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Correction for Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans.

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization. Table of Content (TOC) Highlight Summary Different tubulin isotypes perform different functions in the regulation of MT structure and neurite growth, and missense mutations of tubulin genes have three types of distinct effects on MT stability and neurite growth. One α-tubulin isotype appears to induce relative instability due to the lack of potential post-translational modification sites.


Introduction 111
Microtubules (MTs) play important roles in many aspects of neurite development, being 112 involved in the formation, extension, guidance, and maintenance of neurites (reviewed in Dent et 113 al., 2011;Prokop, 2013;Sainath and Gallo, 2015). MTs constantly explore the growth cone 114 periphery until they are captured by stabilized actin filaments or membrane receptors enriched at 115 the side of the growth cone responding to a guidance cue (Tanaka et al., 1995;Challacombe et 116 al., 1996;Schaefer et al., 2008;Qu et al., 2013). This capture transiently stabilizes MTs and 117 enables MT elongation in the direction that the growth cone has turned. In addition to providing 118 physical support for the neurite growth that follows the changes in actin dynamics, MTs also 119 play an instructive role in neurite guidance. Since local application of the MT-stabilizing drug 120 paclitaxel (also known as taxol) induced growth cone attraction, of the MT-destabilizing drug 121 nocodazole induced repulsion, and of the MT-depolymerizing drug colchicine resulted in 122 branching (Bray et al., 1978;Buck and Zheng, 2002), signals that act by altering MT stability 123 appear to directly initiate growth cone turning. Indeed, the guidance molecule Wnt can induce 124 growth cone remodeling by changing the organization of MT structure through the inactivation 125 of MT-plus end binding protein Adenomatous Polyposis Coli (Purro et al., 2008). These results 126 indicate that the regulation of MT dynamics is crucial for neurite growth and guidance, but how 127 MT stability is controlled locally at specific sites of the growth cone and globally in the entire 128 neuron is not well understood. 129 As the building blocks of MTs, α -and β -tubulins are crucial determinants of MT stability. 130 In fact, eukaryotic genomes contain multiple tubulin genes encoding different isotypes (Sullivan,131 throughout the molecules, many are found in region predicted to mediate either GTP binding, 157 heterodimer stability, inter-dimer interaction, or association with motor proteins and other MAPs 158 (Tischfield et al., 2011). Despite some in vitro studies on the effects of the mutations on tubulin 159 folding, heterodimer assembly, and MT growth (Jaglin et al., 2009;Tian et al., 2010;Tischfield 160 et al., 2010), very few in vivo studies can systematically test how these different tubulin 161 mutations impact axon guidance and extension in living organisms. Moreover, the complexity of 162 the mammalian nerve system makes the analysis of the developmental consequences of these 163 tubulin mutations very difficult. 164 Here we use the morphologically simple and well-defined touch receptor neurons (TRNs) 165 in the nematode Caenorhabditis elegans to model the effects of tubulin mutations on neurite 166 growth. By analyzing a large collection of missense mutations in several tubulin genes, we found 167 that these mutations caused three morphologically distinct defects in TRN neurite outgrowth: 1) 168 the shortening of all TRN neurites; 2) the specific shortening of posteriorly directed neurites; and 169 3) the production of ectopic posteriorly directed neurites. The structural location of the mutated 170 residue correlated with the resulting phenotype. Many tubulin mutations characterized in our 171 study affect the same amino acid residue or region as the disease-causing mutations in humans. 172 We generated several such human mutations in C. elegans tubulin genes through genome editing 173 and found that they also caused distinct neurite growth defects that fall into the above categories. 174 Thus, our system may be used to understand the different effects of the clinically identified 175 tubulin mutations and to facilitate their classification. Moreover, we found that null mutations in 176 two α-tubulin genes led to very different phenotypes, supporting the hypothesis that tubulin 177 isotypes perform specific and often non-overlapping roles in neurite development. 178 Results 179 (Chalfie and Thomson, 1982), led to a similar shortening of the PLM-PN, phenocopying the 246 mec-7 null mutants ( Figure S3A). 247

Antimorphic mutations in mec-7 led to severe neurite growth defects 248
