γtub23c interacts genetically with brahma chromatin ... · 1 γtub23c interacts genetically with...
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γTub23C interacts genetically with Brahma chromatin-remodeling complexes in
Drosophila melanogaster.
Martha Vázquez1, Monica T. Cooper2, Mario Zurita1, and James A. Kennison2.
1Departamento de Fisiología Molecular
y Genética del Desarrollo,
Instituto de Biotecnología, UNAM.
Cuernavaca, Morelos 62250, México.
2Laboratory of Molecular Genetics,
Eunice Kennedy Shriver National Institute of Child Health and Human Development,
National Institutes of Health
Bethesda, MD 20892, USA
Genetics: Published Articles Ahead of Print, published on September 9, 2008 as 10.1534/genetics.108.093492
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Running head: γTub23C interacts genetically with Brahma
Keywords: . γ-tubulin, Brahma, Grip91
Corresponding author: Martha Vázquez
Av. Universidad 2001
Departamento de Fisiología Molecular
y Genética del Desarrollo,
Instituto de Biotecnología, UNAM
Cuernavaca, Morelos 62250, México
Phone: (52)(55)56227631
Fax: (52)(777)3172388
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ABSTRACT
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 γTub23C. γTub23C encodes one of the
two γ-tubulin isoforms in Drosophila and is essential for zygotic viability and normal
adult patterning. γ-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 γTub23C mutations. The genetic interactions between γTub23C
and chromatin remodeling mutations suggest that γ-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 (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 (ARMSTRONG et al.
2005; SHARMA et al. 2003). 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 (GUTIÉRREZ et al. 2003;
VÁZQUEZ et al. 1999). In this work, we describe the characterization of another mutation
isolated in this genetic screen, the Pearl-2 allele of γTub23C. Some γTub23C mutant
phenotypes are modified (enhanced or suppressed) by mutations in genes encoding
subunits of the BRM complexes, and by mutations in Grip91, a γ -tubulin ring complex
subunit. These data suggest a role for γ-tubulin in transcription.
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MATERIALS AND METHODS
Fly strains: Flies were raised at 25oC 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 (LINDSLEY and ZIMM 1992). Mutant stocks carrying l(1)dd4G0122,
l(1)dd42, l(2)23CeA6-2, l(2)23CeA14-9, l(2)23CeA15-2, and γTub23Cbmps1 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 γTub23CPl-2 mutant was first mapped meiotically between the
visible markers al and dp.
The dd4su(Pl) mutation was first mapped by meiotic recombination using visible
markers. Individual recombinant sons from females heterozygous for dd4su(Pl) and a y2
wa ct6 g2 f mutant chromosome were recovered and tested for the survival of
γTub23Cbmps1/γTub23CA6-2 transheterozygotes. 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+.
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P-induced male recombination mapping of γTub23CPl-2: Females with the P-element
insertions shown in Table 1 were crossed to males of the genotype al γTub23CPl-2 KrIf/+;
TMS, P{ry+t7.2=Delta2-3}99B/+. Sons that were P{X}/ al γTub23CPl-2 KrIf; TMS/+ were
crossed to al dp b pr c px sp females and the progeny scored for recombinants between al
and KrIf. 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, γTub23CA6-2, and okra). Although l(2)23Cb has been
renamed l(2)23Dd by Flybase, our deletion mapping places the gene between Rpb9 and
γTub23C, consistent with the original mapping to 23C and the original gene name.
Molecular analyses: After γTub23CPl-2 was mapped between P{EP}Rrp1020 and
P{EPgy2}CG9643EY07345, the DNA sequences of the open reading frames of all four
predicted genes in the region (CG9641, CG3165, CG9643, and γTub23C) were
determined from DNA isolated from homozygotes of γTub23CPl-2 (and the parental
w;red e strain in which it was induced) and γTub23CA6-2 (and the parental cn bw
chromosome in which it was induced). As the only non-synonomous changes found
between the two mutants and their parental chromosomes were in the γTub23C open
reading frame (Figure 2C), we then determined the DNA sequence of γTub23C from
γTub23CA14-9 and γTub23CA15-2 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].
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RESULTS
The γTub23CPl-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)
(GUTIÉRREZ et al. 2003; VÁZQUEZ et al. 1999). From that same genetic screen, we also
isolated the γTub23CPl-2 mutation. In addition to its dominant enhancement of brm (Table
2 and Figure 1C), γTub23CPl-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). γTub23CPl-2 heterozygotes also have
small round eyes. We mapped γTub23CPl-2 to the same chromosomal region as Pearl (Pl)
(ROSIN 1951; ROSIN 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 γTub23C (see below), we have renamed it
γTub23CPl-2.
