensity 0(arbitrary units) 0* * * Signal intensity (arbitrary units) * Signal0 intensity 0(arbitrary units) 0* 0* Signal intensity (arbitrary units) * * 0 0 0* 0* * * 2.5 * * Mouse Mouse 2.5 Mouse Mouse 2.5 α 2.5 α α α 2.5 2.5 * 2.5 3 Mouse Mouse Mouse Mouse 2.5 Mouse Mouse Mouse 2.5 2.5 * 3 α α α * α1.0 α 2.5 α α 3 * 2.5 * 3 3 2.5 * 2.5 * 3 3 1.0 2.5 1.0 * 2.5 3 * * 0.0 3 3 2.5 0.0 2.5 * 2.5 3 * 2.5 3 * * 0.0 0.0 3 3 0.0 0.0 2.5 * 2.5 * 2.5 3 3 3.0 Mock 3.0 3.0 * 3.0 2.5 WT R495G * * 2.5 3 3 3.0 3.0 3.0 3.0 3 2.5 Mock 2.5 WT R495G 2.5 * 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3 1.0 2.5 MockMock WTWT R495GR495G Mock WT R495G Mock * 3.0 3.0 3.0 3.0 WT R495G 3 * 3.0 3.0 3.0 3.0 2.5 * 3 3 2.5 2.5 Mock WT R495G Mock 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 WT R495G 0.5 0.5 0.5 * 0.5 * 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3 3 1.0 2.5 1.0* 2.5 MockMock WTWT R495GR495G MockMock WTWT R495GR495G 3 * * 3 3 2.5 0.0 0.0 2.5 * 3 0.5 0.5 0.50.5 * 0.5 0.5 0.5 0.5 3 * * 0.0 0.0 0.0 0.0 3 3 * * 3 3 3.0 Mock WT3.0 R495G3.0 * 3.0 * 3 3 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock3.0 Mock WT3.0 WT3.0 R495G3.0R495G 3.0 3.0 3.0 3.0 Mock WT3.0 R495G3.0 3.0 3.0 Mock 3.0 3.0 3.0 3.0 3.0 3.0 3.0 WT R495G Mock WT R495G 3.0 Mock 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 WT R495G 0.5 0.5 0.5 0.5 MockMock WTWT R495GR495G 3.0 Mock3.0 Mock WT3.0 WT3.0 R495G3.0R495G 3.0 3.0 3.0 * * * * * * 0.5 0.5 3 30.50.5 3 0.5 3 30.5 3 3 3 0.5 0.5 T * * * * * * * * * 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 S T S * * * * * * 3 3 3 3 3 3 3 3 Signal intensity (arbitrary units) * * * T T * * * * * * 3 3 3 3 3 3 3 33 3 3 3 3 3 3 3 S S T S T S * * * Signal intensity (arbitrary units) * * * Signal intensity (arbitrary units) * * * * * * * * * * * * * * * * * * T S T S 3 3 33 3 3 3 33 3 33 33 3 333 3 3 3 33 333 3 3 3 33 3 T S β-ACTINT S β-ACTINβ-ACTINβ-ACTINT S T Signal intensity (arbitrary units) S 3 3 3 3 3 3 3 33 3 3 3 3 3 3 3 Signal intensity (arbitrary units) oc WT R495G ocN461S Mouse α β-ACTIN Mouse β-ACTINβ-ACTINα β-ACTINβ-ACTINβ-ACTINMouse β-ACTINα β-ACTINβ-ACTINβ-ACTIN0.6 0.6 0.6β-ACTIN0.6 0.6 0.6 0.6 0.6 Signal intensity (arbitrary units) 0.6 0.6 0.6 0.6 ocococWTWT R495G R495G ococN461SWTocN461SocWTWTR495G R495G WTR495G Mouse N461SocN461SR495G N461SocαWTocN461SocWTR495G WTocR495G Mouse N461SococR495G β-ACTINWTN461SαWT WTN461SR495G ocR495G R495G β-ACTINN461SocMouse WTN461SN461Socβ-ACTINWTR495G α WTR495G β-ACTINN461SocR495G β-ACTINN461SocWTN461Socβ-ACTINWTR495G 0.6 0.6 0.6WTR495G 0.6β-ACTINN461SocR495G N461SWTN461SR495G 0.6 0.6 0.6 0.6 N461S 0.6 0.6 0.6 0.6 WT R495G N461S 2.0 WT R495G ocN461S WT R495G ocN461S 3.0 2.0 oc oc WT ocR495G N461S WT R495G N461S 3.0 2.0 2.0 WT R495G ocN461S2.0 WT R495G N461S WT R495G N461S 3.0 3.0 oc 2.0 ocWT R495G N461S2.0 3.0 3.0 oc 3.0 2.0 WT R495G N461S2.0 WT 2.0 R495G ocN461S WT R495G N461S 3.0 oc 3.0 3.0 oc WT oc2.0 R495G N461S WT R495G N461S 3.0 3.0 R495G WT 2.0 R495G 0.5 N461S 2.0 WT R495G N461S 2.0 R495G WT N461S 2.0 WT N461S 2.0 WT N461S R495GWT N461S2.0 R495G WT N461S R495GWT N461S R495G 2.0 0.5 2.0 0.5 3.0 2.0 3.0 2.0 2.0 2.0 3.0 3.0 2.5 2.5 2.5 2.5 3.0 3.0 2.0 2.0 2.0 2.5 2.5 2.5 2.5 3.0 3.0 2.0 2.0 * * * 2.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3.0 3.0 2.5 2.5 2.5 3.0 3.0 2.5 2.5 2.5 2.5 2.5 2.0 0.5 * * * 2.0 2.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 WT N461S R495G WT N461S R495G * * * * * * 2.0 * * * 3 2.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 WT N461S R495GWT N461S R495G WT N461S R495GWT * N461S0.0 R495G* 0.0 2.0 * * * 0.5 0.0 0.0 * * * 32.0 3* * * 30.5 * * * * * * 3 3 0.0 0.0 0.0 0.0 * 0.0 0.0 3 * * * 0.0 0.0 3 * * 2.5 * * * 32.5 3 2.5 * * * 2.5 3 3 * * * * * 2.5 * 2.5 2.5 2.5 * * * * * * * * 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 * * * 2.5 2.5 2.5 * 2.5 * * * 2.5 2.5 2.5 * * 2.5 2.5 2.5 2.5 * 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3 * 2.5 2.5 2.5 2.5 2.5 2.5 2.5 * 2.5 * 0.0 0.0 0.0 0.0 3 * * 2.0 * 33 3 3 2.0 0.4 * * 0.0 0.0 0.00.0 0.0 0.0 3 0.0 0.0 3 2.0 E * 2.0 0.4 2.0 E * * 3 3 3 3 β-ACTIN 0.4 0.4 2.0 0.4 2.0 0.4 * 2.0 E E * * 2.0 2.0 0.4 0.4 E E * β-ACTIN 0.4 0.4 0.42.0 0.4 E E * * β-ACTIN 2.0 * 2.0 E E 2.0 E E * * 2.0 2.0 * * β-ACTIN 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0E E E E 00.4 0.4 2.0 0 E E 0 0 2.0 0 E E 0 0 0 0 Mouse Mouse Mouse α α α Mouse Mouse Mouse α Mouse α α Mouse Mouse α 2.0 Mouse α 2.0 α Mouse Mouse Mouse α 2.0 αβ-ACTIN α2.0 Mouse 2.0 * * Mouse α * * Mouse α 2.0 β-ACTINα2.0 * * Mouse 2.0 * * Mouse α 2.0 Mouse α 2.0 2.0 α Mouse 02.0 * * α 0.42.0 0.4 2.0 0 E E 02.0 2.0 0 2.0 0 E E 02.0 0 0 0 0 Mouse α 2 * * * * Mouse Mouse 2 * * * * α 1.5 * * oc oc oc oc α Mouse 2 Mouse * * α 2 Mouse α Mouse Mouse 1.5 2 oc ocWT * * ocR495G WTocN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S α 2 2 * * 1.5 * * α 1.5 Mouse 1.5 ococ ococWT ococR495G ocWTocN461SWTR495G WTR495G N461SR495G 2.5 N461SN461S α 2 oc oc oc oc α 2 oc oc oc oc 2 * * * * 1.5 1.5 WTWT R495G R495G WTWT N461SWTR495G N461SWTR495G WTR495G WTN461SR495G N461SR495G 2.5 R495G N461S2.5 N461SN461SN461S Mouse 2.5 α WT R495G WT N461SWTR495G WTR495G N461SR495G N461SN461S 2.5 Mouse Mouse 2 oc oc * * oc ococ ocWT ocR495G WTocN461SWTR495G