We next examined 55 mec-7 alleles that represent 48 different missense mutations (Table  249 1) and found three categories of TRN morphological defects. First, 12 mutations caused neurite 250 growth defects similar to the knockout allele and were thus classified as loss-of-function (lf) 251 mutations. Data from u1020 (G34S) animals is shown in Figure 2 as an additional lf example. 252 When mapped to the structure of a bovine αβ tubulin dimer (Figure 3;Nogales et al., 1998), lf 253 mutations were found throughout the molecule and presumably affected protein folding. Second,254 19 mutations caused severe shortening of all TRN neurites; the ALM-AN did not reach the 255 pharynx, the PLM-AN terminated before reaching the PVM cell body and the PLM-PN was 256 significantly shortened ( Figure 2B and D). Because of the dominant-negative nature of these 257 alleles (all, except u430, were either dominant or semi-dominant), we classified them as 258 antimorphic (anti) gain-of-function mutations. We also identified 4 weak mec-7(anti) mutants 259 (Table 1), in which ALM-AN did not extend beyond the nerve ring and PLM-AN did not reach 260 the vulva. Third, 6 mutations led to the growth of an extra posteriorly-directed neurite in ALM 261 neurons, as well as the overextension of the PLM-PN. Among the six alleles, u278 (C303Y) 262 produced the strongest phenotype; the ectopic posterior neurite of ALM neurons often extended 263 posteriorly to the PLM cell body ( Figure 2C). Because this phenotype was novel and different 264 from the phenotype of either mec-7 lf or anti mutations, we classified these alleles as neomorphic 265 (neo) gain-of-function mutations. Except two semi-dominant ones (u278 and ky852), most of the 266 neo alleles were recessive. Because of the antagonism between anteriorly and posteriorly 267 directed outgrowth (Zheng et al., 2016), some neo alleles, especially u1017, caused mild 268 shortening of the ANs, i.e. PLM-AN terminated slightly posterior to the vulva ( Figure 3C). 269 EM studies of the mec-7(anti) allele, e1343 (P171L), found that the mutants contained 270 very few (2.8 ± 0.5 in a cross section) MTs (Chalfie and Thomson, 1982), which suggests that 271 the anti mutations block MT polymerization and thereby cause the severe neurite outgrowth 272 defects. In fact, when mapped to the tubulin structure, amino acid residues mutated in the anti 273 alleles are clustered in three regions: 1) the GTP/GDP binding pocket, 2) the intradimer interface, 274 and 3) the lateral or longitudinal interdimer interface. All of which are important for tubulin 275 polymerization. For example, u430 (A97V), u911 (P171S), u957 (P171L), and u262 (N226Y) 276 mutations alter amino acids in direct contact with the GTP molecule, and u48 (S176F) and u449 277 (V179A) change amino acids in the B5 (the fifth β-strand)-to-H5 (the fifth α-helix) loop, which 278 is crucial for forming the GTP/GDP binding pocket ( Figure 3C). Two other mutations u445 279 (M300V) and u98 (M300T) substitute M300, which is located in a loop near the GTP binding 280 site and may participate in positioning H7 (the seventh α-helix) with its critical N226, and thus 281 indirectly affect GTP binding. 282 The second group of mutated residues in mec-7(anti) alleles is located in the intradimer 283 interface, where β-tubulin makes contact with α-tubulin to form the heterodimer (red residues in 284 Figure 3D). u283 (P243L), u958 (G244S), n434 (N247I), and u162 (D249N) mutations all affect 285 the residues on the H7-to-H8 loop, which interacts extensively with residues on H1 and H2 of α-286 tubulin. Two other dominant mutations gk286000 (A314V) and u955 (A352T) changed residues 287 that located on B8 and B9, respectively, which are physically adjacent to the H7-to-H8 loop at 288 the interface. In addition, two prolines mutations, u910 (P357L) and u956 (P358L) possibly 289 disrupted the B9-to-B10 loop (a.a. 356-361) critical for the positioning of B9. 290 The third group of mec-7(anti) alleles include e1527 (V286D), u18 (A393T), and 291 gk285997 (A393V), which affect residues involved in the lateral (V286) and longitudinal (A393) 292 interdimer interaction between one α/β-tubulin heterodimer and the neighboring one on the MTs. 293 These mutations may interfere with the interaction between tubulin dimers. 294 Overall, the MEC-7/β-tubulin anti mutants likely act in a dominant-negative manner by 295 forming poisonous α/β dimers, whose incorporation into MTs could terminate MT 296 polymerization and induce instability. The mutated β-tubulin either cannot properly bind to GTP 297 (the first group), form misshaped α/β heterodimers that block the growing end of MTs (the 298 second group) or disrupt the stacking of tubulin dimers (the third group). Therefore, changes in 299 tubulin structure led to compromised MT elongation, which caused severe defects in neurite 300 outgrowth, highlighting the importance of functional MTs in neuronal morphogenesis. 301

Neomorphic mutations in mec-7 led to ectopic neurite growth by increasing MT stability 302
