The brahma gene encodes the catalytic subunit of the Drosophila melanogaster BRM chromatin-remodeling complexes. Screening for mutations that interact with brahma, we isolated the dominant-negative Pearl-2 allele of Tub23C. Tub23C encodes one of the two -tubulin isoforms in Drosophila and is essential for zygotic viability and normal adult patterning. -Tubulin is a subunit of microtubule organizer complexes. We show that mutations in lethal (1) discs degenerate 4, which encodes the Grip91 subunit of microtubule organizer complexes, suppress the recessive lethality and the imaginal phenotypes caused by Tub23C mutations. The genetic interactions between Tub23C and chromatin-remodeling mutations suggest that -tubulin might have a role in regulating gene expression.

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THE trithorax and Polycomb group genes encode positive and negative factors required for the proper function of homeotic genes. KENNISON and TAMKUN (1988) identified brahma (brm) as a trithorax group gene required for the maintenance of homeotic gene expression, but brm also regulates the expression of many developmental regulators and facilitates global transcription from RNA polymerase II (ARMSTRONG et al. 2002). The Brm protein is a SWI2/SNF2 family ATPase and is the catalytic subunit of BRM chromatin-remodeling complexes. These complexes modify nucleosome structure; they can also act to generate Z-DNA structures (reviewed in FLAUS and OWEN-HUGHES 2004).

Drosophila BRM complexes and related mouse and human SWI/SNF complexes have roles in a variety of processes, including cell proliferation, differentiation, viral infection, and cancer (reviewed by ROBERTS and ORKIN 2004). Targeting of the BRM complexes for transcriptional regulation involves contact with members of the basal transcription machinery and gene-specific transcriptional activators (for examples, see SHARMA et al. 2003; ARMSTRONG et al. 2005). To identify proteins that are required for proper function of homeotic genes, we screened for mutations that showed genetic interactions with brm mutations to cause a held-out-wings phenotype. This approach allowed us to isolate mutations in the trithorax group genes osa, tonalli, and taranis (VÁZQUEZ et al. 1999; GUTIÉRREZ et al. 2003). In this work, we describe the characterization of another mutation isolated in this genetic screen, the Pearl-2 allele of Tub23C. Some Tub23C mutant phenotypes are modified (enhanced or suppressed) by mutations in genes encoding subunits of the BRM complexes and by mutations in Grip91, a -tubulin ring complex subunit. These data suggest a role for -tubulin in transcription.

Fly strains:

Flies were raised at 25° on a yeast–sucrose–agar medium with either Nipagin or propionic acid or on a cornmeal–molasses–yeast–agar medium with Tegosept. Unless otherwise noted, all mutations and chromosome aberrations are described in LINDSLEY and ZIMM (1992). Mutant stocks carrying l(1)dd4^G0122, l(1)dd4², l(2)23Ce^A6-2, l(2)23Ce^A14-9, l(2)23Ce^A15-2, and Tub23C^bmps1 were provided by the Bloomington stock center.

Mutant phenotypes:

The viability (in percentage) of homozygous or heteroallelic combinations of alleles was determined by dividing the observed number of flies by the expected number and multiplying by 100%. The expected numbers were calculated by counting the numbers of progeny in the crosses that received the balancer chromosomes and dividing by half.

Genetic mapping:

The Tub23C^Pl-2 mutant was first mapped meiotically between the visible markers al and dp.

The dd4^su(Pl) mutation was first mapped by meiotic recombination using visible markers. Individual recombinant sons from females heterozygous for dd4^su(Pl) and a y² w^a ct⁶ g² f mutant chromosome were recovered and tested for the survival of Tub23C^bmps1/Tub23C^A6-2 trans-heterozygotes. After the initial mapping, 28 recombinants between ct⁶ and g² and 13 recombinants between g² and f were recovered and tested. None of these recombinants separated the suppressor from g⁺.