WTR495G N461SR495G N461SN461S 1.5 * * α 1.5 1.5 2.5 2.5 α Mouse 2 Mouse ococ ococWT ococR495G ocWTocN461SWTR495G WTR495G N461SWT R495G N461SR495G WTN461SN461SWTR495G WTR495G N461SR495G N461SN461S α 2 ococ ococococococ 1.5 2 2.5 2.5 Mouse α Mouse WTWT R495G R495G WTWT N461SWTR495G N461SWTR495G WTR495G WTN461SWTR495G N461SWTR495G R495G N461SR495G N461SR495G WTWTN461SN461SN461SWTR495G N461SWTR495G WTR495G WTN461SR495G N461SR495G 2.5 R495G N461S2.5 N461SN461SN461S α 2 α 1.5 Mock1.5 WT MockR495GMockWT 1.5 MockR495GWT WT R495G2 R495G α 2 2 1.5 2 1.5 0.0 4 2 1.5 1.5 1.5 oc ocMockoc ocWT MockR495GMockWT MockR495GWT Mock WT R495GMockWT R495G Mock R495G WT WT R495G R495G 2 2 1.5 2 0.0 WT R495G WT N461SWTR495G 0.0 WTR495G N461SR495G 2.5 N461SN461S 4 oc oc oc oc 1.5 1.5 2 4 2 WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S 1.5 ococ ococococococ 2 0.4 oc oc oc ococ oc oc oc 2 1.5 2 WTWT R495G R495G WTWT N461SWTR495G N461SWTR495G WTR495G WTN461SR495G N461SR495G 2.5 R495G N461S2.5 N461SN461S2.0 N461S 2.0 2.0 2.0 1.5 WT R495G WT N461SWTR495G WTR495G N461SR495G 2.5 N461SN461S 2.5 0.4 2 oc oc oc ococ ocWT ocR495G WTocN461SWTR495G WTR495G N461SR495G N461SN461S 1.5 1.5 1.5 WT 2.0 R495G WT 2.0 N461SWTR495G WT2.0 R495G N461S2.0 R495G 2.5 N461SN461S 2.5 2 0.4 0.4 ococ ococococococ WT R495G WT N461SWTR495G WTR495G N461SR495G N461SN461S 2 0.4 ococ ococococococ 2 0.4 1.5 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 WTWT R495G R495G WTWT N461SWTR495G N461SWTR495G WTR495G WTN461SWTR495G N461SWTR495G 2.5 R495G N461SR495G 2.5 N461SR495G WTWTN461SN461SN461SWTR495G N461SWTR495G WTR495G WTN461SR495G N461SR495G 2.5 R495G N461S2.5 N461SN461SN461S 0.4 2 0.4 2 1.5 Mock1.5 Mock Mock1.5 Mock 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2 WT0.4 0.4 R495GWT R495GWT WT R495G R495G 2 0.4 0.4 1.5 1.5 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 4 0.0 1.5 Mock 1.5 WT Mock R495GMockWT MockR495GWT Mock WT R495GMockWT R495G Mock R495G WT WT R495G R495G 2.5 4 4 0.4 0.0 0.0 2.5 2.5 0.4 2.0 2.0 2.0 2.0 0.4 0.4 0.4 2.0 2.0 2.0 2.0 2.0 2.0 0.4 2.0 2.0 2.0 2.0 2.0 2.0 0.4 0.4 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.4 0.4 0.4 0.4 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 FLAGFLAGFLAG FLAGFLAGFLAG FLAG FLAG FLAG2.5 FLAG FLAGFLAGFLAG 2.5 FLAG FLAG FLAG 2.5 FLAG FLAG FLAG0 0 FLAG 0 0 0 0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 FLAG FLAG FLAG F FLAG FLAG FLAG FLAGF FLAGFLAG FLAG FLAGF FLAG FLAG FLAGTime (Time (Time (FLAGhrsTime (hrs) 0 4 8 MockhrsTime () 0 4 8 ochrs) 0 4 8 oc) 0 4 8 ochrsFLAGoc) 0 4 8 WTWTocWTWTTime (WTR495G Time (R495G 0 4 8R495G Time (hrsR495G 0 4 8Time (hrs 0 4 8) 0 4 8 R495G hrsN461S 0 4 8) 0 4 8 oc hrs N461S) 0 4 8 oc 0 4 8N461S) 0 4 8 ocN461SocWT0 4 8WT0 4 8WT0 4 8Time (WT0 4 8R495G Time ( 0 4 8R495G Time (0 4 8hrsR495G 0 4 8Time (hrs 0 4 8R495G ) 0 4 8 0 4 8 hrsN461S 0 4 8) 0 4 8 oc0 4 8 hrsN461S) 0 4 8 0 4 8 ocN461S) 0 4 8 oc0 4 8 N461Soc0 4 8 WT0 4 8WT0 4 8WT0 4 8WT0 4 8R495G 0 4 8R495G R495G 0 4 8 0 4 8R495G 0 4 8 N461S 0 4 80 4 8 N461S0 4 8 N461S0 4 8 N461S0 4 80 4 80 4 80 4 80 4 8 0 4 8 0 4 8 0 4 8 3.0 Time (hrs) 0 4 8 oc WTMock 0 4 8R495G WTN461S 0 4 8R495GMock 0 4 8 WT R495G 1.0 F Time (Time (hrsMouse hrs) 0 4 8 ) 0 4 8 ococMouse WTMouse 0 4 8Mouse R495G 0 4 8Time (hrsN461S) 0 4 8 oc0 4 80 4 8Time (0 4 8 0 4 80 4 8 hrs) 0 4 8 0 4 8 0 4 80 4 8 0 4 8 0 4 8 1.0 oc Mouse αMouse Mouse α Mouse α α 2.0 3.0 WTWT R495G R495G N461SN461S 3.0 Time ( hrs) 0 4 8 oc WTTime ( 0 4 8R495G hrs) 0 4 8 N461S 0 4 8WT 0 4 8R495G 0 4 8 N461S 0 4 8 0 4 8 1.0 oc 1.0 Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse 2.0 F 1.0 α α Time (Time (α hrsMouse hrs) 0 4 8 α ) 0 4 8 ococMouse WTMouse Time (Time ( 0 4 8Mouse hrsR495G 0 4 8Mouse hrs) 0 4 8 ) 0 4 8 ococN461SMouse 0 4 80 4 8WTMouse 0 4 8Mouse R495G 0 4 80 4 8 0 4 8 N461S0 4 80 4 8 0 4 8 0 4 8 F 2.0 2.0 1.0 1.0 α α α α α α α α 2.0 Mouse αMouse WTMouse WTα R495G Mouse R495G Mouse α N461SαN461SαMouse WTMouse WTα R495G Mouse R495G α N461SαN461S 2.0 1.0 1.0 1.0 Mouse Mouse Mouse αMouse Mouse Mouse αMouse Mouse Mouse αMouse α Mouse αMouse Mouse Mouse αMouse Mouse α2.0 α 2.0 1.0 α α α α α α α αα α α α α2.0 α2.0 α α 2.0 2.0 1.0 Mock 1.0 WT R495G 1.0 1.0 3.0 1.0 Mock WT1.0 R495G1.0 Mock WT R495G 1.0 Mouse Mouse Mouse Mouse 1.0 Mouse αMouse Mouse α Mouse α α 2.0 3.0 3.0 1.0 Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse 2.0 1.0 1.0 α α α Mouse α Mouse Mouse Mouse Mouse Mouse Mouse Mouse 1.5 1.5 1.5 1.5 1.0 1.0 α α α α α α α α 2.0 2.0 2.0 Mouse αMouse Mouse α Mouse Mouse α α αMouse Mouse α Mouse α α 2.0 1.5 1.5 1.5 1.5 1.0 1.0 1.0 Mouse Mouse Mouse αMouse Mouse Mouse αMouse Mouse Mouse αMouse α Mouse αMouse Mouse Mouse αMouse Mouse α2.0 α 2.0 1.0 α α α α α 1.5 α 1.5 α αα α1.5 1.5 1.5 α 1.5 α 1.5 α1.5 2.0 α2.0 α α1.5 1.5 1.5 2.0 2.0 1.5 1.5 1.5 1.5 1.5 3 1.0 1.0 1.0 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 0.2 1.0 1.0 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 3 0.2 0.2 0.2 3 0.2 0.2 0.2 0.2 1.0 1.0 0.2 0.2 0.2 0.2 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 3 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 0.2 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Signal intensity (arbitrary units) 0.2 Signal intensity (arbitrary units) 3 0.2 0.2 3 0.2