The mec-7(neo) mutations appear to induce the growth of ectopic ALM-PNs by forming 303 hyperstable MTs, as first suggested by Kirszenblat et al. (2013). Supporting this hypothesis, 304 pharmacological stabilization of MTs with paclitaxel induced the growth of ALM-PN, although 305 at a low frequency, and destabilizing MTs with colchicine can partially suppress this ectopic 306 growth in mec-7(ky852 neo) mutants (Kirszenblat et al., 2013) and in mec-7(u278 neo) and mec-307 7(u1017 neo) animals ( Figure S3A). We found that mec-7(neo) animals had increased resistance 308 to colchicine compared to the wild-type animals. More of this MT-destabilizing drug was needed 309 to produce the same level of reduction in both TRN function (touch sensitivity) and PLM-PN 310 length in neo mutants than in wild-type animals ( Figure S3B-C). Moreover, these mutants also 311 showed increased tubulin acetylation ( Figure 4C), an indication of stable MTs (Song and Brady, 312 2015). 313 expression 396 In addition to neurite growth, MTs are important for many other functions in TRNs 397 (Bounoutas et al., 2009a;Hsu et al., 2014). Deletion of either mec-12 or mec-7 resulted in touch 398 insensitivity, defects in the localization of pre-synaptic vesicles, and severe loss of MT 399 acetylation, a mark for stable MTs (Figure 4 and S4A). Since MEC-12 is the only α-tubulin 400 isotype that contains the acetylation site (lysine 40) in C. elegans, the TRN-specific 15-p MTs 401 are likely the only MTs that can be acetylated. The loss of mec-7 and mec-12 also induced a 402 global reduction in protein levels through a mechanism dependent on the MAPK pathway dual 403 leucine zipper-bearing kinase DLK-1 (Bounoutas et al., 2011). Interestingly, mutations in dlk-1 404 restored the normal protein expression in mec-7 and mec-12 null mutants but failed to rescue the 405 PLM-PN growth defects in mec-7(lf) animals ( Figure 4D and S4B). Therefore, this PLM-PN 406 defect is most likely a direct effect of the loss of MEC-7 and not the result of secondary changes 407 in protein levels. 408 Compared to the mec-7(lf) mutants, the mec-7(anti) mutations caused stronger defects not 409 only in neurite outgrowth, but also in mechanosensation, synaptic vesicle transport, tubulin 410 acetylation, and protein expression levels (Figure 4 and S4B). Those phenotypes in mec-12(anti) 411 mutants were more severe than the phenotypes in the mec-12(lf) alleles and were comparable to 412 those produced by mec-7(lf) mutations but not as strong as the mec-7(anti) mutations ( Figure 4). 413 The mec-7 and mec-12 neo alleles also caused touch insensitivity, synaptic vesicle transport 414 defects, and reduced TRN protein levels, as found in the lf mutants; but the neo mutants had 415 markedly increased MT acetylation ( Figure 4). Moreover, the fact that both impaired (in anti 416 mutants) and excessive (in neo mutants) neurite growth can occur in conjunction with other TRN 417 defects suggests that the role of MTs in regulating neurite development is genetically separable 418 from other MT functions. 419 Furthermore, several mec-12 partial lf mutations affected only a subset of MT functions. 420 mec-12(e1605) animals carrying the H192Y missense mutation were defective in 421 mechanosensation but had retained normal 15-p MT structures, tubulin acetylation, axonal 422 transport, protein levels, and neurite growth patterns ( Figure S5; Bounoutas et al., 2009a). 423 Similarly, mec-12 mutants carrying the u63 (E415K) or gm379 (G416E) alleles were partially 424 touch-insensitive but kept the large dimeter MTs; however, these animals showed mistargeting 425 of synaptic vesicles presumably because alterations in the EEGE (amino acid 414-417) motif 426 increased affinity with the motor dynein (Hsu et al., 2014). u63 and gm379 mutations also 427 caused a slight decrease in tubulin acetylation and a partial reduction in protein production; 428 gm379 allele led to very mild defects in the growth of PLM-PN ( Figure S5). These mutations 429 may be useful in understanding some specific aspects of MT functions. 430

Modeling the effects of tubulin mutations using the TRN neurites 431
Our genetic analysis shows that missense mutations in tubulin genes can have distinct 432 effects on MT stability and neurite growth pattern, and those different phenotypes appear to 433 correlate with the positions of the altered residues in the tubulin structure. Such structure-434 function analyses have been carried out for the yeast α-tubulin TUB1 and the Drosophila testis-435 specific β-tubulin βTub85D (Fackenthal et al., 1995;Richards et al., 2000) but not for tubulins 436 expressed in the nervous system. Moreover, recent discovery of over a hundred missense 437 mutations in tubulin genes in patients with a range of neurological disorders prompted us to 438 examine the cellular impact of specific tubulin mutations on neurons. Combining the 439 convenience of genome editing in C. elegans and the ease of observing TRN morphology as the 440 readout of MT stability and neurite growth, we could systematically study the effects of those 441 human tubulin mutations. To provide a proof of concept, we generated five clinically observed 442 β-tubulin mutations (S172P, P173L, A302T, P380S, and E410K) in mec-7 through 443 CRISPR/Cas9-mediated gene editing (Materials and Methods; Figure 5A) and found that these 444 mutations indeed caused distinct phenotypes on TRN morphogenesis. 