Chlamydomonas reinhardtii Tubulin-Gene Disruptants for Efficient Isolation of Strains Bearing Novel Tubulin Mutations

The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two β-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 24 strains bearing 12 different β-tubulin mutations. Some of these mutations are known to confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an efficient means of isolating novel C. reinhardtii tubulin mutants. IMPORTANCE Chlamydomonas reinhardtii is a useful organism for the study of tubulin function; however, only five kinds of tubulin mutations have been reported to date. This scarcity is partly due to C. reinhardtii possessing two tubulin genes each for α- and β-tubulin. Here, we obtained several strains in which one of the α- or β-tubulin genes was disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel tubulin mutations that conferred differing degrees of resistance to two anti-tubulin compounds. The majority of the tubulin mutations were located outside of the drug-binding sites in the three-dimensional tubulin structure, suggesting that structural changes underlie the drug resistance conferred by these mutations. Thus, single-tubulin-gene disruptants are an efficient means of generating tubulin mutants for the study of the structure–function relationship of tubulin and for the development of novel therapies based on anti-tubulin agents.

encoding identical amino acid sequences. Here, we used an aphVIII gene cassette 23 insertional library to establish eight disruptants with defective tua2, tub1, or tub2 24 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar 25 length, or flagellar regeneration after amputation. Because few tubulin mutants of C. 26 reinhardtii have been reported to date, we then used our disruptants, together with a tua1 27 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel 28 tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result 29 of several trials, we obtained 8 strains bearing 7 different -tubulin mutations and 24 30 strains bearing 12 different -tubulin mutations. Some of these mutations are known to 31 confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an 32 efficient means of isolating novel C. reinhardtii tubulin mutants. 33 34 IMPORTANCE: Chlamydomonas reinhardtii is a useful organism for the study of tubulin 35 function; however, only five kinds of tubulin mutations have been reported to date. This 36 scarcity is partly due to C. reinhardtii possessing two tubulin genes each for and 37 -tubulin. Here, we obtained several strains in which one of the or -tubulin genes was 38 disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel 39 tubulin mutations that conferred differing degrees of resistance to two anti-tubulin 40 compounds. The majority of the tubulin mutations were located outside of the drug-binding 41 sites in the three-dimensional tubulin structure, suggesting that structural changes underlie 42 48

INTRODUCTION 50
Microtubules are fundamental cytoskeletal filaments that play pivotal roles in eukaryotic 51 cell functions such as cell division, intra-cellular transport, cell shape development, and 52 cilia and flagella assembly. Microtubules are produced by polymerization of -tubulin 53 heterodimers. Most eukaryotic cells possess multiple genes encoding and -tubulin. For 54 example, humans possess seven genes that encode-tubulin and eight genes that encode 55 -tubulin, with each gene encoding a slightly different amino acid sequence. The presence 56 of multiple genes for the two types of tubulin makes it difficult to study the properties of a 57 particular tubulin species by genetic analysis, because the effects arising from mutation of 58 one of the genes can be masked by the expression of the remaining intact genes. 59 The single-cell green alga Chlamydomonas reinhardtii is a useful experimental 60 organism for studying tubulin function because it possesses a small number of tubulin 61 genes and it produces microtubule-based organelles, flagella. In addition, there is a wide 62 range of genetic tools available and a large amount of biological data has been 63 accumulated for this species. In contrast to the majority of eukaryotes, C. reinhardtii 64 possesses only two genes (tua1 and tua2) encoding -tubulin and two genes (tub1 and 65 tub2) encoding -tubulin (1, 2). The two genes for each type of tubulin encode the same 66 amino acid sequence (2, 3), and the expression of all four genes is up-regulated after 67 flagellar excision (4). Whether the two genes in each pair are expressed independently of 68 each other has not yet been firmly established, but the genes do appear to be expressed 69 indiscriminately during flagella formation (4). 70 Although C. reinhardtii possess only two genes for each tubulin, the presence of more 71 than one gene expressing the same protein still makes it difficult to isolate tubulin mutants. 72 disruption (tub1-A, tub1-B), and three showing tub2 disruption (tub2-A, tub2-B, tub2-C). 97 None of the disruptants exhibited any apparent defects in growth rate (data not 107 shown), tubulin expression (Fig. S2C), or flagellar regeneration after amputation (Fig.  108 S2D), suggesting that the disruptants still produced sufficient /-tubulin heterodimer for 109 their cellular functions via the remaining intact genes. The mean flagellar length was 110 comparable among the disruptants (see Fig. S2D). The five -tubulin disruptants showed 111 some difference in their sensitivity to colchicine: tub1-A and tub1-B showed stronger 112 resistance while tub2-A showed weaker resistance than wild type ( Fig. 1), although the 113 sensitivity somewhat varied among alleles (Fig. 1). 114 115

