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First published online 10 January 2007
doi: 10.1242/dev.02766
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1 Departments of Genetics, Yale University School of Medicine, 333 Cedar Street,
New Haven, CT 06520, USA.
2 Departments of Cell Biology, Yale University School of Medicine, 333 Cedar
Street, New Haven, CT 06520, USA.
3 Department of Molecular, Cellular and Developmental Biology, Yale University,
260 Prospect Avenue, New Haven, CT 06511, USA.
* Author for correspondence (e-mail: lynn.cooley{at}yale.edu)
Accepted 30 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fusome, Ring canals, Drosophila oogenesis, Polyprotein
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In some systems, such as oogenesis in mammals, cytoplasmic
bridges disappear early and cells differentiate separately. However, in most
known forms of spermatogenesis and in insect oogenesis, cytoplasmic bridges
persist and contribute to differentiation. In insect oogenesis, one cell in
each cluster becomes the oocyte and is nourished by the nurse cells through
cytoplasmic bridges. In all cases, germline cells must undergo specific
changes in gene expression, morphology and function when making the transition
from proliferation to differentiation.
Drosophila oogenesis is an attractive model to study the
transition from germline cell expansion to differentiation and the formation
of intercellular bridges called ring canals (RCs). In the most anterior
compartment of the ovary, the germarium, germline stem cells (GSCs) undergo
division to form another GSC and a daughter cystoblast
(Huynh and St Johnston, 2004).
The cystoblast undergoes exactly four mitoses with incomplete cytokinesis
forming a 16-cell cyst in which cells are connected by arrested cleavage
furrows. Once mitosis is complete the cells differentiate into 15 nurse cells
and one oocyte. The proliferating cells and differentiated cells can be easily
identified through subcellular structures. During proliferation, an organelle
called the fusome is present. After mitosis is complete, the fusome begins to
disappear and RCs form at the arrested cleavage furrows.
The fusome is derived from a precursor structure called a spectrosome
(Lin et al., 1994), which is
present in GSCs. A portion of the spectrosome is inherited by the daughter
cystoblast, after which it grows and branches during cystoblast divisions to
form the fusome (de Cuevas and Spradling,
1998). Spectrosome- and/or fusome-like structures have been
described in the germline of insects, Xenopus and mouse
(Pepling et al., 1999). A
potentially analogous structure is in mammalian lymphocytes
(Dubielecka et al., 2003). The
spectrosome and fusome in Drosophila are areas of highly condensed
vesicles including modified endoplasmic reticulum
(de Cuevas et al., 1997;
Snapp et al., 2004). Fusome
membranes are associated with a cytoskeleton that includes the Adductin
homolog Hu-li tai shao (Hts), 
-Spectrin, ß-Spectrin, Filamin
(Cheerio - Flybase), a Spectraplakin homolog named Short Stop (Shot), and
Ankyrin (de Cuevas et al.,
1996; Lin et al.,
1994; Roper and Brown,
2004; Sokol and Cooley,
2003). Hts and -Spectrin are necessary for fusome structure
because in mutants the fusome is absent (de
Cuevas et al., 1996; Lin et
al., 1994) causing dramatic defects in nurse cell number and
oocyte specification.
RCs allow the movement of essential proteins and RNA from nurse cells into
the oocyte; thus, mutations that result in their disruption are female
sterile. Formation of RCs at arrested cleavage furrows follows an organized
pattern of RC protein accumulation. F-actin, Filamin and Ovhts-RC, a novel
product of the hts locus, begin to accumulate on RCs starting
immediately after mitosis ends (Robinson
et al., 1994). Mutations in the gene for Filamin
(cheerio) or hts result in a failure of F-actin accumulation
and arrested RC development (Robinson et
al., 1997).
hts was isolated as a female sterile mutant
(Ding et al., 1993;
Yue and Spradling, 1992), and
products of the hts gene are present on both the fusome and RCs,
making Hts an attractive candidate for participating in the transition from
fusome-containing proliferating cells to RC-containing differentiating cells.
Subsequent work showed that there are four distinct proteins made from splice
variants of the hts gene in the ovary
(Whittaker et al., 1999) (this
article). All predicted Hts proteins share identical N-terminal Head and Neck
domains homologous to mammalian Adducin proteins
(Fig. 1), but have unique
C-termini. Add1 and Add2 contain the Adducin Tail and MARCKS domains. ShAdd
protein has 23 novel C-terminal amino acids and no MARCKS domain. Ovhts
contains 80% of the Adducin Tail domain, and a large C-terminal domain unique
to Drosophila Adducins that we call the Ring Canal domain.
Antibodies made against Hts proteins label the fusome and RCs in the
germline as well as the follicle cell membranes
(Fig. 1)
(Robinson et al., 1994;
Zaccai and Lipshitz, 1996).