445 Heterozygous S172P and P173L mutations in human TUBB3 result in microcephaly, 446 polymicrogyria, cortical dysplasia, and agenesis of the corpus callosum, all of which are defects 447 in neuronal migration and axon growth (Jaglin et al., 2009;Poirier et al., 2010;Bahi-Buisson et 448 al., 2014). In vitro studies found that tubulin heterodimer containing the β-tubulin S172P mutant 449 could not be incorporated into MTs (Jaglin et al., 2009), indicating that the mutated protein is 450 nonfunctional. Consistent with these findings, the u1056 (S172P) mutation of mec-7 led to a 451 recessive phenotype similar to mec-7(lf) in the TRNs, which showed significantly shortened 452 PLM-PN and slightly shortened PLM-AN with branching defects ( Figure 5B). Surprisingly, the 453 u1057 (P173L) allele did not cause any TRN morphological or functional defects ( Figure 5B). 454 The difference between the mutant phenotypes in humans and worms is puzzling, since both 455 S172 and P173 are located on the GTP binding B5-to-H5 loop ( Figure 3C), which is identical in 456 C. elegans MEC-7 and human TUBB3 ( Figure S6). Perhaps the differences arise from 457 differences in MT structure and organization: the C. elegans TRNs have 15-p MTs that are 458 bundled, whereas human neurons have 13-p MTs that are not bundled. In fact, when 15-p MTs 459 were converted to 13-p MTs and the bundle was disrupted in mec-17 (α-tubulin acetyl-460 transferase) mutants (Topalidou et al., 2012), both MEC-7 S172P and P173L mutations led to 461 decreased MT stability and the loss of ALM-PN ( Figure 5B and S7). This result suggests that 462 P173L mutation indeed compromises MT stability in TRNs under sensitized conditions. 463 Heterozygous A302T and R380C mutations in human TUBB3, which affect residues on 464 the external surface of the MTs, were associated with moderate congenital fibrosis of the 465 extraocular muscles 3 (CEFOM3), anterior commissure hypoplasia, and corpus callosum 466 hypoplasia. Mutation of A302 and R380 in yeast led to the formation of highly stable, benomyl-467 resistant MTs (Tischfield et al., 2010). Consistent with these observations, we found that the 468 same mutations of MEC-7 produced a neo phenotype (the generation of an ectopic ALM-PN; 469 Figure 5C). 470 TUBB3 E410K mutation was also found in patients with severe CEFOM3 and hypoplasia 471 of anterior commissure and corpus callosum, but these patients also suffer from facial weakness 472 and progressive axonal sensorimotor polyneuropathy (Tischfield et al., 2010). In yeast, MTs 473 containing β-tubulin with the E410K substitution were less stable and less resistant to benomyl 474 and had markedly decreased plus-end accumulation of kinesin-like motor proteins, compared to 475 MTs with A302T and R380C mutations (Tischfield et al., 2010). In C. elegans, MEC-7(E410K) 476 produced a distinct neurite growth phenotype: ALM-AN and PLM-AN, but not PLM-PN were 477 significantly shortened, which indicates reduced MT stability; but the PLM-PN was not affected, 478 making the phenotype also different from that of the mec-7(anti) alleles, which cause general 479 defects in MT polymerization ( Figure 5D). Since E410 is also exposed to the exterior of MTs 480 like A302 and R380 ( Figure 3E), these results suggest that the cellular impact of the missense 481 mutations would depend on the specific amino acid change instead of the location of the affected 482 residues alone. 483 The loss of tba-7/α-tubulin also caused excessive posterior neurite growth 484 In addition to MEC-7 and MEC-12, we identified a second α-tubulin gene tba-7 that also 485 regulates neurite growth in the TRNs. In the course of a mutagenesis for mutants with abnormal 486 TRNs (Supplemental Results), we isolated a missense tba-7 allele u1015 (G92D), which caused 487 the growth of an ectopic ALM posterior neurite, resembling the phenotype of mec-7 and mec-12 488 neo alleles ( Figure 6A and B). This tba-7 allele is likely a lf allele, because it failed to 489 complement with gk787939 (Q230*), which is presumably a null and produced a similar 490 phenotype as u1015 ( Figure S1C). We also examined mutants of other tubulin isotypes, and did 491 not find morphological defects in the TRNs (we did not test for redundancy among the genes; 492 Table S2). 