Mapping of γTub23CPl-2: We first mapped γTub23CPl-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 γTub23CPl-2 by P-induced male-recombination (CHEN et al. 1998). Of 63
recombinants recovered, all except one appear to have resulted from recombination at the
P-element insertion site. These 62 recombinants all place γTub23CPl-2 in the 7kbp region
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between P{EP}Rrp11020 and P{EPgy2} CG9643EY07345 (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}CG9643EY07345 is Df(2L)3G, which
behaves as a deletion of γTub23CPl-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].
l(2)23Ce is allelic to γTub23CPl-2: We mapped all of the phenotypes associated with
γTub23CPl-2 (the held-out wing phenotype in the presence of brm alleles and the
dominant phenotypes in the wing blade) to polytene chromosome bands 23CD. We then
tested mutants previously mapped to 23CD. We found that all alleles of l(2)23Ce that
we tested [l(2)23CeA6-2, l(2)23CeA14-9, and l(2)23CeA15-2] failed to complement
γTub23CPl-2 for viability (Table 3). We have renamed the l(2)23Ce alleles as γTub23C A6-
2, γTub23CA14-9, and γTub23CA15-2. When transheterozygous to other alleles, γTub23CA15-
2 is similar to the two deficiencies tested [Df(2L)JS17 and Df(2L)3G], and is probably a
null allele. The γTub23CA14-9 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
γTub23CPl-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 γTub23CA14-9 eclosed at 70 % to 83% of the expected numbers, no
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γTub23CA14-9/γTub23CPl-2 flies eclosed. γTub23CPl-2 is an antimorphic, or dominant-
negative, allele and the phenotypes observed in γTub23CPl-2 heterozygotes are loss-of-
function phenotypes for γTub23C caused by interference of the γTub23CPl-2 mutant
protein with the wild-type γTub23C protein. Consistent with this interpretation is the
suppression of the dominant phenotypes of γTub23CPl-2 by an additional wild-type copy
of γTub23C (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 γTub23C
allelic series, being in order of decreasing activity: γTub23CA14-9>γTub23CA6-2>
γTub23CA15-2=Df(2L)JS17=Df(2L)3G>γTub23CPl-2.
Molecular characterizations: We mapped the γTub23CPl-2 mutation to a 7 kbp
genomic region that includes four predicted genes, γTub23C, CG9641, CG3165, and
CG9643 (Figure 2B). Our analyses showed changes in the γTub23C open-reading frame
for all four alleles that we sequenced (Figure 2C). The γTub23C gene encodes a protein
of 475 residues. The γTub23CA15-2 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 γTub23CA15-2 behaves as a null mutation.
γTub23CA14-9 changes arginine 217 to histidine (R217H), γTub23CA6-2 changes serine 233
to phenylalanine (S233F), and γTub23CPl-2 changes methionine 382 to isoleucine
(M382I).
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Differential suppression of γTub23C lethality by an X-linked suppressor:
Subsequent to our molecular analyses, we obtained the γTub23Cbmps1 mutant (MAHONEY
et al. 2006). While γTub23Cbmps1 and γTub23CPl-2 both change the same methionine to
isoleucine (M382I), the description of γTub23Cbmps1 differed from our observations for
γTub23CPl-2 in two important aspects. The first difference was the much lower
penetrance of the dominant phenotypes for γTub23Cbmps1. The second important
difference was the eclosion of many γTub23Cbmps1 hemizygotes. We observed these
same differences when we began experiments with γTub23Cbmps1, but noticed striking
differences in the results of the reciprocal crosses with the γTub23Cbmps1 mutant flies.