P-induced male recombination mapping of Tub23C^Pl-2:

Females with the P-element insertions shown in Table 1 were crossed with males of the genotype al Tub23C^Pl-2 Kr^If/+; TMS, P{ry^+t7.2=Delta2-3}99B/+. Sons that were P{X}/al Tub23C^Pl-2 Kr^If; TMS/+ were crossed to al dp b pr c px sp females and the progeny scored for recombinants between al and Kr^If. Recombinants were recovered and balanced for further testing. To determine which recombinants carried flanking deletions that removed essential genes, each recombinant chromosome was crossed to deletions in 23CD and to known mutants in 23C [lilli, l(2)23Cb, l(2)23Cd, Tub23C^A6-2, and okra]. Although l(2)23Cb has been renamed l(2)23Dd by FlyBase, our deletion mapping places the gene between Rpb9 and Tub23C, consistent with the original mapping to 23C and the original gene name.

View this table: In this window In a new window   TABLE 1 P-induced male recombination

 

Molecular analyses:

After Tub23C^Pl-2 was mapped between P{EP}Rrp1¹⁰²⁰ and P{EPgy2}CG9643^EY07345, the DNA sequences of the open reading frames of all four predicted genes in the region (CG9641, CG3165, CG9643, and Tub23C) were determined from DNA isolated from homozygotes of Tub23C^Pl-2 (and the parental w;red e strain in which it was induced) and of Tub23C^A6-2 (and the parental cn bw chromosome in which it was induced). As the only nonsynonomous changes found between the two mutants and their parental chromosomes were in the Tub23C open reading frame (Figure 2C), we then determined the DNA sequence of Tub23C from Tub23C^A14-9 and Tub23C^A15-2 homozygotes. Sequencing was done from PCR-amplified genomic fragments. Mutant homozygotes were identified using a GFP-expressing balancer chromosome [CyO, P{w^+mC=ActGFP}JMR1].

View larger version (47K): In this window In a new window Download PPT slide   FIGURE 2.— Genetic and molecular characterization of the Pearl region in 23C. (A) Deficiency mapping of the Pearl region. (B) Genomic map of region 23C. Predicted transcriptional units in the region are shown by arrows indicating their transcriptional direction. P-element transposons in the region are indicated by inverted triangles. EP1020 is P{EP}Rrp11020, KG01159 is P{SUPor-P}Rrp1KG01159, and EY07345 is P{EPgy2}CG9643EY07345. The thickest line marks the 7-kbp region where Tub23CPl-2 was localized by P-induced male recombination. (C) The amino acid sequences for the two -tubulin proteins (labeled 23C and 37C) in Drosophila (WILSON et al. 1997) and the changes found in the Tub23C mutant alleles. The arrows indicate the changes in the Tub23C mutant alleles. At the top of each arrow are the allele name and the amino acid substitution. The numbered areas indicate peptides with known or presumed functions (taken from BURNS 1995 and references therein): 1 is a peptide implicated in the autoregulation of β-tubulin translation; peptides 2–10 are implicated in the binding or the hydrolysis of GTP by β-tubulin. Peptide 10 is also implicated in the release of -, β-, and -tubulin from the TCP1 chaperonine. Note that none of the Tub23C mutations are in any of the peptides with known or presumed functions and that all the changed residues in the Tub23C mutants are identical in both Tub23C and Tub37C wild-type genes.

 

The Tub23C^Pl-2 mutation enhances brahma mutants:

Flies heterozygous for some combinations of mutations in trithorax group genes have held-out wings (VÁZQUEZ et al. 1999). On the basis of this phenotype we isolated several dominant enhancers of brm, including alleles of the trithorax group genes osa (osa), tonalli (tna), and taranis (tara) (VÁZQUEZ et al. 1999; GUTIÉRREZ et al. 2003). From that same genetic screen, we also isolated the Tub23C^Pl-2 mutation. In addition to its dominant enhancement of brm (Table 2 and Figure 1C), Tub23C^Pl-2 has additional dominant phenotypes in the wing blade (Figure 1B), including pearl-like structures [predominantly in the second (L2) and/or third (L3) wing vein(s)], blisters in the wing blade, and notches or gaps in the ventral and dorsal margins in one or both wings (Figure 1B). Tub23C^Pl-2 heterozygotes also have small round eyes. We mapped Tub23C^Pl-2 to the same chromosomal region as Pearl (Pl) (ROSIN 1951, 1952), a dominant mutation that had the same unique combination of phenotypes. The original Pl mutant is no longer extant. We originally called our mutation Pl², but since it is allelic to Tub23C (see below), we have renamed it Tub23C^Pl-2.