Signal intensity (arbitrary units) 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) 0.2 0.2 Signal intensity (arbitrary units) 0.2Signal intensity 0.2(arbitrary units) Signal intensity (arbitrary units) 0.2 0.2 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) GFP+/DAPI+ GFP+/DAPI+ Signal intensity (arbitrary units) 0.2 GFP+/DAPI+ Signal intensity (arbitrary units) Mouse GFP+/DAPI+ Signal intensity (arbitrary units)

0.2 GFP+/DAPI+ α Mouse GFP+/DAPI+ Signal intensity (arbitrary units) Signal intensity (arbitrary units)

0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ Signal intensity (arbitrary units) Mouse Mouse α Mouse * * *0.2 * GFP+/DAPI+ GFP+/DAPI+ 0.2 Signal intensity (arbitrary units) α α Mouse Mouse 0.2 0.2 Signal intensity (arbitrary units)

α Signal intensity (arbitrary units) Mouse α α Mouse α Mouse α α Mouse 0.2 0.2 Signal intensity (arbitrary units) 0.2 0.2 * * * * * * * *

Mouse α GFP+/DAPI+ Mouse Mouse Proportion of *

α GFP+/DAPI+ α Mouse α Proportion of Mouse FLAG FLAG FLAG FLAG GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ Mouse Proportion of Proportion of 0.2

α Mouse GFP+/DAPI+ FLAGα FLAG FLAG FLAGProportion of * Mouse Proportion of *

0.2 GFP+/DAPI+

α GFP+/DAPI+ α FLAGFLAG FLAGFLAGFLAGFLAGFLAGProportionFLAG of α Mouse FLAG FLAG FLAGProportion of FLAGFLAG FLAG FLAG FLAG

0.2 0.2 GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+ GFP+/DAPI+

Proportion of Proportion of 0.2 Mouse α Mouse α Mouse α Mouse Mouse α Mouse α Mouse Mouse α α Mouse Mouse Mouse Mouse Mouse α FLAG0.2FLAGFLAG0.2 0.2 FLAGFLAGFLAGFLAGFLAGProportion of FLAGFLAGProportion of FLAGFLAGFLAG0.2FLAGFLAG0.2 0.2 0.2FLAGFLAGFLAGFLAGFLAGFLAGFLAGFLAGFLAG α α Mouse Proportion of α Mouse Mouse

Mouse α Proportion of Proportion of Mouse α α Mouse * α Mouse 1 α

α Mouse Proportion of Proportion of Proportion of α Mouse FLAG Mouse Mouse FLAG FLAG FLAG Proportion of α α Mouse Mouse

1 Proportion of α

Mouse Mouse Proportion of Proportion of α FLAG FLAG FLAG FLAGProportion of Proportion of Proportion of α α α

Proportion of * 1 α α * 1 1

α FLAGFLAG FLAGFLAGFLAGFLAGFLAGProportionFLAG of Proportion of Proportion of Proportion of α Proportion of Proportion of FLAG FLAG FLAG FLAGFLAG FLAG FLAG FLAG 1 1 Proportion of Proportion of 1 FLAG FLAG Proportion of FLAGProportion of FLAGFLAG FLAG FLAG FLAG 1 1 Mouse 1 FLAGFLAG FLAGFLAGFLAGFLAGFLAGFLAGFLAGFLAG FLAGFLAGFLAGFLAGFLAGFLAG 1 Mouse 1 α 1 0.5 Mouse Mouse 1 Proportion of α Mouse Mouse 1 2.5 0.5 1 1 1.5 Proportion of α α Mouse α Mouse α 1 0.5 0.5 0.5 1.5

Proportion of Proportion of Mouse Mouse

Proportion of α Mouse Mouse

Proportion of α 1 2.5 0.5 2.5 0.5 1.5 1.5 1.5 1.5

Proportion of α α

Proportion of α α 1 1 0.5 1 0.5 0.5 1.5 1.5 Proportion of Proportion of 0.5 Proportion of Proportion of * 3 1 1 1.5 1.5 1.5 1.5 1 0.5 1 1 1 0.5 0.5 0.5 * 0.5 * 3 3 0.5 1 1 0.5 1 0.5 0.5 1 1.5 1 2.5 0.5 0.5 0.5 1 1 1.5 0.5 0.5 1.5 1.5 1.0 1.0 1.0 1.0 1.5 1.5 2.5 0.5 2.5 0.5 0.5 1.0 1.0 1.0 1.0 1.5 1.5 0.5 * 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 3 1.5 1.5 1.5 1.5 1.0 1.0 1.0 1.0 0.5 0.5 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.5 * 0.5 * 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 3 3 0.5 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 β-ACTIN 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 FLAG+GFP+/GFP+ β-ACTIN 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 β-ACTIN FLAG+GFP+/GFP+ β-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN FLAG+GFP+/GFP+ β-ACTIN FLAG+GFP+/GFP+ β-ACTIN FLAG+GFP+/GFP+ β-ACTIN β-ACTIN FLAG+GFP+/GFP+ β-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN β-ACTIN β-ACTINβ-ACTINβ-ACTIN β-ACTINβ-ACTINβ-ACTIN β-ACTINFLAG+GFP+/GFP+ FLAG+GFP+/GFP+ * * * * * * * * * β-ACTIN FLAG+GFP+/GFP+ β-ACTIN FLAG+GFP+/GFP+ β-ACTIN FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN1.5 FLAG+GFP+/GFP+ β-ACTIN FLAG FLAG+GFP+/GFP+ β-ACTIN1.5 FLAG+GFP+/GFP+ β-ACTIN β-ACTIN FLAG FLAG+GFP+/GFP+ 1.5 FLAG+GFP+/GFP+ FLAG+GFP+/GFP+ β-ACTIN Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) 1.5 β-ACTIN 1.5 FLAGFLAG FLAG+GFP+/GFP+ FLAG+GFP+/GFP+

FLAG Signal intensity FLAG(arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) β-ACTIN β-ACTIN1.5 1.5 β-ACTIN1.5 FLAG Signal intensity (arbitrary units) Signal intensity FLAG(arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) 1.5 1.5 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) β-ACTIN Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) β-ACTIN1.5 FLAGFLAG FLAGFLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) β-ACTIN Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) β-ACTIN1.5 1.5

1.5 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAGFLAG 1.5 1.5 1.5 1.5 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAGFLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG Signal intensity FLAG(arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units)

FLAG Signal intensity (arbitrary units) Signal intensity FLAG(arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) FLAGFLAG FLAGFLAG Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) oc WT R495G oc N461SWT R495G oc N461SWT1.5 1.5 R495G 1.5 N461S1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Mouse Mouse Mouse Mouse αMouse 1.5 αMouse Mouse αMouse αMouse 1.5 Mouse ααMouse αMouse Mouse αMouse 1.5 Mouse αMouse Mouse Mouse Mouse αMouse αMouse αMouse Mouse αMouse αMouse αMouse αMouse αα1.5 αMouse ααMouse ααMouse 1.5 Mouse α αMouse αMouse 1.5 Mouse αMouse Mouse Mouse αMouse αMouse Mouse αMouse ααMouse αα1.5 αMouse ααMouse αMouse Mouse α αMouse αMouse Mouse αMouse Mouse αMouse αMouse ααα α α Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) oc 0.0 Mouse Mouse Mouse αMouse Mouse Mouse αMouse Mouse α Mouse α Mouse Mouse Mouse Mouse Mouse Mouse Mouse WT R495G N461S 0.0 α α α α α α αMouse α αMouse Mouse α Mouse 1.0 Mouse α α αMouse Mouse α Mouse α α oc oc 0.0 0.0 0.0 Mouse Mouse Mouse αMouse Mouse Mouse αMouse Mouse 1.0 Mouse αMouse α Mouse Mouse Mouse Mouse Mouse Mouse 2.0 α α α α WT R495G N461S 0.0 1.0 1.0 WT R495G N461S 0.0 1.0 α α α α α α α αα α α α α α α α 1.0 0.0 0.0 0.0 0.0 1.0 0.0 1.0 2.0 2.0 0.0 0.0 1.0 1.0 0.0 1.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 2.0 0.0 0.0 0.0 1.0 1.0 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.5 0.5 0.5 0.5 1.0 1.0 1.0 0.0 1.0 2.0 2.0 0.0 0.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1.0 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.0 1.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * * * 0.0 0.0 0.0 * * * * * * 0 0 0 * 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

(0 hrs post treatment) 0 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (0 hrs post treatment) 0 0 * * 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (0 hrs post treatment) (0 hrs post treatment) (0 hrs post treatment) 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 T 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (0 hrs post treatment) S (0 hrs post treatment)

T (0 hrs post treatment) T 0 0 (0 hrs post treatment) S (0 hrs post treatment) S Signal intensity (arbitrary units) 0 T T S S T S (0 hrs post treatment) T 0S T 0 S Signal intensity (arbitrary units)