Mutant phenotypes are consistent with expression data and include loss of
oocyte specification, too few nurse cells, and RC deformities
(Yue and Spradling, 1992).
Protein localization and mutant phenotype analyses were done prior to knowing
that the hts gene produces multiple proteins; therefore, further
characterization of hts is needed to understand its roles during
oogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
wild-type control was w1118
(Lindsely and Zimm, 1992).
hts
G was from the Zuker
collection (Koundakjian et al.,
2004) and htsW532X was from Trudi
Schüpbach (Princeton University, Princeton, NJ). Expression of
P{UASH} transgenes was done by crossing to one of the following
lines: P{bamP-bam5-Gal4:VP16}
(Chen and McKearin, 2003),
P{tub-Gal4}LL7 (Lee and Luo,
1999), P{nos-Gal4-VP16}
(Van Doren et al., 1998) or a
maternal triple driver, MTD-Gal4, containing the
P{Gal4-nos.NGT}40 (Tracey et al.,
2000), P{COG-GAL4:VP16}
(Rorth, 1998) and
P{nos-Gal4-VP16} (Van Doren et
al., 1998) germline drivers.
Sequencing of mutant alleles
The hts gene was amplified from homozygous mutants by PCR, and the
products were sequenced (W. M. Keck Foundation Biotechnology Resource
Laboratory, Yale University).
Generation of transgenes and transgenic animals
The pCOH vector was made by removing the K10 3' UTR fragment from
pCOG (Robinson and Cooley,
1997) and replacing it with the first 301 nucleotides of the
ovhts 3' UTR (representing base pairs 3868 to 4168)
(Whittaker et al., 1999). The
pUASH vector was made using the same methodology to replace the K10 3'
UTR of pUASP (Rorth, 1998)
with that of ovhts.
The coding region of Ovhts from the cDNA (bp 397-3864) was subcloned into
the pCOH or pUASH vectors. The GFP (S65T) gene was modified from the
pCS2*mt-GFP (gift of Michael Klymkowsky, University of Colorado,
Boulder) vector by PCR to add an 8 Ala linker on the N-terminus of GFP and
cloned into Bluescript KS. GFP (S65T) was then subcloned into
P{COH-Ovhts} and P{UASH-Ovhts} to make
P{COH-Ovhts::GFP} and P{UASH-Ovhts::GFP}.
P{COH-Ovhts-3::GFP} was made by subcloning the
StuI to BglII restriction fragment from pMT-Ovhts-3
(see below) into P{COH-Ovhts::GFP}. To make
P{COH-Cer::Ovhts}, the Cerulean gene was modified using PCR from the
mCerulean-C1 vector (Rizzo et al.,
2004) to add flanking EcoRI sites, and then subcloned in-frame
directly upstream of Ovhts in P{COH-Ovhts}.
To make P{COH-ShAdd::Ven}, ShAdd was cloned into pCOH. Venus
(Nagai et al., 2002) was
modified using PCR to add a 5' XhoI site and 3'
7xHis tag followed by a stop the another XhoI site. Venus was
then subcloned in-frame directly downstream of ShAdd in
P{COH-ShAdd}.
Embryo injections to generate transgenic animals were either performed as
previously described (Robinson and Cooley,
1997), or were done at Duke University, Model System Genomics.
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) using
a peptide representing amino acids 689-718 of Add1, GSPKKDKKK -
KKGLRTPSFLKKKKEKKKAEA (W. M. Keck Foundation Biotechnology Resource
Laboratory, Yale University). Peptide conjugation, injection and antibody
production was done at Cocalico Biological, Inc. (Cocalico Biological, Inc.,
Reamtown, PA). htsM antibody purification was done using the SulfoLink Kit
(Pierce #44895).
S2 cell vectors and transfection
All S2 cell vectors were made in the pMT V5 His copper-inducible system
(Invitrogen). Full-length pMT-Ovhts contained the entirety of the Ovhts coding
region (bp 397-3864). Nucleotides were removed by two-step PCR in each
deletion construct resulting in the loss of the following amino acids from
Ovhts: Ovhts-1 amino acids 632 to 651, Ovhts-2 amino acids
639-658, Ovhts-3 amino acids 650-669 and Ovhts-4 amino acids 785
to 794.
S2 cells were cultured, transfected with 1 µg of DNA using Lipofectin (Invitrogen #50503) and induced with 0.7 mM CuSO4.
Immunoblots
Protein samples were prepared as previously described
(Robinson et al., 1997).