493 tba-7 was expressed in the TRNs, and the excessive growth phenotype of tba-7(u1015) 494 could be rescued by expressing tba-7(+) from the TRN-specific mec-17 promoter, indicating that 495 TBA-7 acts cell-autonomously in the TRNs ( Figure S8). tba-7(lf) mutants did not exhibit touch 496 insensitivity, synaptic vesicle mistargeting, or reduced protein levels, suggesting that the general 497 function of TRN MTs was not affected and that the primary function of TBA-7 is to regulate 498 neurite growth ( Figure S9). We hypothesized that the loss of TBA-7 led to the formation of 499 hyperstable MTs, similar to the mec-7 and mec-12 neo mutations. Supporting this hypothesis, 500 electron microscopy studies revealed that, like mec-7(neo) mutants, tba-7(u1015) animals 501 retained the large diameter 15-p MTs and have closely packed MT bundles, which occupied a 502 larger-than-normal area of a neurite cross-section ( Figure 1). tba-7(lf) mutants also retained 503 normal tubulin acetylation levels ( Figure S9C) and had increased resistance to colchicine ( Figure  504 S3), which confirmed the presence of stable MTs. Treatment with 1 mM colchicine fully 505 suppressed in the growth of ALM-PN in tba-7(u1015) animals, as did either lf or anti mutations 506 in mec-7 or mec-12 ( Figure 6B). 507 The amino acids sequences of α-tubulin TBA-7 and MEC-12 are 82% identical and 94% 508 similar; they mainly differ in a N-terminal region and the C-terminal tail ( Figure 6C). Domain 509 swapping experiments showed that changing L31 of TBA-7 to Q found in MEC-12 or replacing 510 the C-terminal DANDNGD (a.a. 435-441 in TBA-7) sequence with MEDNGEEG (a.a. 440-447 511 in MEC-12) partially impaired the function of TBA-7 proteins to restrict excessive neurite 512 growth; and TBA-7 proteins with both L31Q mutation and the C-terminal replacement 513 completely lost the ability to rescue the tba-7(lf) phenotype ( Figure 6D). Q31 in MEC-12 is a 514 potential site for polyamination (Song et al., 2013) and E445 of the GEE motif (a.a. 444-446 in 515 MEC-12, but absent in TBA-7) is a target for polyglutamination (Edde et al., 1990). Post-516 translational modifications at these two sites could increase the stability of neuronal MTs (Song 517 and Brady, 2015). Interestingly, replacing the a.a. 35-47 region of TBA-7 with the sequence 518 from MEC-12 that contains lysine 40 (the site for α-tubulin acetylation) did not affect the TBA-7 519 function ( Figure 6D), suggesting that the absence of this MT acetylation site in TBA-7 was not 520 responsible for its activity in preventing ectopic neurite growth. 521 Since TBA-7 lacks some modification sites (e.g. Q31 and E445) that could stabilize MTs, 522 our results suggest that TBA-7 may be a MT-destabilizing tubulin isotype, as compared to MEC-523 12. In wild-type animals, TBA-7 incorporation into the 15-p MTs with MEC-7 and MEC-12 may 524 reduce MT stability; when TBA-7 is not available, more MEC-12 and possibly other α-tubulin 525 isotypes replace TBA-7 and this replacement led to the formation of hyperstable MTs. 526 The tba-7(u1015 lf); mec-7(u1017 neo) double mutant had an ALM-PN that was similar 527 in length to that in mec-7(u1017) single mutants ( Figure 6A) but additionally produced up to 4 528 and 5 short ectopic neurites sprouting from the ALM and PLM cell bodies, respectively; this 529 phenotype was rarely observed in mec-7(u1017) animals ( Figure 6E Figure 6G). KLP-7 belongs to the kinesin-13 family of catastrophe factors that bind to 538 MT plus-ends and promotes depolymerization, thus generating dynamic microtubules (Han et al., 539 2015). Importantly, we found that, similar to the tubulin neo alleles, the growth of the ectopic 540 ALM-PN in klp-7(lf) mutants was suppressed by the loss of mec-7 or mec-12, but the klp-7 541 phenotype was not enhanced by mec-7(neo) mutations. These results suggest that the mutated 542 MEC-7 proteins may render MTs hyperstable by reducing their interaction with KLP-7 or by 543 making the MTs insensitive to the action of KLP-7. 544

Discussion 545
Neuronal morphogenesis requires optimal MT stability 546 MTs both support and regulate neurite growth. As the building block of MTs, αand β-547 tubulins determine the structural properties of MTs and mediate their interaction with motor 548 proteins and MAPs. As a result, any alteration in the tubulin proteins can potentially lead to 549 changes in MT stability, which then affects neurite formation, guidance, and extension. In this 550 study, we analyzed the effects of 67 tubulin missense mutations on neurite development in C. 551 elegans touch receptor neurons. Based on the phenotypes, we categorized these mutations into 552 three classes: loss-of-function (lf), antimorphic (anti), and neomorphic (neo)  The correlation between the amount of neurite growth and the stability of MTs suggests 562 that MT structure and dynamics are major determinants of neurite growth. Given the great 563 number of proteins involved in both positively and negatively regulating MT assembly and 564 disassembly (Mimori-Kiyosue, 2011), our results support the hypothesis that an optimal level of 565 MT stability is required for proper neuronal morphogenesis. The finding that negative regulators 566 of MT stability, such as the α-tubulin isotype TBA-7 and the MT-destabilizing kinesin-13 KLP-567 7, prevent excessive TRN neurite growth further supports this hypothesis. 568

Structure-function analysis of tubulin mutations 569