(0 hrs post treatment) T T

T (0 hrs post treatment) T 0 S S S T (0 hrs post treatment) S T S Signal intensity (arbitrary units) Signal intensity (arbitrary units) S Signal intensity (arbitrary units) Signal intensity (arbitrary units)

T T (0 hrs post treatment) T 0 0 0 S S T T S S S (0 hrs post treatment) T S Signal intensity (arbitrary units) Signal intensity (arbitrary units) (0 hrs post treatment) T T * 2.0 0 0 S S (0 hrs post treatment) T T S S Signal intensity (arbitrary units) Signal intensity (arbitrary units) 0 0 0 0 * * Signal intensity (arbitrary units) Signal intensity (arbitrary units) 2.0 T T S2.0 S 0 0 T S 0 0 0 T T S S T β-ACTINS T β-ACTIN0Sβ-ACTINT β-ACTIN 0 Signal intensity (arbitrary units) T β-ACTINS T Tβ-ACTINS β-ACTINS T β-ACTINS T S Signal intensity (arbitrary units) T S Signal intensity (arbitrary units) Signal intensity (arbitrary units) S Signal intensity (arbitrary units) T T β-ACTINS β-ACTINS β-ACTINβ-ACTINβ-ACTINβ-ACTINT Tβ-ACTINβ-ACTINS S β-ACTIN T β-ACTINSβ-ACTINβ-ACTINβ-ACTIN T β-ACTINSβ-ACTINβ-ACTIN Signal intensity (arbitrary units) Signal intensity (arbitrary units) β-ACTINT Tβ-ACTINS β-ACTINS β-ACTINβ-ACTINT Tβ-ACTINS β-ACTINS β-ACTIN Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) 2.0 2.0 2.0 Mouse Mouse Mouse Mouse α β-ACTINαMouse αβ-ACTINβ-ACTINα β-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINMouse Mouse β-ACTINMouse β-ACTINβ-ACTINβ-ACTINMouse β-ACTINαβ-ACTINβ-ACTINβ-ACTINαβ-ACTINαβ-ACTINβ-ACTINβ-ACTINαβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINMouse β-ACTINβ-ACTINMouse β-ACTINβ-ACTINMouse β-ACTINβ-ACTINβ-ACTINMouse αβ-ACTINβ-ACTINβ-ACTINαβ-ACTINαβ-ACTINβ-ACTINβ-ACTINαβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTIN Signal intensity (arbitrary units) Signal intensity (arbitrary units) 0 Mock WT R495G Mouse α 0 Mock WT R495G Mouse Mouse α Mouse 0 Mouse Mock WT R495G0 Mock WT R495G 0 Mock WT R495G α α Mouse α Mouse 0 α Mock WT R495G 0 Mock WT R495G 0 Mouse Mouse α Mouse Mouse 0 α Mouse Mock WT R495G 0 Mock WTMock R495GWT R495G α 0 Mock WT R495G αα α α Mouse Mouse 0 Mock WT R495G0 Mock WT R495G 0 Mock WT R495G α α 0 Mock WT R495G 0 Mock WT R495G 0 4 8 0 Mock 0 0 WT R495G Mock WT R495G Mock WT R495G 0 4 8 0 0 Mock WT R495G 4 8 0 Mock WT R495G0 0 0 0 4 4 8 8 Mock WT R495G 0 4 8 * * Mock WT R495G 0 4 8 0 0 Mock WT R495G 4 0 0 8 4 8 0 Mock WT R495G 4 8 Mouse 0 Mock WT R495G 0 Mock 0 WTMock R495GWT R495G 0 4 0.5 8 R495G * * 0 WT N461S R495G* * WT N461S 0 Mock WT R495G 4 0.5 8 α 0 0 4 4 8 8 2 0 0 R495G R495G WT N461S 0 WT N461S Mock WT R495G 0.5 0.5 Mock 0 4 0.5 8 WT R495G 0.5 Mouse Mouse Mock WT R495G 0 4 8 R495GR495G 0 1.5 WTWT N461SN461S R495GR495G WTWTR495GN461SN461S WT N461S R495G 0 R495G WT N461S 0 R495G Mock 4 WT N461S 0.5 8 WT R495G 0.5 α WT N461S 0 Mock WT R495G 4 8 α 2 0 4 2 8 R495G 0 WT N461S 0 R495G R495G WT N461S Mock R495GWT R495G WT N461S 0.5 0.5 WT N461S Mock WT R495G 0.5 0.5 0 4 8 R495GR495G 1.5 R495GR495G WTWT N461SN461S R495GR495G 1.5 WTWT N461SN461S WTWT N461SN461S R495GR495G WTWT N461SN461S 0 0 4 0 4 8 4 8 8 0 0 0 4 0 0 4 4 8 4 4 8 8 8 8 R495G WT N461S0 R495G 4 0.5 8 WT N461S 0.5 0 0 4 0 4 8 4 8 8 Fold change to first %me point 2 R495G WT N461S R495G 0.0 0.0 0.0 0.0 WT N461S 0.5 0.5 0 0 4 0.5 4 8 0.5 8

Fold change to first %me point WTWT N461SN461S R495GR495G WTWT N461SN461S R495GR495G R495G0.0 0.0 0.0 0.0 1.5 R495G WT N461S R495G WT N461S 0.5 0 R495G 4 WT N461S 8 WT N461S 0.5 0 4 8 Fold change to first %me point

Fold change to first %me point 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2 Fold change to first %me point 2 R495G WT N461S R495G R495G 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 WT N461S R495G WT N461S 0.5 0.5 WT N461S 0.5 0.5

Fold change to first %me point Fold change to first %me point WTWT N461SN461S R495GR495G R495GR495GWTWT N461SN461S R495GR495G WTWT N461SN461S R495GR495G 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.5 WTWT N461SN461S