Primary antibodies were diluted in PBT with 5% milk as follows: polyclonal rat
htsF at 1:5000 (Lin et al.,
1994; Robinson et al.,
1997), monoclonal htsRC at 1:10
(Robinson et al., 1994)
(Developmental Studies Hybridoma Bank), monoclonal 1B1 at 1:10
(Zaccai and Lipshitz, 1996)
(Developmental Studies Hybridoma Bank), polyclonal rabbit anti-GFP antibody
1:1000 (Torrey Pines TP401). Horseradish-peroxidase-conjugated secondary
antibodies were used at 1:30,000 (Pierce #31430, #31460 and #31470). Proteins
were detected using SuperSignal West Dura ECL Substrate (Pierce #34076).
Immunofluorescence and image collection
Ovaries were dissected and fixed as described previously
(Robinson and Cooley, 1997).
To visualize actin, egg chambers were incubated with 2 U rhodamine-conjugated
phalloidin in PBT. For antibody labeling, ovaries were incubated either
overnight at 4°C or 2 hours at room temperature with htsRC antibody at
1:10, 1B1 antibody at 1:7, htsF antibody at 1:500, purified htsM antibody at
1:250 or anti-GFP antibody at 1:750 (Molecular Probes A11122). Secondary
antibodies conjugated to Alexafluor 488 or 568 (Molecular Probes) were used at
1:500 and were incubated with ovaries for 2 hours at room temperature. Ovaries
were stored in Antifade (0.23% DABCO in 0.1 M Tris-HCL 90% glycerol) overnight
at 4°C and then mounted. All images were taken on either a ZEISS LSM-510
or a ZEISS LSM-510 META microscope (Center for Cell and Molecular Imaging,
Yale University School of Medicine), and images were processed using Adobe
Photoshop 7.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Robinson et al.,
1997) recognizes ShAdd, Add1 and Add2 (Add
) and Ovhts. In
germaria, htsF antibody labeled the fusome in the germline
(Fig. 2A, arrow) and plasma
membranes in follicle cells (Fig.
2A, arrowhead). 1B1 antibody
(Zaccai and Lipshitz, 1996),
which recognizes Ovhts and Add, had an identical germariumlabeling
pattern to htsF (Fig. 2B). htsM
antibody, which recognizes only Add, labeled follicle cell membranes
(Fig. 2C) and showed no
labeling of the germline. In later-stage egg chambers, htsF, 1B1 and htsM
antibodies continued to show specific labeling of lateral follicle cell
membranes but no germline labeling (Fig.
2E-G). Consistent with RNA in-situ data
(Whittaker et al., 1999),
Ovhts is germline-specific and Add are follicle-cell-specific. As
shadd mRNA is also exclusively found in the germline, and the htsF
antibody only labels the fusome in the germline, ShAdd is likely a fusome
component (see below). Although both shadd and ovhts mRNAs
are expressed in the germline throughout oogenesis
(Whittaker et al., 1999),
their protein products detected with 1B1 and htsF antibodies were only present
in the germarium. This suggests that the proteins are either not translated or
are not stable once egg chambers are formed.
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).
When ShAdd::Ven was expressed with the ovarian tumor (otu)
promoter in the germline of wild-type flies, it localized specifically to
spectrosomes and fusomes (Fig.
2I). As the fusome began to degrade in Region 2, the localization
of ShAdd::Ven also became more dispersed
(Fig. 2I). Thus, ShAdd::Ven
provided additional evidence that ShAdd is a fusome protein.
HtsRC antibody (Robinson et al.,
1994), which recognizes the C-terminus of Ovhts, had a completely
different localization pattern. Starting in Region 2a of germaria, htsRC
labeled discrete puncta, which resolved into RCs in Region 2
(Fig. 2D). htsRC antibody
labeled RCs throughout the rest of oogenesis
(Fig. 2H).
In order to verify the different labeling patterns of antibodies against the N- and C-termini of Ovhts, we made tagged Ovhts transgenes that were expressed specifically in the germline. In separate constructs containing the native ovhts UTRs, the N-terminus of Ovhts was tagged with Cerulean, a modified ECFP, and the C-terminus was tagged with GFP. The Cer::Ovhts transgene did not produce a fluorescent product; however, upon labeling with anti-GFP antibodies, Cer::Ovhts was detected on the fusomes in germaria (Fig. 2J). Like the N-terminus of Ovhts as seen by antibody labeling, Cer::Ovhts localized to both spectrosomes and branched fusomes. Co-staining with 1B1 showed that as the fusome began to break down in Region 2, Cer::Ovhts became dispersed and lost colocalization with 1B1.
GFP fluorescence from Ovhts::GFP localized specifically to RCs (Fig. 2K). As with htsRC antibody, protein was first detected in Region 2a as puncta that appeared to be near, although not within the fusome (Fig. 2K,L', arrowhead). By Region 2b Ovhts::GFP was in rings (Fig. 2K,L', arrow). Ovhts::GFP was seen on RCs in all subsequent stages until stage 13 (data not shown). Thus, localization of tagged Ovhts transgenes recapitulated antibody labeling, with the N-terminus present on fusomes and the C-terminus localizing to RCs.