The large number of missense mutations analyzed in this study allowed us to perform a 570 structure-function analysis of neuronal tubulin proteins in living animals. Mapping the altered 571 amino acid residues onto the Bos taurus tubulin α/β heterodimer structure 1JFF (Nogales et al., 572 1998), we found that lf mutations mostly affect residues located in the interior of the structure 573 and possibly disrupt protein folding. anti mutations affected residues that participate in GTP 574 binding, intradimer interaction, or interdimer interactions. neo mutations mostly altered residues 575 on the exterior surface and probably perturb the association of motor proteins and MAPs with 576 MTs (Figure 3 and 7). 577 Our findings are consistent with similar structure-function relationship studies conducted 578 in yeast α-tubulin TUB1 and β-tubulin TUB2 and Drosophila testis-specific β2-tubulin (Reijo et 579 al., 1994;Fackenthal et al., 1995;Richards et al., 2000) and provide another model to study the 580 properties of tubulin proteins. For example, yeast TUB1 mutations that caused supersensitivity to 581 the MT-destabilizing drug benomyl mainly altered amino acids involved in GTP binding (e.g. 582 D70) and intradimer (e.g. E98) and interdimer interaction; the change of equivalent residues in 583 MEC-12 (D69 and E97) were identified in mec-12(anti) mutants. Similarly, some Drosophila β2-584 tubulin mutations (S25L and G96E) that made MTs less stable (Fackenthal et al., 1995) affected 585 the same or the adjacent residue in mec-7(anti) alleles [u319 (S25F) and u430 (A97V)]. 586 Moreover, yeast TUB1 mutations conferring benomyl-resistance mostly affected the residues on 587 the outer surface of the MTs (Richards et al., 2000) as did the mec-7(neo) mutations similarly 588 that were resistant to colchicine. Thus, comparative studies using the three different organisms 589 can help identify key residues in the tubulin structure responsible for various MT functions. 590

Modeling the effects of neuronal tubulin mutations 591
We have used the C. elegans TRNs as a model to study neuronal MTs, which are much 592 more stable and have different types of post-translational modification than the MTs in dividing 593 cells (Baas et al., 2016). Clinical studies in the past five years identified more than 100 missense 594 mutations in tubulin genes that cause a wide spectrum of neurodevelopmental disorders, such as 595 microcephaly, lissencephaly, polymicrogyria, and cortical malformation, along with severe 596 defects in axon guidance and growth, including the complete or partial absence of corpus 597 callosum, defects in commissural fiber tracts, and degeneration of motor and sensory axons 598 (Tischfield et al., 2011;Chakraborti et al., 2016). Patients carrying different tubulin mutations 599 often display distinct symptoms, but the complexity of the human nervous system makes it 600 difficult to analyze the impact of specific mutations at the cellular level. 601 Combining CRISPR/Cas9-mediated genome editing with in vivo examination of TRN 602 morphology, we were able to create several disease-causing human β-tubulin mutations in the C. 603 elegans mec-7 gene and examined their effects on neurite growth at a single neuron resolution. 604 Our phenotypic evaluation was largely consistent with previous characterization of those 605 mutations, suggesting that the TRN system could be instrumental to model the effects of human 606 tubulin mutations identified in the clinic. 607 In fact, many disease-causing tubulin mutations alter amino acids that are physically 608 adjacent to or located in the same region as the ones affected in our mutants, suggesting that they 609 may affect MT stability in similar ways. By mapping 51 TUBA1A, 24 TUBB2B, and 19 610 TUBB3B mutations that were clinically identified onto the tubulin structure, we could correlate 611 the location of some of the affected residues with the resulting clinical manifestation (Table S1). 612 For example, 7/8 (87.5%) mutations that alter residues involved in GTP binding caused complete 613 agenesis of corpus callosum, an indication of severe defects in axonal growth, whereas only 5/23 614 (21.7%) changes of residues in the interior of the structure and 10/32 (31.2%) mutations of 615 residues involved in MAP binding did so (Table S1). 616 Position of the mutated residues alone, however, could not determine the effects of the 617 missense mutation. For example, although the β-tubulin B5-to-H5 loop (from V170 to V179) 618 contacts GTP, and four strong mec-7(anti) mutations (P171S, P171L, S176F, and V179A) 619 mapped to this loop, the S172P mutation led to a lf phenotype and P173L mutation caused no 620 defects in TRNs. One possible explanation is that S172 and P173 are further away from the GTP 621 binding site, thus their mutations interfere less with microtubule function. 