For example, when γTub23CA6-2 heterozygous females were mated to γTub23Cbmps1
heterozygous males, no γTub23Cbmps1/γTub23CA6-2 mutant progeny eclosed. In the
reciprocal cross in which γTub23Cbmps1 heterozygous females were mated to γTub23CA6-2
heterozygous males, 73% of the expected γTub23Cbmps1/γTub23CA6-2 sons eclosed (Tables
3 and 4), while less than 2% of the expected γTub23Cbmps1/γTub23CA6-2 daughters eclosed
(Table 4). The eclosion of mutant sons only when the mothers were from the
γTub23Cbmps1 stock suggested the presence of an X-linked suppressor in this stock. We
replaced the X chromosome in the γTub23Cbmps1 stock with an X chromosome marked
with w1 and repeated the crosses. The penetrance of the dominant phenotypes in
γTub23Cbmps1 heterozygotes was much greater and the differential eclosion of mutant
sons in reciprocal crosses disappeared. When we replaced the X chromosomes in the
other γTub23C mutant and deficiency stocks with the X chromosome from the original
γTub23Cbmps1 stock, we found that more mutant flies eclosed. The γTub23Cbmps1 stock
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contains a recessive X-linked suppressor that we named l(1)dd4su(Pl) (see the following
section for the allelism to l(1)dd4). We will refer to the suppressor as dd4su(Pl) for the
remainder of this work. The survival of γTub23C mutant genotypes with the suppressor
[dd4su(Pl)] and without [dd4+] are given in Table 3.
While most heteroallelic γTub23C combinations were lethal, dd4su(Pl) rescued some
genotypes to eclosion (Table 3). Based on the ability to be rescued by dd4su(Pl), we
separated heteroallelic γTub23C combinations into two classes (Class I and Class II,
Table 3). For example, for the Class I genotype γTub23Cbmps1/γTub23CA6-2 no flies
eclosed when they were dd4+ but 73% eclosed when they were dd4su(Pl). The same effect
was observed for γTub23CPl-2/γTub23CA6-2 and γTub23CA14-9/γTub23CA15-2 flies, where
6% and 25% eclosed, respectively, with dd4+, but 74% and 94% eclosed, respectively,
with dd4su(Pl). For Class II genotypes, there were almost no differences in the eclosion
rates between dd4+ and dd4su(Pl).
The Class I genotypes include one, and only one, of the following alleles: γTub23CA14-9,
γTub23CPl-2, and γTub23Cbmps1. 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, γTub23CPl-2 or γTub23Cbmps1,
do not eclose when also heterozygous for a deficiency or for the weak hypomorphic allele
γTub23CA14-9. The dd4su(Pl) flies that are heterozygous for γTub23CPl-2 or γTub23Cbmps1,
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) γTub23CA14-9 allele.
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The X-linked suppressor of γTub23C lethality is in the gene encoding the Grip91 γ-
tubulin-interacting protein: We used meiotic recombination to map the X-linked
suppressor in the γTub23Cbmps1 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 will refer to as dd4. dd4
encodes the Grip91 protein, a subunit of the γ-tubulin γTuSC and γTuRC 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
dd4G0122, and the test genotype γTub23Cbmps1/γTub23CA6-2. The data are in Table 4.
Rescue of γTub23Cbmps1/γTub23CA6-2 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
dd4G0122) complemented the suppressor (dd4su(Pl)) for viability, with the mutant females
eclosing at the expected numbers. However, neither of the lethal alleles complemented
dd4su(Pl) for the suppression of γTub23Cbmps1/γTub23CA6-2 lethality, i.e., both lethal alleles
of dd4 also suppressed the γTub23C lethality. We conclude that the suppressor, dd4su(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.
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γTub23CPl-2 genetically interacts with BRM complex mutations, and with tna and
tara: γTub23CPl-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 γTub23CPl-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 γTub23CPl-2 to cause the held-out wings
phenotype. The genetic interactions between osa1 and γTub23CPl-2 (Table 2 and Figure
1F-G) are even stronger than the genetic interactions between brm2 and γTub23CPl-2
(Table 2 and Figure 1C). Flies heterozygous for all three mutations (γTub23CPl-2, brm2,
and osa1) have poor survival and twisted and blistered wings even more severe than the
phenotypes of the γTub23CPl-2/+; osa1/+ double heterozygotes (Figure 1C-D). Other
mutations that showed weaker but significant interactions with γTub23CPl-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 γTub23CPl-2
could be explained by reduced transcription of γTub23C. If heterozygous brm mutants
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have reduced transcription of γTub23C, then all loss-of-function γTub23C phenotypes
should be enhanced in all mutant genotypes. To test this prediction, we chose several
combinations of hypomorphic γTub23C alleles to examine in brm2 heterozygotes.
CyO/γTub23CA14-9 ; TM6C/brm2 females were crossed to males with mutations or
deletions of γTub23C. A reduction in transcription would be expected to reduce viability
and enhance all of the γTub23C mutant phenotypes in progeny that receive the brm2
mutant when compared to their TM6C siblings. While γTub23CA14-9/γTub23CA6-2,
γTub23CA14-9/γTub23CA15-2, and γTub23CA14-9/ Df(2L)3G transheterozygous flies all have
reduced viability, the viability was not further reduced in brm2 heterozygous flies. As
expected, the held-out wings phenotype of γTub23CA14-9/ 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 between brm2, dd4su(Pl) , and γTub23C.