View this table: In this window In a new window   TABLE 2 Tub23CPl-2 interactions with general transcription machinery and trithorax-group mutants

 

View larger version (65K): In this window In a new window Download PPT slide   FIGURE 1.— Tub23CPl-2-dependent phenotypes. (A) Wild-type fly with the wings held back parallel to the body axis. (B) Tub23CPl-2/+ flies; note the notches and the pearl-like structures in the wings (indicated by arrows, one of them pointing to the area shown in the inset). (C) Tub23CPl-2/+; brm2/+ (shown in C) and Tub23CPl-2/+; osa1/+ double heterozygous flies have held-out wings in addition to the wing notches and pearl-like structures. (D) Tub23CPl-2/+; brm2/osa1 triple heterozygous fly with twisted wings (in addition to held-out and notched wings with pearl-like structures). (E) Wild-type wing from Oregon-R stock. (F) Wing from Tub23CPl-2/+; osa1/+ double heterozygous fly. The arrow indicates a pearl-like structure along the third wing vein shown in the inset (G).

 

Mapping of Tub23C^Pl-2:

We first mapped Tub23C^Pl-2 by meiotic recombination and then by complementation with available chromosome deletions to polytene chromosome subdivisions 23CD (Figure 2A). We next used the P-element insertion lines in the 23CD region to map Tub23C^Pl-2 by P-induced male recombination (CHEN et al. 1998). Of 63 recombinants recovered, all except 1 appear to have resulted from recombination at the P-element insertion site. These 62 recombinants all place Tub23C^Pl-2 in the 7-kbp region between P{EP}Rrp1¹⁰²⁰ and P{EPgy2} CG9643^EY07345 (Figure 2B). Inverse PCR with recombinants that still retained the P insertion and genetic complementation with mutants in the region were both used to identify recombinants with flanking deletions and duplications in the 23BD region. The flanking deletions were useful for determining the order of the essential genes in the region. One of the flanking deletions recovered by P-induced male recombination from P{EPgy2}CG9643^EY07345 is Df(2L)3G, which behaves as a deletion of Tub23C^Pl-2 (Table 3), but does not delete any of the other essential genes in 23C that have been identified [lilli, Rpb9, or l(2)23Cb].

View this table: In this window In a new window   TABLE 3 Survival to eclosion (%) of Tub23C mutant trans-heterozygotes with dd4+ or dd4su(Pl)

 

l(2)23Ce is allelic to Tub23C^Pl-2:

We mapped all of the phenotypes associated with Tub23C^Pl-2 (the held-out-wings phenotype in the presence of brm alleles and the dominant phenotypes in the wing blade) to polytene chromosome band 23CD. We then tested mutants previously mapped to 23CD. We found that all alleles of l(2)23Ce that we tested [l(2)23Ce^A6-2, l(2)23Ce^A14-9, and l(2)23Ce^A15-2] failed to complement Tub23C^Pl-2 for viability (Table 3). We have renamed the l(2)23Ce alleles as Tub23C ^A6-2, Tub23C^A14-9, and Tub23C^A15-2. When trans-heterozygous to other alleles, Tub23C^A15-2 is similar to the two deficiencies tested [Df(2L)JS17 and Df(2L)3G] and is probably a null allele. The Tub23C^A14-9 allele behaves as a hypomorph, often eclosing when heterozygous to the deficiencies and most other alleles. All of the eclosing mutant flies (regardless of which combination of alleles) have the same phenotypes observed for Tub23C^Pl-2 heterozygotes (small round eyes and held-out and blistered wings with pearl-like structures), but with far fewer notches in the wing margins. While flies hemizygous for Tub23C^A14-9 eclosed at 70–83% of the expected numbers, no Tub23C^A14-9/Tub23C^Pl-2 flies eclosed. Tub23C^Pl-2 is an antimorphic, or dominant-negative, allele and the phenotypes observed in Tub23C^Pl-2 heterozygotes are loss-of-function phenotypes for Tub23C caused by interference of the Tub23C^Pl-2 mutant protein with the wild-type Tub23C protein. Consistent with this interpretation is the suppression of the dominant phenotypes of Tub23C^Pl-2 by an additional wild-type copy of Tub23C [observed with both Dp(2;1)JS13 and with tandem duplications recovered from the P-induced male recombination]. These tests allowed us to establish a Tub23C allelic series in order of decreasing activity: Tub23C^A14-9>Tub23C^A6-2>Tub23C^A15-2=Df(2L)JS17=Df(2L)3G>Tub23C^Pl-2.