1.5 Fold change to first %me point

Fold change to first %me point Fold change to first %me point 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Fold change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %me point 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 β-ACTINβ-ACTINβ-ACTINβ-ACTIN β-ACTINβ-ACTINβ-ACTINβ-ACTIN β-ACTINβ-ACTINβ-ACTINβ-ACTIN 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 FLAG FLAG FLAG 0 4 0 4 0 4 0 4 0 4 8 0 4 8 β-ACTIN 8 β-ACTINβ-ACTIN0 4 β-ACTIN 8 β-ACTIN 8 β-ACTINβ-ACTINβ-ACTIN 8 β-ACTIN 8 0β-ACTINβ-ACTIN0 00β-ACTIN00 0 00 0 00 000 00 000000000000000000 0000 00 0 00 000 00 000000000000000000 0000 00 0 00 000 00 0000000000000 0 0 FLAG FLAG FLAG 0 4 0 4 0 4 0 4 0 4 0 4 0 4 8 0 4 0 4 8 0 4 0 4 0 4 8 0 4 0 4 0 4 8 8 8 0 4 0 4 0 4 0 4 8 0 4 0 4 8 8 8 0 4 8 0 4 0 4 8 8 0 4 8 0 4 8 0 4 Mock 8 0 4 8 WT 8 0 4 Mock 8 R495G 8 MockWT 8 MockR495GWT 8 WT R495G 8 8 R495G 8 8 8 8 8 0.0 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 0.0 MockMock WTWTMockMockR495GR495GMockWT MockWT MockR495GWTMockR495GWT WT R495G WT MockR495G R495G R495G Mock Mock Mock 4 WT R495GWT R495GWTMock WT R495GWT Mock R495G R495G MockWT MockR495GWT WT R495G R495G 0.0 0.0 0.0 Mock Mock Mock Mock 4 4 WT R495GWT R495GWT WT R495G R495G 0.0 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 4 0.0 MockMock WTWTMockMockR495GR495GMockWT MockWT MockR495GWTMockR495GWT WT R495G WT R495G R495G R495G 0.0 MockMock WTWTMockMockR495GR495GMockWT MockWT MockR495GWTMockR495GWT WT R495G WT R495G R495G R495G 1.0 4 4 4 4 4 4 0.0 0.0 0.0 0.0 1.0 1.0 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 0.0 Mock WT MockR495GMockWT MockR495GWT WT R495G R495G 4 0.0 MockMock WTWTMockMockR495GR495GMockWT MockWT MockR495GWTMockR495GWT WT R495G WT MockR495G R495G R495G Mock Mock Mock 4 WT R495GWT R495GWTMock WT R495G Mock R495G Mock Mock 2.5 WT R495GWT R495GWT WT R495G R495G 0.0 0.0 0.0 Mock Mock Mock Mock 4 4 WT R495GWT R495GWT WT R495G R495G 0.0 Mock Mock Mock Mock 2.5 WT R495GWT R495GWT WT R495G R495G 4 4 0.0 MockMock WTWTMockMockR495GR495GMockWT MockWT MockR495GWTMockR495GWT WT R495G WT R495G R495G R495G 0.0 MockMock WTWTMockMockR495GR495GMockWT MockWT MockR495GWTMockR495GWT WT R495G WT R495G R495G R495G 2.5 2.5 4 2.5 4 2.5 1.0 2.54 2.54 2.5 2.54 2.54 2.5 0.0 0.0 0.0 0.0 1.0 1.0 2.5 2.5 2.5 2.5 2.5 2.5 0 0 00 00 0 2.5 0 2.5 0 0 0 0 0 0 0 0 0 0 Signal intensity (arbitrary units) 2.5 2.5 2.5 2.5 0 0 0 0 0 0 Signal intensity (arbitrary units) 0me (0me (hrsSignal intensity ) (arbitrary units) hrs) 0me (0me (hrs) hrs) 0me (hrs) Signal intensity (arbitrary units) 0me (hrs) 0me (hrs) 0me (hrs0me () 0me (hrs) hrs) 0me (hrs) Signal intensity (arbitrary units) 1.0 Signal intensity (arbitrary units) 0me (hrs) 0me (hrs) 0me (hrs) 1.0 0me (hrs) 0me (hrs) Mouse 1.0 1.0 0me (hrs) 1.0 F 0me (hrs) 0me (hrs) α 1.0 1.0 F 0me (1.0hrs) Mouse Mouse 1.0 1.0 F F F F α α 0me (hrs) 0me (hrs) 1.0 0me (hrs) F F 1.0 0me (1.0hrs) F F 0me (0me (hrs) 1.0hrs) F F 0me (hrs) 0me (hrs) 1.0 0me (hrsMock) 1.0 WT R495G 1.0 0me (hrs) 1.0 MockMock0me (Mock hrsWT) R495G 0me (hrs) 3.0 WTWT R495GR495G 0me ( hrsMock) WT R495G0me ( hrsMock) WT R495G Mouse 3.0 F 0me (hrs) Mock 0me (WT hrsR495G) Mock WT R495G 3.0 3.0 MockMock 3.0 WTWT R495GR495G MockMock α F 3.0 WTWT R495GR495G Mouse Mouse F F 3.0 F 3.0 F α α 1.0 1.0 1.0 1.0 1.0 1.0 F 3.0 F 3.0 F F 3.0 F 3.0 F MockMock WT WT R495GR495G 1 1.0 MockMock 1.0 1.0 1.0 3.0 WTWT R495GR495G Mock WT R495G Mock WT R495G 1.0 3.0 Mock WT R495G Mock WT R495G 1 1 3.0 3.0 MockMock 3.0 WTWT R495GR495G MockMock 1.0 1.0 1.0 3.0 WTWT R495GR495G 1.0 3.0 1.0 3.0 1.0 3.0 3.0 1.0 3.0 3.0 0.5 3 3 1 0.5 0.5 3 3 3 3 3 3 1 1 3 3 3 3 0.5 3 3 β-ACTIN β-ACTIN β-ACTIN 0.5 0.5 3 3 3 3 3 3 3 3 3 3 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * β-ACTIN β-ACTIN 0 0 2 0 2 0 4 2 4 2 6 0 4 6 4 8 2 6 8 6 4 8 8 6 8 * * * * β-ACTIN 1.5 0 2 0 4 2 6 4 8 6 8 1.5 0 2 1.5 0 4 0 0 2 6 2 0 2 4 0 8 4 2 4 6 2 6 4 6 8 4 8 6 8 6 * 8 * 8 0 0 2 0 0 2 4 2 0 2 4 * 6 * 4 2 4 6 8 6 4 6 * 8 * 8 6 8 * 8 * time * * * * time 2.5 time 2.5 time 2.5 2.5 time 2.5 time 2.5 time 2.5 time 2.5 * time 3 time 2.5 2.5 2.5 2.5 * 1.5 3 time time * * * 3 3 * 3 time 3 time * * 1.5 1.5 3 3 time * * time * * 3 3 3 3 time time 2.5 2.5 0 4 8 2.5 2.5 2.5 2.5 2.5 2.5 * 0 4 8 3 2.5 2.5 2.5 2.5 * 0 4 8 0 4 8 0 4 8 3 * * * 3 3 * 0 4 8 0 4 8 3 3 * * 0 4 8 3 0 4 8 3 0.0 0 4 8 * * * * 0 4 8 3 3 0 4 8 0 4 8 3 3 0 4 8 0.0 0.0 0 4 8 0 4 8 0 4 8 0 4 8 0.0 * * * 0 0 0.0 0 0.0 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * (0 hrs post treatment) (0 hrs post treatment) 0 %me (hrs%me post MG1320 (hrsoc post MG132ocococ treatment)oc%meococWT (hrsocWT treatment)%meWT post MG132WTR495G (0 hrs post treatment) (hrsWTR495G %meWTR495G post MG1320R495G (hrsR495G N461S treatment)ocR495G N461S post MG132ocN461SocN461S treatment)%meocN461SWTN461S (hrsocWT treatment)%meWT post MG132WTR495G (hrsR495G %meR495G post MG132R495G (hrsN461S treatment)ocN461S post MG132%meocN461SocN461S treatment) (hrs%meoc%meWT post MG132 (hrsocWT treatment) (hrsWT post MG132WTR495G post MG132R495G R495G treatment)%meR495G N461S treatment) (hrsN461S treatment)%meN461S post MG132N461S (hrs%me post MG132 (hrs treatment) post MG132 treatment)%me (hrs treatment)%me post MG132 (hrs%me post MG132 (hrs treatment) post MG132 treatment)%me (hrs treatment) post MG132 treatment) WTWT R495G R495G N461SN461Soc WT 2.0 R495G ocN461S WT R495G N461S ococ WT 2.0 R495G ococN461S WT R495G N461S WTWT 2.0 R495G 2.0 R495G N461SN461S WTWT R495G R495G 2.0 N461SN461S 2.0 2.0 0 Mock WT R495G 2.0 2.0 2.0 2.0 2.0 0 Mock WT 0 R495GMock WT R495G 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0 Mock 0 WT R495G 4 8 2.0 2.0 2.0 2.0 2.0 * * * * * * 0 0 0 4 4 8 * * * 8 * * * Mock WT 0 R495GMock WT R495G * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 0 4 8 0 0 4 4 8 8 * 2.0 * 2.0 * * * 2.0 2.0 * Fold change to first %me point 2.0 2.0 * * 2.0 2.0 * * * * 2.0 2.0 Fold change to first %me point 2.0 2.0 Mouse Mouse Mouse Mouse αMouse αMouse α α α Fold change to first %me point Mouse Mouse Mouse Mouse α α α α Mouse Mouse 2.0 Mouse 2.0 Mouse α 2.0 α 2.0 α α 2.0 2.0 2.0 2.0 * * * * * * * * 2.0 2.0 2.0 2.0 * * * * Mouse Mouse α Mouse 2 * * * * 2 Mouse * * * * α α Mouse 1.5 * * * * α 2 2 Mouse α 2 Mouse Mouse α Mouse Mouse 1.5 2 α 2 0 4 1.5 1.5 8 2 α α 1.5 1.5 α α 2 2 2 2 0 4 1.5 1.5 1.5 8 1.5 0 4 1.5 1.5 8 2 2 2 2 1.5 1.5 2 2 0 4 1.5 1.5 8 2 1.5 2 1.5 2 2 1.5 2 2 0 4 0 4 1.5 1.5 8 1.5 1.5 1.5 8 FLAGFLAGFLAGFLAGFLAGFLAGFLAGFLAG FLAGFLAGFLAGFLAGFLAGFLAG0me (FLAGFLAG hrs) FLAGFLAGFLAGFLAGFLAGFLAG0me (FLAGFLAG hrs) 0me (hrs) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0me (hrs) 0me (hrs) 0me (hrs1.0 1.0 ) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Signal intensity (arbitrary units) Mouse Mouse Mouse Mouse Mouse Mouse αMouse αMouse αα ααα α Mouse Mouse Mouse Mouse Mouse Mouse αMouse αMouse αα ααα α Mouse Mouse Mouse Mouse Mouse Mouse αMouse αMouse αα ααα α Signal intensity (arbitrary units) Signal intensity (arbitrary units) 1 1 1 1 1 1 1 1 1 1 1 1 0 2 4 6 0.5 8 0.5 0 2 0.5 0.5 4 1 6 0.5 8 0 2 4 6 0.5 8 1 1 1 0.5 0.5 0.5 1 0.5 1 1 1 1 0.5 0.5 1 1 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 time 0.5 0.5 0.5 0.5 β-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTINβ-ACTIN0 β-ACTINβ-ACTIN β-ACTINβ-ACTINβ-ACTIN 4 β-ACTIN0 β-ACTIN 8 β-ACTINβ-ACTINβ-ACTINtime 4 β-ACTIN0 8 time 4 8 β-ACTIN β-ACTIN1.5 β-ACTINβ-ACTIN β-ACTIN1.5 β-ACTIN1.5 β-ACTIN1.5 1.5 1.5 1.5 1.5 1.5 1.51.5 1.5 1.5 1.51.5 1.5 1.5 1.51.5 1.5 1.5 1.51.5 1.5