Mutant isolation using tubulin-gene disruptants 116
Next, we used the disruptants to isolate C. reinhardtii strains expressing tubulins with 117 missense mutations. Three parent strains were used: tub2-A, a double disruptant generated 118 by crossing tua1-A with tub1-B, and a double disruptant generated by crossing tua2-A and 119 tub1-B. As a result of 1-3 trials with each parental strain against oryzalin or propyzamide, 120 32 strains showing a total of 19 different tubulin missense mutations were isolated. Table 1  121 shows the obtained mutants classified by the gene affected, as well as the results of a 122 qualitative assessment of each strain's resistance to oryzalin and propyzamide. Most of the 123 oryzalin-resistant strains, as well as a propyzamide-resistant mutant (pyz532), had a 124 missense mutation in an -tubulin gene. In contrast, most of the propyzamide-resistant 125 strains, other than pyz532, had mutations in a -tubulin gene. 126 Figure 2 shows a predicted three-dimensional structure of C. reinhardtii /-tubulin 127 heterodimer labeled with the site of each missense mutation reported here and in previous 128 studies (3, 5, 6). Five of the isolates had mutations that have been reported previously: ory2 129 had a tua1 Y24H mutation as did upA12 (3); pyz8, pyz9, and pyz523 had a tub2 K350E 130 mutation as did col R 4 (6); and pyz6 had a tub2 K350M mutation as did col R 15 (6). The five 131 mutants isolated in the present study exhibited stronger drug-resistance than the three 132 previously reported mutants (data not shown). This stronger drug-resistance may reflect the 133 fact that the mutants isolated here express only mutated or -tubulin from a single gene, 134 whereas previously reported mutants express a mutated tubulin together with a wild-type 135

counterpart. 136
Some of the identified mutations involved the substitution of amino acids with 137 different charges. For example, the propyzamide-resistant missense strains pyz2/pyz524, 138 pyz503, and pyz530/pyz534/pyz502/pyz525/pyz526/pyz527 expressed -tubulins with the 139 mutations Q134H, E198L, and E198K, respectively. The isoelectric point (pI) values of 140 these -tubulins predicted from their amino acid sequences were 4.59, 4.58, and 4.63, 141 respectively, which were greater than the pI of wild-type -tubulin (4.55). We confirmed 142 the expression of -tubulins with different pIs in those strains by two-dimensional 143 polyacrylamide gel electrophoresis (2D-PAGE) of axonemal proteins from the mutants and 144 wild type (Fig. 2). As expected, the spot of -tubulin appeared at higher pH values in the 145 analysis also verified that each mutant expressed -tubulin from only a single gene, since it 147 detected no -tubulin spots with the wild-type pI in mutant samples. 148 149