The rescue of γTub23CPl-2/γTub23CA6-2 by dd4su(Pl) was reduced by about half in brm2
heterozygotes. The enhancement of the held out wings phenotype in γTub23CPl-2/+;
brm2/+ double heterozygotes was reduced from 76% to 14% in dd4su(Pl) males.
DISCUSSION
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
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chromatin achitecture and interactions between complexes or function as histone
chaperones (SHEN et al. 2003; SZERLONG et al. 2003). Actin is also part of pre-initiation
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 α- 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). γ-tubulin is essential for
microtubule function, but unlike α- and β-tubulin, it is not a component of microtubules.
Rather, it is located at microtubule-organizing centres (MTOCs) and functions in the
initiation of microtubule nucleation and in the establishment of microtubule polarity
(LUDERS and STEARNS 2007; OAKLEY 1992). γ-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, γ-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 γ-tubulin exists in two related complexes: a large complex
similar to the Xenopus γTuRC (γ-Tubulin Ring Complex) (36.9S, ~2000 kDa) and a
small soluble complex called γTuSC (γ-tubulin Small Complex) (8.5S ~240kDa)
(OEGEMA et al. 1999). The Drosophila γTuRC consists of ~8 polypeptides, including γ-
tubulin, Grip163, Grip128, Grip91, Grip84, Grip75, and GP71WD (GUNAWARDANE et al.
2000; GUNAWARDANE et al. 2003; OEGEMA et al. 1999). The γTuRC has a lockwasher-
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like structure and a cap at one of the ends of the complex. The Drosophila γTuSC is a
tetramer of two γ-tubulin molecules and one molecule each of Grip91 and Grip84.
Several γTuSCs form the γTuRC 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 γTuSC
and γTuRC complexes have been functionally studied using genetic approaches (for
recent examples BARBOSA et al. 2003; COLOMBIÉ et al. 2006; GUNAWARDANE et al.
2003; VOGT et al. 2006). Null mutations in dd4 (which encodes Grip91) and 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 γ-tubulin genes, γTub23C and γTub37C. They encode very
similar (but not identical) proteins, but they have different expression patterns and mutant
phenotypes. γTub37C is largely restricted to the female germ line 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 γTub23C is in the soluble small γTuSC
fraction (RAYNAUD et al. 2001) and is absent at the centrosome (WILSON et al. 1997). At
this stage, γTub37C is found in both the γTuSC and γTuRC fractions (RAYNAUD 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). γTub37C mutants are female sterile
(TAVOSANIS and GONZÁLEZ 2003; TAVOSANIS et al. 1997).
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The γTub23C 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.
γTub23C is required for meiosis in males and for spermatogenesis (SAMPAIO et al. 2001).
γTub23C is essential for zygotic viability and for development of imaginal tissues:
We isolated the γTub23CPl-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, γTub23CPl-2 mutants are homozygous lethal, while the heterozygotes have defects in
imaginal eye and wing development. We showed that γTub23CPl-2 is a dominant-
negative mutation and that l(2)23Ce alleles are loss-of-function mutations in γTub23C
with recessive phenotypes similar to the dominant phenotypes of γTub23CPl-2. γTub23C
has 30% identity to α and β tubulins, which are structural components of microtubules. It
is known which parts of the β tubulin protein are involved in auto-regulation for
translation and for binding and hydrolisis of GTP. The γ-tubulin protein shares some of
these regions with β-tubulin. The γTub23C mutations characterized in this work do not
map to any of these known regions, with the exception of the truncated form in the
γTub23CA15-2 allele (Figure 2C). This suggests that the proteins synthesized from the
γTub23CA14-9, γTub23CA6-2, γTub23CPl-2, and γTub23Cbmps1 alleles might be affecting
other γ-tubulin functions (see below).
We were surprised during the course of this work to identify a dd4 allele with no
discernable phenotype except the suppression of some γTub23C mutant phenotypes
(including zygotic lethality). Since dd4 encodes Grip91, a protein that physically
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interacts with γ-tubulin, we believe that the genetic interactions have important
implications.