Molecular characterizations:

We mapped the Tub23C^Pl-2 mutation to a 7-kbp genomic region that includes four predicted genes, Tub23C, CG9641, CG3165, and CG9643 (Figure 2B). Our analyses showed changes in the Tub23C open reading frame for all four alleles that we sequenced (Figure 2C). The Tub23C gene encodes a protein of 475 residues. The Tub23C^A15-2 mutation changes tryptophan 104 to a stop codon, predicting the formation of a truncated protein. This is in agreement with our complementation analyses that suggested that Tub23C^A15-2 behaves as a null mutation. Tub23C^A14-9 changes arginine 217 to histidine (R217H), Tub23C^A6-2 changes serine 233 to phenylalanine (S233F), and Tub23C^Pl-2 changes methionine 382 to isoleucine (M382I).

Differential suppression of Tub23C lethality by an X-linked suppressor:

Subsequent to our molecular analyses, we obtained the Tub23C^bmps1 mutant (MAHONEY et al. 2006). While both Tub23C^bmps1 and Tub23C^Pl-2 change the same methionine to isoleucine (M382I), the description of Tub23C^bmps1 differed from our observations for Tub23C^Pl-2 in two important aspects. The first difference was the much lower penetrance of the dominant phenotypes for Tub23C^bmps1. The second important difference was the eclosion of many Tub23C^bmps1 hemizygotes. We observed these same differences when we began experiments with Tub23C^bmps1, but noted striking differences in the results of the reciprocal crosses with the Tub23C^bmps1 mutant flies. For example, when Tub23C^A6-2 heterozygous females were mated to Tub23C^bmps1 heterozygous males, no Tub23C^bmps1/Tub23C^A6-2 mutant progeny eclosed. In the reciprocal cross in which Tub23C^bmps1 heterozygous females were mated to Tub23C^A6-2 heterozygous males, 73% of the expected Tub23C^bmps1/Tub23C^A6-2 sons eclosed (Tables 3 and 4), while <2% of the expected Tub23C^bmps1/Tub23C^A6-2 daughters eclosed (Table 4). The eclosion of mutant sons only when the mothers were from the Tub23C^bmps1 stock suggested the presence of an X-linked suppressor in this stock. We replaced the X chromosome in the Tub23C^bmps1 stock with an X chromosome marked with w¹ and repeated the crosses. The penetrance of the dominant phenotypes in Tub23C^bmps1 heterozygotes was much greater and the differential eclosion of mutant sons in reciprocal crosses disappeared. When we replaced the X chromosomes in the other Tub23C mutant and deficiency stocks with the X chromosome from the original Tub23C^bmps1 stock, we found that more mutant flies eclosed. The Tub23C^bmps1 stock contains a recessive X-linked suppressor that we named l(1)dd4^su(Pl) [see the following section for a discussion of the allelism to l(1)dd4]. We will refer to the suppressor as dd4^su(Pl) for the remainder of this work. The survival of Tub23C mutant genotypes with the suppressor [dd4^su(Pl)] and without [dd4⁺] are given in Table 3.