0.0 0.0 0.0 0.0 0.0 0.0 0.0 %me (hrs post MG132 treatment) %me (hrs post MG132 treatment) %me (hrs post MG132 treatment) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0 0 0 0 00.0 00.0 0.0 00.0 0.0 (0 hrs post treatment) 0 0 0 (0 hrs post treatment) 0 0 (0 hrs post treatment) (0 hrs post treatment) (0 hrs post treatment) (0 hrs post treatment) (0 hrs post treatment) (0 hrs post treatment) (0 hrs post treatment) (0 hrs post treatment) 0 0 0 0 0 0 0 0 (0 hrs post treatment) (0 hrs0 post treatment) 0 0 0

0 Mock WT R495G 0 Mock WT R495G 0 0 MockMock WTWT R495GR495G 0 Mock WT R495G 0 Mock WT R495G 0 Mock WT R495G 0 Mock WT R495G 0 0 MockMock WTWT R495GR495G 0 0 MockMock WTWT R495GR495G 0 Mock0 0 WT R495G 4 4 8 8 0 Mock0 0 WT R495G 4 4 8 8 0 0 MockMock WTWT R495GR495G 0 0 0 0 4 4 8 8 Mock0 WT R495G 4 8 Mock0 WT R495G 4 8 0 Mock0 0 WT R495G 0 4 4 8 8 Mock0 0 WT R495G 4 4 8 8 0 0 MockMock WTWT R495GR495G 0 0 MockMock WTWT R495GR495G 0 4 8 0 0 0 4 4 4 0 8 8 8 0 4 4 8 8 0 0 0 0 4 0 4 0 4 4 8 4 8 4 8 8 8 8 Fold change to first %me point Fold change to first %me point Fold change to first %meFold point change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %me point Fold change to first %meFold point change to first %me point 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 0 4 Fold change to first %meFold point change to first %me point 8 8 0 4 8 8 0 4 8 8 0 4 0 4 8 0 4 0 4 8 0 4 0 4 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 0me (0me (0me (0me (hrshrs) hrs) hrs) ) 0me (0me (0me (0me (hrshrs) hrs) hrs) ) 0me (0me (0me (0me (hrshrs) hrs) hrs) ) 1.0 1.0 1.0 1.0 1.0 1.0 0me (hrs) 1.0 1.0 1.0 0me (hrs) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0me (0me (hrs1.0 hrs) 1.01.0 ) 1.0 1.0 1.0 0me (0me (0me (0me (hrshrs) hrs) hrs) ) 0me (0me (0me (0me (hrshrs) hrs) hrs) )

0 0 0 0 2 2 2 2 0 0 0 0 4 4 4 4 2 2 2 2 6 0 6 0 6 0 6 0 4 4 4 4 8 2 8 2 8 2 8 2 6 6 6 6 4 4 4 4 8 8 8 8 6 6 6 6 8 8 8 8 0 4 8 0 4 8 0 4 8 0 4 8 time timetime time 0 4 8 0 4 8 0 4 8 0 4 8 time timetime time 0 4 8 0 4 8 0 4 8 0 4 8 time timetime time %me%me (hrs%me (hrs%me post MG132 (hrs post MG132 (hrs post MG132 post MG132 treatment) treatment) treatment) treatment)%me%me (hrs%me (hrs%me post MG132 (hrs post MG132 (hrs post MG132 post MG132 treatment) treatment) treatment) treatment)%me%me (hrs%me (hrs%me post MG132 (hrs post MG132 (hrs post MG132 post MG132 treatment) treatment) treatment) treatment) leenta ige 8

GFP FLAG MERGE

uCP

mCP shRNA + WT Zbtb18 Zbtb18 lCP

uCP

mCP shRNA+N461S Zbtb18 Zbtb18 lCP

uCP

mCP shRNA+R495G Zbtb18 Zbtb18 lCP leenta ige 9

Control Zbtb18Zbtb18 shRNA Zbtb18Zbtb18 shRNA +WT Zbtb18Zbtb18 shRNA + N461S Zbtb18 shRNA + R495G

CP

IZ

VS/ SVZ

Zbtb18 shRNA + Zbtb18 shRNA + CP Zbtb18 shRNA + WT Zbtb18 shRNA * # IZ * # Control

* # VZ/SVZ # Embryonic subcompartments *

0 10 20 30 40 50 60 70 80 Percentage of GFP+ cells leenta ige 10

wildtype B Zbtb18 suppression N461S rescue R495G rescue

MZ

Disrupted radialglial-guided locomotion CP

Defective Exacerbated IZ multipolar- multipolar- to-bipolar to-bipolar transition transition SVZ

VZ SUPPLEMENTARY DATA

Supplementary data are available at Human Mutaon online.

Legends to Supplementary Figures

Supplementary Figure S1. (A) Planar representaon of the ZBTB18 polypepde featuring an N- terminal BTB domain (dark blue) and four zinc fingers (in yellow) at its C-terminus, with pathogenic missense mutaons indicated (Cohen et al., 2017; Depienne et al., 2017; Rauch et al., 2012). (B) CLUSTAL Omega (ver1.2.4) alignment of the C-terminal region (aa371-531) of ZBTB18 comprising four zinc finger regions (ZF1 to ZF4). Amino acids highlighted with yellow and red represent conservave and non-conservave changes, respecvely. Disease-associated missense variants are idenfied with an arrow. Affected residues are highly conserved among vertebrates.

Supplementary Figure S2. Representave structures of ZBTB18-DNA complexes from molecular dynamics simulaons. (A) Wildtype-E1mut complex. (B) Wildtype-E2 complex. (C) Wildtype-E2mut complex. (D) N461S-E1mut complex. (E) N461S-E2 complex. (F) N461S-E2mut complex. (G) R495G- E1mut complex. (H) R495G-E2 complex. (I) R495G-E2mut complex.

Supplementary Figure S3. RMSDs over me for ZBTB18-DNA complexes in the three selected simulaons for each complex. The grey region indicates the regions of the simulaons used for MMGB/SA calculaons. (A) Wildtype-E1 complex. (B) N461S-E1 complex. (C) R495G-E1 complex. (D) Wildtype-E1 mutant complex. (E) N461S-E1 mutant complex. (F) R495G-E1 mutant complex. (G) Wildtype-E2 complex. (H) N461S-E2 complex. (I) R495G-E2 complex. (J) Wildtype-E2 mutant complex. (K) N461S-E2 mutant complex. (L) R495-E2 mutant complex.

Supplementary Figure S4. Luciferase reporter assays performed with the Rnd2 3’ enhancer in the presence of 125 ng of ZBTB18 WT construct alone, or in combinaon with 125ng of N461S (A) or R495G (B). Quanes of expression plasmid were balanced to 250ng per well of transfected cells with an empty construct. Signals represent fold change relave to baseline control (reporter transfected with empty mammalian constructs encoding only the FLAG epitope tag). All transfecon condions were balanced for equal quanes of plasmid DNA. Bars represent average ± SEM. A Kruskal-Wallis analysis was employed to test mulple condions, while a Mann-Whitney U test was applied for stascal analyses between 2 treatments. **p < 0.01 compared to control.

Supplementary Figure S5. An Illustrave summary of the transcriponal acvity and DNA binding of ZBTB18, and its missense variants, in the context of the Rnd2 3’ enhancer reporter. The strength of reporter signal is represented by the number of arrows, as well as the size of the arrows. (A) WT ZBTB18 binds E1 and E2 mofs to repress reporter acvity, but repressor acvity is aenuated when these mofs are mutated to E1mut or E2mut. (B) In contrast, the N461S variant binds E1, E1mut and E2 to mediate reporter acvity; however, the intensity of augmented reporter signal is dependent on the contextual presentaon of each of these DNA mofs, since N461S has reduced capacity for repression in the context of the E1mut+E2 and E1mut+E2mut motif pairs. (C) On the other hand, the R495G variant binds E1, E1mut and E2 and significantly augments reporter acvity, possibly through the formaon of a protein complex with addional factors, as evidenced in our EMSA assays. Supplementary Figure S6. Localisaon of FLAG-tagged wildtype ZBTB18, as well as the N461S and R495G variants in transiently transfected HeLa cells. (A) Representave images of the two main loca lisaon paerns (Pancellular [a’] and Exclusively Nuclear), and two unique nuclear localisaon pae rns (Uniform [a’’] and Punctate [a’’’]). Outline of visibly transfected cells (dashed green lines) are marked by GFP co-expression (not shown). (B) The nuclear localisaon of N461S was significantly different from WT 24 hours post-transfecon. (C) The pancellular localisaon of N461S was significantly different from WT 24 hours post-transfecon. (D) For the proporon of cells exhibing exclusively nuclear localisaon, punctate distribuon of R495G was significantly increased compared to WT at 24 hours. (E-G) The nuclear localisaon and nuclear immunostaining paerns for WT, N461S and R495G were not significantly different at 48 hours post-transfecon. Bars represent mean ± SEM n=3. Scale bar for a’’’ represents 12.5μm. ***p<0.001 compared to WT.