622 The identity of the residue replacing the wild type one is also important. For instance, 623 u278 (C303Y) was the strongest mec-7(neo) mutant, but gk286001 (C303S) had no effect at all; 624 since both tyrosine and serine have the polar hydroxyl group, the bulky phenyl group of the 625 tyrosine may be responsible for disrupting some MT functions. Moreover, the u1058 (A302T) 626 mutation also caused a strong mec-7(neo) phenotype. A302 and C303 on the H9-to-B8 loop are 627 exposed to the exterior of MTs but are not located on the major landing surface (H11 and H12) 628 for motor protein and MAPs (Nogales et al., 1998). Our results suggest that A302 and C303 may 629 represent a previously unrecognized site of interaction between the MTs and their associated 630 proteins. Even for residues located on H11 and H12, the effects of their mutation can be different. 631 u1059 (R380S) is a mec-7(neo) mutation, whereas u1060 (E410K) allele produced an anti-like 632 phenotype, suggesting that the impact of the missense mutation depends on how the interaction 633 of MTs with the motor proteins or MAP is affected by the amino acid change. 634

MT-destabilizing tubulin isotype and "multi-tubulin hypothesis" 635
The presence of multiple tubulin genes in eukaryotic genomes, their subtle differences in 636 the protein sequences, and their functional differences led to the "multi-tubulin hypothesis." In 637 multicellular organism, the tubulins may have distinct expression patterns, e.g. Drosophila β2-638 tubulin is only expressed in the male germline (Kemphues et al., 1980); human TUBB3B is 639 highly enriched in the nervous system, whereas TUBB2B is expressed in many tissues (Sullivan 640 and Cleveland, 1986). In single-cell organisms like Tetrahymena, different β-tubulin isotypes are 641 spatially separated; some are only used to make the nuclear MTs of the mitotic apparatus, and 642 some are only detected in the MTs of somatic cilia and basal bodies (Pucciarelli et al., 2012). 643 Our studies, however, found that two distinct α-tubulin isotypes (MEC-12 and TBA-7) could 644 have different functions when both incorporated into the same MTs in the same neurons; MEC-645 12 promotes stability and TBA-7 promotes instability. 646 The abundance of large diameter 15-p MTs in the TRNs requires MEC-12, but the role of 647 TBA-7 is to increase MT dynamics and to prevent the ectopic growth of posteriorly directed 648 neurites in ALMs. Thus, TBA-7 is a MT-destabilizing tubulin isotype, and this destabilizing 649 activity depends on the absence of potential polyamination and polyglutamination sites that 650 could increase MT stability. Our results are consistent with early in vitro experiments suggesting 651 that TUBB3 (also known as β III ) also promotes MT dynamics; removal of ΤUΒΒ3 from the brain 652 extract resulted in a tubulin mixture that assembled much more rapidly than the unfractionated 653 control (Banerjee et al., 1990), and MTs assembled from the purified αβ III heterodimers were 654 considerably more dynamic than MTs made from the αβ II and αβ IV dimers (Panda et al., 1994). 655 However, the long-standing question is whether some tubulin isotypes do destabilize MTs in vivo. 656 Our findings suggest the answer is yes. A balanced incorporation of multiple tubulin isotypes 657 into the same MT structure is critical to generate MTs with the optimal stability. 658 The absence of post-translational modification sites in tubulin isotypes may lead to 659 functional diversity. In addition to TBA-7, among the other eight C. elegans α-tubulin isotypes, 660 TBA-8 and TBA-5 also lack the Q31 for polyamination, and TBA-6 lacks the E445 for 661 polyglutamination; all nine human α-tubulins have Q31, but TUBAL3 has a short C-terminal tail 662 and does not contain potential polyglutamination sites ( Figure S10). The incorporation of those 663 tubulin isotypes may be a general regulatory mechanism to control MT stability. Despite the 664 functional differences between MEC-12 and TBA-7, the fact that both mec-12(lf) mutants and 665 mec-12(lf); tba-7(lf) double mutants retained the ability to grow out a normal amount of neurites 666 suggests also genetic redundancy among the α-tubulins in supporting neurite growth. 667 Different contributions of tubulin isotypes to MT dynamics were also observed in C. 668 elegans embryogenesis (Honda et al., 2017). The spindle MTs required two β-tubulins (TBB-1 669 and TBB-2), but TBB-2 was incorporated into the MTs twice as much as TBB-1; the loss of 670 TBB-2 caused a dramatic decrease in MT growth rate and an increase in catastrophe frequency, 671 leading to highly unstable MTs, whereas the loss of TBB-1 only slightly reduced growth rate. 672 These data support the "multi-tubulin concept" that different tubulin isotypes have distinct 673 functions in the same cells. 674

Electron microscopy 731