View this table: In this window In a new window   TABLE 4 Suppression of Tub23Cbmps1/Tub23CA6-2 lethality by dd4 mutations

 
While most heteroallelic Tub23C combinations were lethal, dd4^su(Pl) rescued some genotypes to eclosion (Table 3). On the basis of the ability to be rescued by dd4^su(Pl), we separated heteroallelic Tub23C combinations into two classes (class I and class II, Table 3). For example, for the class I genotype Tub23C^bmps1/Tub23C^A6-2, no flies eclosed when they were dd4⁺ but 73% eclosed when they were dd4^su(Pl). The same effect was observed for Tub23C^Pl-2/Tub23C^A6-2 and Tub23C^A14-9/Tub23C^A15-2 flies, where 6 and 25% eclosed, respectively, with dd4⁺, but 74 and 94% eclosed, respectively, with dd4^su(Pl). For class II genotypes, there were almost no differences in the eclosion rates between dd4⁺ and dd4^su(Pl).

The class I genotypes include one, and only one, of the following alleles: Tub23C^A14-9, Tub23C^Pl-2, and Tub23C^bmps1. Genotypes that do not include one of these three suppressible alleles (or which include two of the suppressible alleles) are class II. The failure of suppression in flies with two suppressible alleles is interesting. For example, dd4⁺ flies heterozygous for one of the suppressible alleles, Tub23C^Pl-2 or Tub23C^bmps1, do not eclose when also heterozygous for a deficiency or for the weak hypomorphic allele Tub23C^A14-9. The dd4^su(Pl) flies that are heterozygous for Tub23C^Pl-2 or Tub23C^bmps1, however, eclose at 16–24% of the expected frequencies if also heterozygous for one of the deficiencies, but at only 1–2% of the expected frequency if also heterozygous for the hypomorphic (and suppressible) Tub23C^A14-9 allele.

The X-linked suppressor of Tub23C lethality is in the gene encoding the Grip91 -tubulin-interacting protein:

We used meiotic recombination to map the X-linked suppressor in the Tub23C^bmps1 stock to a region that includes the garnet gene. Because we recovered no recombinants between the suppressor and garnet in our mapping experiments, the suppressor is very close to garnet. The gene next to garnet in the genome is lethal (1) discs degenerate 4 [l(1)dd4], which we refer to as dd4. dd4 encodes the Grip91 protein, a subunit of the -tubulin TuSC and TuRC complexes (BARBOSA et al. 2000). Some dd4 mutant alleles cause zygotic lethality, while flies with other mutant alleles survive in dry media and have held-up wings, absence of some bristles, defects in abdominal segments, and male sterility (BARBOSA et al. 2000). Although the suppressor had no mutant phenotype in either sex, we decided to test for allelism to dd4 because of the biochemical data. We used two lethal alleles, dd4² and dd4^G0122, and the test genotype Tub23C^bmps1/Tub23C^A6-2 (see Table 4). Rescue of Tub23C^bmps1/Tub23C^A6-2 lethality by the suppressor is almost completely recessive (only 2% of the expected flies eclosed if heterozygous for the suppressor, while 77% survived if homozygous for the suppressor). Both lethal alleles of dd4 (dd4² and dd4^G0122) complemented the suppressor [dd4^su(Pl)] for viability, with the mutant females eclosing at the expected numbers. However, neither of the lethal alleles complemented dd4^su(Pl) for the suppression of Tub23C^bmps1/Tub23C^A6-2 lethality; i.e., both lethal alleles of dd4 also suppressed the Tub23C lethality. We conclude that the suppressor dd4^su(Pl) is an allele of dd4. The suppression is a loss-of-function phenotype, but the suppressor must retain some functions necessary for zygotic viability and fertility.