Supplementary Figure S7. Low steady-state levels of N461S in transiently transfected cells. (A-B) There was a significant decrease in the proporon of FLAG+GFP+/GFP+ cells transfected with the N461S construct, yet the proporon of GFP-transfected cells across condions was not significantly different. (C) Western blot to immunodetect WT, N461S and R495G as FLAG-epitope tagged proteins. (D) There was a significant decrease in the steady-state levels of FLAG-N461S signal compared to WT (n=3 biological replicates per condion). (E) Western blot to immunodetect WT, N461S and R495G as FLAG-epitope tagged proteins 0, 4, and 8 hrs post treatment with 10µM MG132. (F) There was a significant increase in the levels of FLAG-N461S signal during the me course; with the trend not significantly different to either WT or R495G, but significantly different to control (n=3 biological replicates per condion). **p<0.05, ***p<0.001, ****p<0.0001 compared to control, while ##p<0.05, ###p<0.001 compared to WT.

Supplementary Figure S8. Representave coronal secons of E17.5 embryonic cerebral corces describing the Corcal Plate (CP) distribuon and immunoreacvity of Zbtb18 shRNA-treated neurons co-electroporated with expression constructs encoding FLAG-tagged WT, N461S and R495G. The lower- (lCP), medial- (mCP) and upper CP (uCP) subdivisions are defined. Co-delivery of N461S augments the uCP accumulaon of Zbtb18 shNRA-treated cells, compared to cells rescued with WT construct. In contrast, co-delivery of R495G failed to rescue the radial posioning of shRNA-treated neurons (see Figure 5G for quanficaon). Robust immunoreacvity was observed for WT and R495G electroporated cells, but N461S immunoreacvity was not detected on any secon for all brains tested (n=6-8 per condion). Asterisk represents spurious immunoreacvity adjacent to a blood vessel.

Supplementary Figure S9. The effects of ZBTB18 and its disease-associated variants on radial migraon E14.5-born corcal cells within the embryonic E16.5 mouse cerebral cortex. (A) In utero electroporaon of E14.5 mouse cortex with either; control shRNA together with an empty expression vector; Zbtb18 shRNA and an empty vector; Zbtb18 shRNA and WT; Zbtb18 shRNA and N461S; or Zbtb18 shRNA and R495G. Brains were collected two days later (E16.5), white bars delineate the corcal plate (CP), intermediate zone (IZ), and ventricular zone/subventricular zone (VS/SVZ). (B) Quanficaon of distribuon of GFP-labelled cells within the CP, IZ, and VS/SVZ. *P<0.05, **P<0.01 compared to control; #P<0.05, ##P<0.01 compared to Zbtb18 shRNA. Scale bar in (e) represents 200 μm.

Supplementary Figure S10. Summary illustraon of the effects of ZBTB18 pathogenic variants on radial migraon in our rescue assays. (A) Bipolar-shaped corcal neurons leave the VZ/SVZ and adopt a mulpolar morphology to iniate somal translocaon within the IZ before transioning to a bipolar shaped neuron to migrate into the CP by radialglial-guided locomoon. Zbtb18 is crical to the development of embryonic corcal project neurons at mulple steps. For example, Zbtb18 expression is crical for mediang neuroprogenitor cell cycle exit within the VZ/SVZ (Okado et al., 2009), as well as for the generaon of appropriate numbers of neurons, rather than glia (Xiang et al., 2011). As immature neurons transit within the SVZ through to the IZ and CP, Zbtb18 regulates downstream target genes including Neurog2 and Rnd2 to drive mulpolar-to-bipolar transion (Ohtaka-Maruyama et al., 2013) and radial migraon into the CP. (B) Knockdown of Zbtb18 disrupts the radial migraon of newborn corcal neurons, culminang in their mulpolar-to-bipolar transion within the IZ and lower CP, and this migraon defect is corrected by co-delivery of ZBTB18 (Figure 5A-C,F,G; and Ohtaka- Maruyama et al., 2013; Heng et al., 2013). (C) In contrast, co delivery of N461S leads to exuberant radial migraon into the CP, with the accumulaon of mulpolar-shaped cells which is suggesve of disrupted radialglial-guided locomoon (Figure 5D,F,G). (D) Co-delivery of R495G leads to an exacerbaon of the migraon defect of shRNA treated cells, culminang in a failure in mulpolar-to-bipolar transion of cells and their accumulaon in the IZ (Figure 5E-G).

Supplementary Video Legend

Supplementary Video S1. Three-dimensional rotang model of the WT ZBTB18-E1 complex, with zinc finger domains highlighted.

Supplementary Tables

Supplementary Table S1. Summary of disease-associated missense variants of ZBTB18, their predicted impact on the polypeptide product, and their associated neuropathological traits. Intellectual neuropathological Variant Effect of missense variant PolyPhen2 and SIFT scores disability? traits(reference) (Y/N) Non-polar hydrophobic to PolyPhen2: 0.999 (probably damaging) Epilepsy and flapping of L434P non-polar hydrophobic Y SIFT:0.00 (damaging) hands(11) (conserved) Polar neutral hydrophilic to PolyPhen2: 0.969 (probably damaging) Seizures, Microcephaly (OFC Y447H charged basic hydrophilic Y SIFT:0.00 (damaging) 52 cm [-2.0])(13) (non-conserved) polar neutral hydrophilic to Global CC hypoplasia, inferior PolyPhen2: 0.996 (probably damaging) N461S polar neutral hydrophilic Y cerebellar vermis SIFT:0.48 (tolerated) (conserved) hypoplasia(15) Hyperactive, tick disorder, Charged basic hydrophilic to obsessive–compulsive PolyPhen2: 0.999 (probably damaging) R464H Charged basic hydrophilic Y behaviour, short and dysgenic SIFT:0.00 (damaging) (conserved) CC, likely microcephaly (OFC 50 cm [−2.25])(11) Charged basic hydrophilic to Limited speech and likely PolyPhen2: 1.00 (probably damaging) R464C polar neutral hydrophilic Y macrocephaly SIFT:0.00 (damaging) (non-conserved) (OFC 58cm [90th])(8) Polar neutral hydrophilic to PolyPhen2: 0.954 (probably damaging) Q486E Charged acidic hydrophilic Y Unknown(8) SIFT:0.00 (damaging) (non-conserved) Charged basic hydrophilic to PolyPhen2: 0.996 (probably damaging) R495G non-polar neutral hydrophobic Y Unknown(14) SIFT:0.00 (damaging) (non-conserved) PolyPhen2 run using http://genetics.bwh.harvard.edu/cgi-bin/ggi/ggi2.cgi SIFT run using http://sift.jcvi.org/www/SIFT_enst_submit.html Supplementary Table S2. Templates for each of the C2H2 subdomains of the ZBTB18 zinc finger domain. Region Template (PDB ID) (Reference) Sequence alignment/region modelleda C2H2 1 3UK3 FMCPLCNKVFPSPHILQIHLSTH RECSYCGKFFRSNYYLNIHLRTH C2H2 2 1MEY (Kim and Berg, 1996) PTCSLCGKTFSCMYTLKRHERTH YKCPECGKSFSQSSNLQKHQRTH C2H2 3 1MEY (Kim and Berg, 1996) YTCTQCGKSFQYSHNLSRHAVVH YKCPECGKSFSQSSDLQKHQRTH C2H2 4 5K5H (Hashimoto et al., 2017) HACKWCERRFTQSGDLYRHIRKFH YECYICHARFTQSGTMKMHILQKH aIn this column, the first row indicates the ZBTB18 sequence; the second row indicates the template sequence. Regions not shown in this table were modelled de novo by Prime during model building.