Implications of the genetic interactions between γTub23C and dd4 (Grip91)
mutations on current structural models of γTuRC and γTuSC complexes: Grip91,
Grip84, and γ-tubulin form the lockwasher region of γTuRC and γTuSC complexes
(MORITZ et al. 2000). Grip91 and Grip84 (or their orthologues in yeast and human)
interact with each other and with γ-tubulin (ALDAZ et al. 2005; MORITZ et al. 2000;
WIESE 2008). The interactions between Grip91 and γ-tubulin facilitates binding of GTP
binding to γ-tubulin (GUNAWARDANE et al. 2003). Grip91 is required for correct bipolar
spindle assembly during mitosis and male meiosis and it helps to locate γ-tubulin to the
centrosome (BARBOSA et al. 2003; BARBOSA et al. 2000).
Grip91 is an essential protein encoded by the dd4 gene (BARBOSA et al. 2003;
BARBOSA et al. 2000). Semi-lethal alleles have held-up wings and other imaginal defects
and are male sterile (BARBOSA et al. 2003; BARBOSA et al. 2000). The dd4su(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 γTub23C.
What is the significance of the two types of γTub23C alleles from the functional point
of view? The defects produced by suppressible alleles may involve γTuSC and/or γTuRC
functions, while the defects produced by non-suppressible alleles may involve γTub23C
functions independent of the γTuSC and γTuRC complexes. It is also possible that
different mutant proteins, although in some cases retaining partial activity, may affect
19
other different functions of γTub23C. Some of these other functions may require Grip91
(and possibly the integrity of γTuRC and/or γTuSC complexes) and some may not. Such
functions could affect the assembly of the γTuSC and/or γTuRC complexes, the transport
of the complex(es) to subcellular compartments, and/or the relationships of γTub23C
with other proteins involved in microtubule-independent processes. We believe that the
new alleles of γTub23C and dd4 that we have characterized can help to test the current
structural models of γTuRC and γTuSC complexes proposed in the biochemical and
crystallographic studies (ERICKSON 2000; MORITZ et al. 2000).
Recent work shows that γ-tubulin has a microtubule-independent role in establishing or
maintaining a mitotic checkpoint block (PRIGOZHINA et al. 2004) and that γTuRCs
proteins may have a centrosome-independent role in the spindle assembly checkpoint.
For this last function γ-tubulin is probably in a complex associated with Cdc20 and
BubR1 kinases (MULLER et al. 2006). We have found that the genetic interactions
between γTub23C and Brm are caused not by reduced γTub23C transcription, but more
probably by the presence of defective γ-tubulin proteins. This suggests roles for γ-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 CONACyT (M.V.) and PAPPyT-UNAM (M.V.
and M. Z.), Howard Hughes Medical Institute (M. Z.). This research was supported in
part by the Intramural Research Program of the National Institutes of Health, NICHD.
20
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26
Table 1. P-induced male recombination.
____________________________________________________
P-transposon Polytene No. of
insertion Location recombinants
____________________________________________________
P{lacW}lilli05431 23C1-3 4
P{SUPor-P}Rrp1KG01159 23C3 9
P{EP}Rrp11020 23C3-4 3
P{EPgy2} CG9643EY07345 23C4 31
P{SUPor-P}CG3558KG02323 23C4 7
P{lacW}v(2)k05816 23C5 3
P{PZ}toc01361 23D2 3
P{lacW}Madk00237 23D3 3
27
Table 2. γTub23CPl-2 interactions with general transcription machinery and
trithorax-group mutants.
____________________________________________________________
Genotype Number of flies with Penetrance
held-out wings/Total (%)
____________________________________________________________
brm2/+ 9/498 2
mor1/+ 0/120 0
osa1/+ 6/208 3
osa2/+ 0/41 0
tna1/+ 19/115 17
tara2/+ 0/129 0
tara20/+ 0/151 0
γTub23CPl-2/+ 5/727 1
γTub23CPl-2/+; brm1/+ 16/46 35
γTub23CPl-2/+; brm2/+ 71/94 76
γTub23CPl-2/+; mor1/+ 10/105 10
γTub23CPl-2/+; mor2/+ 18/43 42
γTub23CPl-2/+; mor6/+ 20/107 19
γTub23CPl-2/+; osa1/+ 307/314 98
28
γTub23CPl-2/+; osa2/+ 17/44 39
γTub23CPl-2/+; tna1/+ 111/148 75
γTub23CPl-2/+; tara2/+ 19/88 22
γTub23CPl-2/+; tara20/+ 19/91 21
When trans-heterozygous with γTub23CPl-2, the following mutations gave no more than
3% penetrance for the held-out wings phenotype: Taf11, Taf41, Taf4S466, Taf61, Taf62,
trxE2, trxB11, ash16, ash1B1, ash21, ash22, kis1, kis2, kto1, kto3, vtd3, vtd14, sls1, dev1, dev2,
Vha5512, Vha5516, urd2, Trl3, Trl62, and skd3.