Tub23C^Pl-2 genetically interacts with BRM complex mutations and with tna and tara:

Tub23C^Pl-2 was isolated because it interacts with brm² to cause a held-out wings phenotype (Table 2). Since several trithorax group mutants were isolated in this same genetic screen, we tested Tub23C^Pl-2 for genetic interactions with a collection of trithorax group mutants and with mutants in some general transcription factors. From the general transcription factors, we chose to test Taf1, Taf4, and Taf6 and the Mediator complex subunits Med12 and Med13 encoded by the trithorax-group genes kohtalo (kto) and skuld (skd), respectively. We tested mutants in subunits of nucleosome-remodeling or histone-modification complexes, including brm, mor, snr1, osa, kismet, ash1, ash2, and trithorax. We also tested the trithorax group genes Trithorax-like (Trl), tna, tara, verthandi (vtd), sallimus (sls), devenir (dev), Vha55, and urdur (urd). The mutations tested and the results are summarized in Table 2. We found that only alleles of brm, osa, and tna showed strong genetic interactions with Tub23C^Pl-2 to cause the held-out wings phenotype. The genetic interactions between osa¹ and Tub23C^Pl-2 (Table 2 and Figure 1, F and G) are even stronger than the genetic interactions between brm² and Tub23C^Pl-2 (Table 2 and Figure 1C). Flies heterozygous for all three mutations (Tub23C^Pl-2, brm², and osa¹) have poor survival and twisted and blistered wings even more severe than the phenotypes of the Tub23C^Pl-2/+; osa¹/+ double heterozygotes (Figure 1, C and D). Other mutations that showed weaker but significant interactions with Tub23C^Pl-2 included brm¹, osa², mor¹, mor², mor⁶, tna¹, tara², and tara²⁰ (Table 2). We did not find significant interactions with any of the other mutations tested. We considered the possibility that the interactions between the BRM complex mutations and Tub23C^Pl-2 could be explained by reduced transcription of Tub23C. If heterozygous brm mutants have reduced transcription of Tub23C, then all loss-of-function Tub23C phenotypes should be enhanced in all mutant genotypes. To test this prediction, we chose several combinations of hypomorphic Tub23C alleles to examine in brm² heterozygotes. CyO/Tub23C^A14-9;TM6C/brm² females were crossed to males with mutations or deletions of Tub23C. A reduction in transcription would be expected to reduce viability and enhance all of the Tub23C mutant phenotypes in progeny that receive the brm² mutant when compared to their TM6C siblings. While Tub23C^A14-9/Tub23C^A6-2, Tub23C^A14-9/Tub23C^A15-2, and Tub23C^A14-9/ Df(2L)3G trans-heterozygous flies all have reduced viability, the viability was not further reduced in brm² heterozygous flies. As expected, the held-out wings phenotype of Tub23C^A14-9/Df(2L)3G flies was enhanced in brm² heterozygotes, but the expressivity of pearl-like structures in the wings was not enhanced. Finally, we examined interactions among brm², dd4^su(Pl), and Tub23C. The rescue of Tub23C^Pl-2/Tub23C^A6-2 by dd4^su(Pl) was reduced by about half in brm² heterozygotes. The enhancement of the held-out-wings phenotype in Tub23C^Pl-2/+; brm²/+ double heterozygotes was reduced from 76 to 14% in dd4^su(Pl) males.

Proteins identified as part of the eukaryotic cytoskeleton may have more direct roles in transcriptional regulation than originally thought (reviewed in OLAVE et al. 2002). Actin and actin-related proteins (ARPs) are found in BRM complexes from yeast to humans, including the BRM complexes in Drosophila (PAPOULAS et al. 1998). The function of actin and ARPs in these complexes is not well understood. Some ARPs interact with DNA-bending proteins and with histones and it was proposed that they facilitate chromatin architecture and interactions between complexes or function as histone chaperones (SHEN et al. 2003; SZERLONG et al. 2003). Actin is also part of preinitiation complexes and is necessary for transcription by RNA polymerases I, II, and III (HOFMANN et al. 2004; HU et al. 2004; PHILIMONENKO et al. 2004). The - and/or β-tubulins are also found with a subset of trithorax-group proteins in the mammalian ASCOM complex (Activating signal cointegrator 2, Asc2 complex), which is required for transactivation by nuclear receptors (GOO et al. 2003; LEE et al. 2006), and in a histone H2A deubiquitinase complex (ZHU et al. 2007). -Tubulin is essential for microtubule function, but unlike - and β-tubulin, it is not a component of microtubules. Rather, it is located at microtubule-organizing centers (MTOCs) and functions in the initiation of microtubule nucleation and in the establishment of microtubule polarity (OAKLEY 1992; LUDERS and STEARNS 2007). -Tubulin contributes to the proper formation of mitotic spindles and cytoplasmic microtubular arrays. There are critical cytoskeletal and nuclear envelope connections, linking, for example, MTOCs to the nuclear lamina (reviewed in TADDEI et al. 2004). In addtion, -tubulin has been proposed to have microtubule- and/or centrosome-independent function(s) in mitosis (PRIGOZHINA et al. 2004) or spindle assembly checkpoints (MULLER et al. 2006).