We re-analyzed previously collected cross-section images of ALM neurites from wild 732 type animals (N501, N631, N933, and N934 from the Hall lab collection) and from mec-7(neo) 733 mutants u170 and u278 (Savage et al., 1994). The wild type animals had been fixed by either 734 chemical immersion fixation without tannic acid (samples N501, N631;Hall, 1995) or by high-735 pressure freezing/freeze substitution (HPF/FS) including tannic acid (samples N933, N934;736 Topalidou et al., 2012). The mec-7(neo) alleles had also been fixed by chemical immersion, 737 without tannic acid. 738 For this study, we fixed mec-7(ok2152 lf), mec-12(tm5083 lf), and tba-7(u1015 lf) adults 739 using an HPF/FS protocol that included a first fixation in 0.5% glutaraldehyde + 0.1% tannic 740 acid and a second fix in 2% osmium tetroxide + 0.1% uranyl acetate (UAc), both at -90 °C, 741 followed by staining in 1% UAc in acetone at 0 °C. We also fixed adults of mec-12(anti) mutants, 742 u950, u76, and u1021, using HPF/FS using a similar protocol, but without tannic acid. Eighty-743 nanometer transverse sections were collected at multiple positions along the ALM anterior 744 neurite and post-stained with uranium acetate and lead citrate. A Philips CM10 electron 745 microscope with an attached Morada digital camera (Olympus) was used to acquire the images. 746 For each animal, we counted the number of MTs in the ALMs and measured the MT 747 diameter on sections collected from at least five different positions along the ALM-AN; data 748 from ALML and ALMR were combined. For each strain, we examined sections from at three 749 different animals. For wild-type animals, tba-7(lf), and mec-7(neo) mutants, we used at least ten 750 sections to measure the distance between two closest MT centers and the proportion of MT-751 occupied area to the cross-sectional area of the neurite. 752 We noticed that our earlier studies found that wild-type TRN MTs had much larger 753 diameters (29.6 ± 0.4 nm; Chalfie and Thomson, 1979) than we find here (21.5 ± 1.6 nm). 754 Fixation method could not explain the difference, because we obtained similar results from N501 755 and N631 (fixed by chemical immersion) and N933 and N934 (fixed by HPF/FS); data obtained 756 using both methods showed smaller MT diameters than the measurements from earlier studies, 757 which used chemical immersion fixation. Although we could not explain this discrepancy, we 758 are very confident in the current calibration of the electron microscope. 759

Phenotype Scoring and Statistical Analysis 760
We measured the length of TRN neurites in at least 30 fourth stage larvae or young adults 761 grown at 20° C, except where otherwise stated. Relative length of posteriorly directed neurites 762 was calculated by dividing the neurite length by the diameter of the cell body. Defects in TRN 763 anterior neurite length were assessed by counting the percentage of cells whose neurites failed to 764 reach the vulva (for PLM) or the posterior pharyngeal bulb (for ALM); at least 50 cells were 765 examined for each strain. 766 Fluorescence intensity of the RFP expressed from the uIs134 transgene in the ALM cell 767 body was used to measure TRN protein levels; intensity was calculated after manual background 768 subtraction using ImageJ as previously described (Chen and Chalfie, 2015). Synaptic vesicle 769 localization was quantified by calculating the percentage of animals in each of the four 770 phenotypical categories: 1) normal localization to the PLM synaptic branch; 2) reduced intensity 771 of the synaptic marker at the synapses as partial defect; 3) complete loss of normal localization to 772 the synapse as complete defect; 4) localization to the PLM-PN as mistargeting. At least 50 adult 773 animals were examined. 774 Acetylated α-tubulin staining using antibody [6-11B-1] (Abcam, Cambridge, MA) was 775 performed and analyzed as previously described (Topalidou et al., 2012). For colchicine 776 treatment, animals were grown in standard NGM agar plates containing different concentrations 777 (from 0.06 mM to 2 mM) of colchicine before phenotypic analysis as described before 778 (Bounoutas et al., 2009a). Similarly, 100 nM paclitaxel was added to the NGM agar plates, L1 779 animals were placed onto the plates, and young adults were examined for TRN morphology. 780 PyMOL (Schrodinger, 2015) was used to view the structure of α/β tubulin dimer (1jff.pdb; 781 Nogales et al., 1998) and to label affected residues in mec-7 and mec-12 mutants,. 782 For statistical analysis, ANOVA and the post hoc Dunnett's test (comparison of each of 783 many treatments with a single control) or Tukey-Kramer test (studentized range test for all 784 pairwise comparison) were performed using the GraphPad Prism (version 5.00 for Windows, 785 GraphPad Software, La Jolla California USA, www.graphpad.com) to identify significant 786 difference between the mutants and wild type animals. Student's t test was used to find 787 significant difference between two samples in paired comparisons. Single and double asterisks 788 indicated p < 0.05 and p < 0.01, respectively. The χ 2 test was used for the categorical data to find 789 significant difference between different strains. 790 facilities for electron microscopy were supported by NICHD P30 HD71593 for the RFK-IDDRC 795