Supplementary Table S3. Templates for the E1 and E2 probe sequences. DNA probe Template (PDB ID) (Reference) Sequence alignmentsa E1 5KE6 (Hashimoto et al., 2017) TAACAGATGTCTGTC GAGGTGTGGC AGGGAAAAGCCCGGG E2 5KL2 (Hashimoto et al., 2016) GGGCAGATGGGAGTA GCGTGGGAGT AGGGAAAAGCCCGGG aIn each row of this column, the first line indicates the DNA probe sequences to be modelled, the second line indicates the DNA sequence contained in the PDB template and the third line indicates the DNA sequence from PDB 2I13. Supplementary Table S4. Binding energy decomposition for all residues making a major contribution to the binding energy in at least one complex.a WT N461S R495G Residue E1 E1mut E2 E2mut E1 E1mut E2 E2mut E1 E1mut E2 E2mut Lys386 -2.9 -4.4 -1.2 -2.3 -2.7 -3.4 -1.4 -1.9 -2.2 -4.0 -1.3 -4.5 Ser390 -1.2 -0.9 -3.3 -2.1 -2.1 -0.4 -1.8 -0.4 -0.6 -1.8 -3.6 -1.9 His392 -2.1 -0.8 -1.7 -5.0 -3.8 -1.5 -3.1 -3.4 -2.0 -1.1 -1.8 -4.2 His397 -3.4 -4.2 -1.5 -1.4 -3.7 -4.6 -0.3 -2.2 -4.6 -3.6 -0.3 -2.0 Thr400 -3.2 -2.6 -1.3 -0.6 -4.4 -3.8 -0.4 -1.9 -4.8 -3.6 -0.7 -0.3 Arg403 -1.5 -3.3 -2.2 -1.9 -4.9 -5.2 -1.8 -3.5 -5.0 -2.1 -0.9 -0.4 Arg409 -0.5 -0.2 -0.3 -0.9 -0.3 -0.7 -2.7 -3.8 -0.2 -1.8 -0.3 -1.7 Lys426 -2.5 -3.8 -4.0 -3.1 -4.2 -3.8 -2.7 -3.2 -4.0 -3.3 -4.4 -3.2 Phe428 -0.2 -3.4 -2.4 -1.4 -0.3 -0.4 -1.9 -4.6 -3.0 -3.4 -2.9 -0.4 Ser429 -0.2 -4.9 -2.8 -1.2 +0.1 +0.1 -1.5 -5.9 -2.1 -3.3 -3.5 -1.0 Cys430 -0.5 +0.0 -0.9 -1.2 -0.4 -0.2 -0.8 -0.4 -0.8 -0.3 -0.7 -2.6 Tyr432 -4.1 -4.7 -5.9 -5.4 -5.3 -4.3 -5.7 -3.6 -4.5 -4.8 -5.5 -5.4 Thr433 -0.5 -4.1 -2.2 -0.9 -0.6 -1.0 -2.4 -5.4 -2.6 -3.2 -4.1 -1.3 Leu434b -0.1 +0.1 -0.0 -0.1 -0.1 -0.1 -0.1 +0.0 -0.0 -0.1 +0.1 -0.1 Arg436 -2.5 -4.1 -6.9 -5.0 -3.4 -4.8 -3.7 -2.4 -5.8 -3.5 -5.1 -3.1 His437 -2.0 -2.6 -4.9 -2.4 -2.1 -3.4 -3.7 -4.3 -4.6 -2.8 -4.1 -3.7 Arg439 -1.4 -2.2 -1.0 -0.9 -1.6 -1.5 -1.3 -3.0 -1.5 -4.0 -1.3 -1.2 Thr440 -2.8 -1.4 -2.8 -1.8 -2.3 -1.5 -3.0 -1.5 -3.0 -1.4 -3.6 -2.5 Lys445 -3.5 -0.7 -2.4 -1.2 -4.0 -0.9 -2.3 -0.6 -1.9 -0.6 -2.1 -2.5 Tyr447 -2.1 -0.5 +0.0 -0.1 -1.6 -2.9 +0.1 -1.7 -0.9 -3.4 +0.1 +0.0 Lys454 -4.5 -3.9 -4.4 -3.9 -4.1 -3.0 -4.0 -2.2 -4.4 -4.2 -4.3 -4.9 Phe456 -4.4 -1.0 -3.4 -1.4 -3.7 -0.6 -2.7 -1.1 -2.8 -1.4 -2.5 -4.0 Gln457 -5.3 -1.1 -4.5 -1.0 -5.4 +0.3 -4.0 -0.5 -3.8 -0.4 -3.5 -5.1 Tyr458 -3.2 -0.7 -3.4 -0.7 -2.2 -2.5 -3.1 -4.3 -1.9 -3.5 -1.1 -4.2 Ser459 -3.3 -3.0 -0.3 -1.2 -2.1 -3.5 -0.6 -0.4 -0.8 -3.9 -1.2 -0.8 His460 -3.3 -4.5 -3.3 -2.7 -3.5 -4.4 -4.2 -4.6 -3.0 -4.8 -3.6 -3.8 Asn/Ser461 -8.6 -2.3 -9.6 -1.4 -2.5 -1.5 -2.3 -3.3 -7.7 -3.9 -3.6 -5.5 Ser463 -0.0 -4.4 +0.2 +0.1 -1.0 -4.7 -0.2 -1.0 +0.2 -4.5 -0.4 -0.1 Arg464 -4.0 -3.1 -4.9 -3.8 -3.9 -2.7 -4.0 -4.3 -4.0 -5.5 -4.5 -3.1 His465 -2.6 -4.6 -4.0 -4.4 -1.9 -2.2 -2.8 -2.7 -4.3 -5.6 -3.9 -2.8 Arg471 -0.7 -5.7 -4.0 -4.7 -6.3 -4.8 -1.4 -4.6 -2.9 -6.8 -7.8 -3.2 Arg482 -6.5 -2.3 -3.8 -4.0 -6.5 -6.1 -7.4 -4.6 -4.0 -3.2 -3.9 -4.6 Gln486 -3.1 -2.7 -4.3 -2.5 -2.8 -2.9 -2.9 -3.4 -3.6 -2.4 -3.5 -2.8 Asp489 +2.4 +0.4 +2.8 +2.0 +3.8 +2.9 +1.4 +1.5 +3.3 -0.0 +1.9 +2.2 Tyr491 -1.6 -4.5 -0.2 -3.9 -1.9 -3.8 -0.8 -2.2 -0.2 -3.2 -1.8 -0.1 Arg492 -4.6 -2.7 -4.6 -3.7 -3.2 -4.0 -5.7 -4.5 -4.7 -2.2 -4.3 -4.3 His493 -5.9 -0.3 -0.7 -2.9 -5.3 -3.9 -5.8 -4.2 -2.3 -1.3 -4.9 -4.2 Arg/Gly495 -0.7 -2.5 -0.3 -2.7 -1.2 -1.4 -0.6 -1.0 +0.0 +0.0 +0.0 -0.0 dA/dG 3 +2.3 +1.2 +0.4 +0.2 -0.0 +0.6 -2.8 -1.2 +3.1 +1.2 -1.8 -1.4 dC 4 -3.8 -5.2 -1.2 -3.8 -2.2 -3.2 -4.3 -4.2 -2.2 -4.1 -5.1 -4.1 dA 5 -2.2 -0.3 -1.7 -0.8 -2.1 -2.3 -1.3 -3.5 -1.2 -2.5 -1.7 -1.1 dG 6 -7.5 -1.8 -5.5 -2.8 -5.4 -3.4 -4.8 -3.3 -5.8 -1.5 -3.7 -6.0 dG/dA 9 -2.9 -1.1 -4.1 -0.2 -6.1 -1.3 -4.7 -2.9 -3.5 -2.5 -4.9 +0.9 dG 4’ -3.3 -5.4 -1.9 -4.5 -4.0 -6.0 -0.5 -1.8 -2.5 -4.0 -1.8 -1.9 dG/dC 11’ +0.9 -4.1 -0.2 +0.7 -0.2 -0.2 +0.2 +0.4 -0.3 +0.1 -0.1 +0.5 dG/dT 15’ -0.9 -0.4 -1.4 -2.2 -0.9 -0.5 -1.4 -1.2 -5.1 -1.3 -3.5 -1.9 aResidues individually accounting for greater than 2.5% of the total binding energy shown in this table. Only total energies (in kcal/mol) are shown. bThis residue is also included here as it is the site of a known disease-associated variant (see Table S1) but has very minor contribution to the binding energy in all complexes generated. Supplementary References

Hashimoto, H., Wang, D., Steves, A. N., Jin, P., Blumenthal, R. M., Zhang, X., & Cheng, X. (2016). Distinctive Klf4 mutants determine preference for DNA methylation status. Nucleic Acids Res, 44(21), 10177-10185. doi:10.1093/nar/gkw774 Kim, C. A., & Berg, J. M. (1996). A 2.2 A resolution crystal structure of a designed zinc finger protein bound to DNA. Nat Struct Biol, 3(11), 940-945.