Drosophila embryonic -tubulin exists in two related complexes: a large complex similar to the Xenopus TuRC (-tubulin ring complex) (36.9S, 2000 kDa) and a small soluble complex called TuSC (-tubulin small complex) (8.5S 240 kDa) (OEGEMA et al. 1999). The Drosophila TuRC consists of approximately eight polypeptides, including -tubulin, Grip163, Grip128, Grip91, Grip84, Grip75, and GP71WD (OEGEMA et al. 1999; GUNAWARDANE et al. 2000, 2003). The TuRC has a lockwasher-like structure and a cap at one of the ends of the complex. The Drosophila TuSC is a tetramer of two -tubulin molecules and one molecule each of Grip91 and Grip84. Several TuSCs form the TuRC lockwasher region. The other Grips (Grip163, 128, and 75) form the cap (MORITZ et al. 2000).

Drosophila is the only metazoan in which the genes encoding subunits of the TuSC and TuRC complexes have been functionally studied using genetic approaches (for recent examples, see BARBOSA et al. 2003; GUNAWARDANE et al. 2003; COLOMBIÉ et al. 2006; VOGT et al. 2006). Null mutations in dd4 (which encodes Grip91) and in Grip84 are lethal and display defects in spindle assembly (BARBOSA et al. 2003; COLOMBIÉ et al. 2006), while null mutations in Grip128 and Grip75 are viable, but sterile (SCHNORRER et al. 2002; VOGT et al. 2006).

In Drosophila there are two -tubulin genes, Tub23C and Tub37C. They encode very similar (but not identical) proteins, but they have different expression patterns and mutant phenotypes. Tub37C is largely restricted to the female germline and early stages of embryogenesis. It is required for bicoid (bcd) mRNA localization at mid-oogenesis (SCHNORRER et al. 2002), female meiosis, and nuclear proliferation (TAVOSANIS et al. 1997; WILSON et al. 1997). In syncytial embryos, Tub23C is in the soluble small TuSC fraction (RAYNAUD-MESSINA et al. 2001) and is absent at the centrosome (WILSON et al. 1997). At this stage, Tub37C is found in both TuSC and TuRC fractions (RAYNAUD-MESSINA et al. 2001). It is localized at the centrosome (TAVOSANIS et al. 1997; WILSON et al. 1997) and over the spindle regions (TAVOSANIS et al. 1997). Tub37C mutants are female sterile (TAVOSANIS et al. 1997; TAVOSANIS and GONZÁLEZ 2003).

The Tub23C isoform is expressed in a variety of tissues in both sexes (WILSON et al. 1997), including larval brains and imaginal discs, and it is required for somatic mitotic divisions. It is also expressed in ovaries and is the only isoform expressed in testes. Tub23C is required for meiosis in males and for spermatogenesis (SAMPAIO et al. 2001).