DISCUSSION 167
By screening an AphVIII insertional library, we isolated three disruptants lacking tua2, two 168 lacking tub1, and three lacking tub2. All were most likely null mutants (Fig. S2A). 169 Although these disruptants lacked one of their tubulin-encoding genes, their cytoplasmic 170 tubulin levels remained normal (Fig. S2C), suggesting the presence of an auto-regulatory 171 mechanism that maintains the tubulin mRNA level, as observed in other eukaryotic cells 172 (10). Indeed, in tua1-A, tub1-B, and tub2-A, the mRNA expression level of the remaining 173 or -tubulin gene was increased approximately 2-fold compared with wild type (Fig.  174 S2B). Also, flagellar length, ability to produce flagella after amputation (Fig. S2D), and 175 overall cell growth rate did not noticeably differ from the wild-type growth rate (data not 176 shown). Thus, although C. reinhardtii possesses two -tubulin genes and two -tubulin 177 genes, a single gene for each type is enough to supply the tubulin necessary for its cellular 178 functions. However, it should be noted that the present findings do not mean that the two 179 genes for each tubulin have exactly the same function; rather, the two genes may differ 180 from each other in a subtle manner. For example, we observed that whereas the tub1 181 disruptants were resistant to colchicine, the tub2 disruptants were sensitive although some 182 allele-specific variation was observed (Fig. 1). This suggests that there is some difference 183 in the regulation of gene expression that is dependent on the concentration of free tubulin 184 in the cytoplasm (11). Thus, how the two genes encoding the two tubulins differ in their 185 function and regulation warrants further investigation, and our single-tubulin-gene mutants 186 established here should be useful for such investigations. 187 Next, we used the disruptants to obtain mutants with resistance to two anti-tubulin 188 agents, propyzamide and oryzalin. Several rounds of trials to isolate mutants resistant to 189 one or both of the agents afforded 8 mutants with 7 different -tubulin gene missense 190 mutations and 24 mutants with 12 different -tubulin gene missense mutations. The 191 number of mutations obtained was much larger than the total number that has been 192 reported previously (i.e., 3 kinds of -tubulin mutations and 2 kinds of -tubulin 193 mutations) (3, 5, 6). In addition, we found that about one-third of the colonies picked from 194 the screening plates harbored a mutation in a tubulin gene (data not shown). Together, 195 these findings suggest that our approach of using single-tubulin-gene disruptants is a 196 highly efficient means of obtaining tubulin mutant strains. 197 How the sensitivity to anti-tubulin agents varied in the mutants is an important issue 198 that warrants clarification. Some of the tubulin mutants that conferred resistance to the 199 anti-tubulin agents had a mutation near to where the anti-tubulin agents bind to the 200 /-tubulin heterodimer (Fig. 3). The binding site of oryzalin, inferred from that of an 201 analogous compound, tubulysin M, is at the intra-dimer interface (12) close to the 202 -tubulin mutation F351L (in strain pyz532). Other ory mutants whose mutation sites  (Table S1). For -tubulin, mutation F49C in ory314, F52L in ory3, and 216 S165A in ory205/ory505 have been reported in a Toxoplasma gondii oryzalin-resistant 217 mutants (14, 15). For -tubulin, mutation Q134H in pyz2/pyz524 has been reported in a 218 Beauveria bassiana benzimidazole-resistant mutant (16); mutations E198K/L in 219 pyz530/pyz534/pyz502/pyz525/pyz526/pyz527 and pyz503 are found in fungi and 220 nematodes that confer benzimidazole resistance and phenylcarbamate hypersensitivities 221 Although the present study selected mutants based only on their resistance to two 231 anti-tubulin agents, use of other agents such as the microtubule-stabilizing agent paclitaxel, 232 or screening for other properties such as hypersensitivity to drugs, resistance to low 233 temperature, or deficiency in flagellar formation and motility will lead to the isolation of a 234 greater variety of mutants. Detailed analyses of many such mutants will deepen our 235 understanding of the structure-function relation of tubulins. Since some of the tubulin 236 mutations identified in the present study corresponded to mutations found in human 237 tubulins that confer drug resistance in cancer cells, we expect that studies of 238 Chlamydomonas tubulin mutants will contribute to the development of improved cancer 239 therapies. 240

Isolation of anti-tubulin drug resistant mutants 263
C. reinhardtii strains whose tub1 or tub2 was disrupted with or without tua2 disruption 264 were grown to the mid-log phase and then irradiated by ultraviolet light until about 50% of 265 the cells were killed. The culture was spread on TAP-agar plates containing 20 M 266 propyzamide or 10 M oryzalin, kept in the dark for 12 h, and then incubated under light 267 for 5-10 days. Colonies that appeared were transferred to liquid TAP medium in 96-well 268 plates containing the same concentration of propyzamide or oryzalin. From each culture 269 that grew, genomic DNA was extracted and subjected to PCR using the following primers: Co., Japan) and cultured for a week at 26°C under 12-h light/12-h dark conditions. A 281 wild-type strain (CC-125) and a colchicine-resistant mutant strain (col R 4 (6)) were used as 282 references. 283 284

Three-dimensional structure prediction of C. reinhardtii/-tubulin heterodimer 285
The three-dimensional structure of the C. reinhardtii/-tubulin heterodimer was 286 predicted by using FAMS software (27) based on a known tubulin tetramer structure 287 obtained from the Protein Data Bank (PDB ID: 1Z2B (28)). To determine the amino acids 288 that most likely interacted with the examined drugs, in silico molecular docking analyses 289 were performed using the ChooseLD program (29). 290

2D-PAGE of isolated axonemes 292
Axonemes were isolated from the C. reinhardtii strains by using standard procedures (30). 293 A small aliquot of axonemal precipitate (~2 or 10 g) was extracted with a buffer 294 containing 5 M urea and 2 M thiourea and analyzed by 2D-PAGE as described previously 295 (31). Since and -tubulin are modified post-translationally, the loading amount was 296 adjusted so that their major forms only were detectable by silver staining. The predicted pI 297 values of the wild-type and mutant tubulins were calculated by using the EMBOSS 298 database and the Sequence Manipulation Suite, which is a collection of JavaScript 299 programs for examining short protein sequences 300    Figure 3 Kato-Minoura, T. et al.