29
Table 3. Survival to eclosion (%) of γTub23C mutant transheterozygotes with dd4+ or dd4su(Pl)
_________________________________________________________________________________
dd4su(Pl) suppressible (Class I) dd4su(Pl) non-suppressible (Class II)
__________________________________________________________________________________
γTub23C genotype dd4+ dd4su(Pl) γTub23C genotype dd4+ dd4su(Pl)
__________________________________________________________________________________
γTub23CA14-9/Df(2L)3G 83 100 γTub23CA6-2/γTub23CA15-2 3* 0
γTub23CA14-9/Df(2L)JS17 70 100 γTub23CA6-2/Df(2L)3G 2* 0
γTub23CA14-9/γTub23C A6-2 65 74 γTub23CA6-2/Df(2L)JS17 0 0
γTub23CA14-9/γTub23CA15-2 25 94 γTub23CA14-9/γTub23Cbmps1 0 2
γTub23CPl-2/γTub23CA6-2 6* 74 γTub23CA14-9/γTub23CPl-2 0 1
γTub23Cbmps1/γTub23CA6-2 0 73 γTub23CA15-2/Df(2L)JS17 0 2
γTub23CPl-2/Df(2L)JS17 0 24 γTub23CA15-2/Df(2L)3G 0 0
γTub23CPl-2/Df(2L)3G 0 19 Df(2L)3G/Df(2L)JS17 0 0
γTub23Cbmps1/Df(2L)3G 0 19 γTub23CPl-2/γTub23Cbmps1 0 0
30
γTub23Cbmps1/Df(2L)JS17 0 16
γTub23CA15-2/γTub23Cbmps1 0 13
γTub23CA15-2/γTub23CPl-2 0 7
*eclosed, but all quickly became stuck in the medium.
31
Table 4. Suppression of γTub23Cbmps1/γTub23CA6-2 lethality by dd4 mutations.
__________________________________________________________
dd4 genotype Survival of γTub23Cbmps1/γTub23CA6-2 flies
# observed/expected %
_________________________________________________________
+/Y 0/351 0
+/+ 0/218 0
+/dd4su(Pl) 8/478 2
+/dd42 0/38 0
+/dd4G0122 2/109 2
dd4su(Pl)/Y 186/255.5 73
dd4su(Pl)/dd4su(Pl) 58/75.5 77
dd4su(Pl)/dd42 102/101 101
dd4su(Pl)/dd4G0122 105/105 100
The expected numbers of γTub23Cbmps1/γTub23CA6-2 flies is half the number of CyO
individuals in the same crosses. For +/Y or dd4su(Pl)/Y genotypes the expected frequency
was calculated with the CyO sons and for the rest of the genotypes it was calculated with
the CyO daughters.
32
Figure legends.
Figure 1.- γTub23CPl-2-dependent phenotypes. (A) Wild-type fly with the wings held
back parallel to the body axis. (B) γTub23CPl-2/+ flies, note the notches and the pearl-like
structures in the wings (indicated by arrows, one of them shown in the inset). (C)
γTub23CPl-2/+; brm2/+ (shown in picture) and γTub23CPl-2/+; osa1/+ double heterozygous
flies have held-out wings in addition to the wing notches and pearl-like structures. (D)
γTub23CPl-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 γTub23CPl-2/+; osa1 /+ double heterozygous fly. The arrow indicate
a pearl-like structure along the third wing vein shown in the inset (G).
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 indicated by arrows according 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 wide line marks the 7 kbp region where γTub23CPl-2 was
localized by P-induced male recombination. (C) The amino acid sequences for the two γ-
tubulin proteins (labeled 23C and 37C) in Drosophila (WILSON et al. 1997) and the
changes found in the γTub23C mutant alleles. The arrows indicate the changes in the
γTub23C mutant alleles. At the top of each arrow are the allele name and the aminoacid
substitution. The numbered blocks indicate peptides with known or presumed functions
(taken from BURNS 1995) and references therein): 1 is a peptide implicated in the
33
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 α, β, and γ
tubulin from the TCP1α chaperonine. Note that none of the γTub23C mutations are in
any of the peptides with known or presumed functions and that all the changed residues
in the γTub23C mutants are identical in both γTub23C and γTub37C wild-type genes.