Tub23C is essential for zygotic viability and for development of imaginal tissues:

We isolated the Tub23C^Pl-2 mutation in a mutant screen designed to identify genes that interact with brm in wing development. In addition to showing genetic interactions with brm, Tub23C^Pl-2 mutants are homozygous lethal, while the heterozygotes have defects in imaginal eye and wing development. We showed that Tub23C^Pl-2 is a dominant-negative mutation and that l(2)23Ce alleles are loss-of-function mutations in Tub23C with recessive phenotypes similar to the dominant phenotypes of Tub23C^Pl-2. Tub23C has 30% identity to - and β-tubulins, which are structural components of microtubules. It is known which parts of the β-tubulin protein are involved in autoregulation for translation and for binding and hydrolysis of GTP. The -tubulin protein shares some of these regions with β-tubulin. The Tub23C mutations characterized in this work do not map to any of these known regions, with the exception of the truncated form in the Tub23C^A15-2 allele (Figure 2C). This suggests that the proteins synthesized from the Tub23C^A14-9, Tub23C^A6-2, Tub23C^Pl-2, and Tub23C^bmps1 alleles might affect other -tubulin functions (see below).

We were surprised during the course of this work to identify a dd4 allele with no discernible phenotype except the suppression of some Tub23C mutant phenotypes (including zygotic lethality). Since dd4 encodes Grip91, a protein that physically interacts with -tubulin, we believe that the genetic interactions have important implications.

Implications of the genetic interactions between Tub23C and dd4 (Grip91) mutations on current structural models of TuRC and TuSC complexes:

Grip91, Grip84, and -tubulin form the lockwasher region of TuRC and TuSC complexes (MORITZ et al. 2000). Grip91 and Grip84 (or their orthologs in yeast and humans) interact with each other and with -tubulin (MORITZ et al. 2000; ALDAZ et al. 2005; WIESE 2008). The interactions between Grip91 and -tubulin facilitate binding of GTP to -tubulin (GUNAWARDANE et al. 2003). Grip91 is required for correct bipolar spindle assembly during mitosis and male meiosis and it helps to locate -tubulin to the centrosome (BARBOSA et al. 2000, 2003).

Grip91 is an essential protein encoded by the dd4 gene (BARBOSA et al. 2000, 2003). Semilethal alleles have held-up wings and other imaginal defects and are male sterile (BARBOSA et al. 2000, 2003). The dd4^su(Pl) allele that we identified is unusual in that it has no defects in viability, fertility, or developmental patterning. Its only phenotype is the suppression of class I (but not class II) genotypes of Tub23C.

What is the significance of the two types of Tub23C alleles from the functional point of view? The defects produced by suppressible alleles may involve TuSC and/or TuRC functions, while the defects produced by nonsuppressible alleles may involve Tub23C functions independent of the TuSC and TuRC complexes. It is also possible that different mutant proteins, although in some cases retaining partial activity, may affect other different functions of Tub23C. Some of these other functions may require Grip91 (and possibly the integrity of TuRC and/or TuSC complexes) and some may not. Such functions could affect the assembly of the TuSC and/or TuRC complexes, the transport of the complex(es) to subcellular compartments, and/or the relationships of Tub23C with other proteins involved in microtubule-independent processes. We believe that the new alleles of Tub23C and dd4 that we have characterized can help to test the current structural models of TuRC and TuSC complexes proposed in biochemical and crystallographic studies (ERICKSON 2000; MORITZ et al. 2000).

Recent work shows that -tubulin has a microtubule-independent role in establishing or maintaining a mitotic checkpoint block (PRIGOZHINA et al. 2004) and that TuRCs proteins may have a centrosome-independent role in the spindle assembly checkpoint. For this latter function, -tubulin is probably in a complex associated with Cdc20 and BubR1 kinases (MULLER et al. 2006). We have found that the genetic interactions between Tub23C and Brm are caused not by reduced Tub23C transcription, but more probably by the presence of defective -tubulin proteins. This suggests roles for -tubulin in transcription and/or chromatin remodeling. This is further supported by the recent description of interactions between Pericentrin (an integral centrosomal component) and CHD3, a Brm-related protein in the NuRD chromatin-remodeling complex (SILLIBOURNE et al. 2007).

This work was supported by grants from the Consejo Nacional de Ciencia y Technológia (M.V.), the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica–Universidad Nacional Autónoma de México (M.V. and M.Z.), and the Howard Hughes Medical Institute (M.Z.). This research was supported in part by the Intramural Research Program of the National Institute of Child Health and Human Development of the National Institutes of Health.

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