Western analysis of Ovhts shows cleavage products
Although the predicted size of the Ovhts protein is 128 kDa, ovary extract
never contained a protein of this size. Instead, htsF
(Fig. 3A) and 1B1 (data not
shown) antibodies detected a doublet of 
90 kDa, which are the Add
proteins. When protein extracts were analyzed from virgin female ovaries,
which contain fewer late-stage egg chambers and therefore are enriched for
germarial tissue, a band of 80 kDa was detected
(Fig. 3A). As reported
previously (Robinson et al.,
1994), the htsRC antibody detected a 60 kDa doublet
(Fig. 3B). These results
suggested that Ovhts is cleaved into two smaller proteins. Moreover, when
ovary extracts of flies expressing Ovhts::GFP or Cer::Ovhts were blotted with
anti-GFP antibody, only bands that would represent the cleavage products (with
the added GFP tag) were seen (Fig.
3C). These results, along with protein localization data, show
that Ovhts is cleaved. Based on the fact that antibodies to the N-terminus of
Ovhts label fusomes and antibodies to the C-terminus label RCs, we designated
the cleavage products Ovhts-Fus and Ovhts-RC, respectively.
To further investigate Ovhts cleavage, we expressed Ovhts in S2 cells,
which do not express endogenous Ovhts. Western analysis with either 1B1 to
visualize the N-terminus or htsRC to visualize the C-terminus, revealed 80 kDa
and 60 kDa proteins like those detected in ovary extracts
(Fig. 3D,E). Additionally, an
140 kDa band that represents full-length Ovhts was apparent in S2 cell
extract (Fig. 3D,E). As the
full-length protein can only be detected when Ovhts is overexpressed in S2
cells and not in ovary extracts, the cleavage process in ovaries must be very
efficient.
Characterization of new hts alleles
In order to elucidate the functions of the individual hts
proteins, new alleles of hts were characterized. All previously
described alleles of hts were P-element insertions or
imprecise excisions that reduce expression of all hts transcripts. We
examined two new EMS-induced hts alleles.
htsW532X (gift from Trudi Schüpbach) contains a
single nonsense mutation, W532X, in the tail domain
(Fig. 1B).
htsG was in the Zuker
collection (Koundakjian et al.,
2004), and DNA sequencing showed a deletion of a single G in the
last part of the Tail domain (G2346 of the ovhts transcript). This
results in a frame shift followed by six novel amino acids and a stop codon.
Conceptual translation of htsG
results in a truncated protein that does not contain any of the normal
C-terminal domains (Fig. 1B).
These mutations are downstream of the entire ShAdd coding sequence.
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G and
htsW532X with Hts antibodies and western analysis showed a
distinct difference between the alleles. Even though both truncation alleles
should encode the epitope for the htsF antibody, protein was detected only in
htsG (see Fig. S1E,I in the
supplementary material). Western analysis showed that whereas
htsG expressed a single
truncation product, htsW532X produced no detectable
protein (see Fig. S1M in the supplementary material) and is therefore a null
allele. Additionally in htsG,
antibodies 1B1 and htsF labeled a cytoplasmic protein that persisted in egg
chambers after they emerged from the germarium, which is never seen in wild
type (see Fig. S2 in the supplementary material; data not shown). Therefore,
the mutant truncated protein was aberrantly stable in germline cells that
normally do not have Ovhts-Fus. Mutant follicle cells labeled with 1B1
antibody showed a significant, but not complete loss of Add
localization to lateral membranes (see Fig. S2D in the supplementary
material).
Tagged transgenes rescue ring canals but not the fusome
To determine the functional requirements of the different Hts proteins in
the germline, we crossed tagged hts transgenes expressed from the
otu promoter into both
htsG and
htsW532X mutant backgrounds for rescue experiments. Both
P{Ovhts::GFP} and P{Cer::Ovhts} rescued recruitment of
Ovhts-RC and actin on RCs (Fig.
4B,C, data not shown). However, other hts phenotypes were
not rescued. Labeling with htsF, 1B1 or -spectrin antibodies showed no
fusome-like structure (Fig.
4B,C, data not shown). Anti-GFP labeling in mutants expressing
P{Cer::Ovhts} only showed cytoplasmic labeling
(Fig. 4A). Additionally, the
egg chambers still had too few cells and degenerated. We tested whether the
addition of ShAdd would improve rescuing activity. When P{ShAdd-Ven}
was expressed alone in htsG,
Venus fluorescence was diffuse in the cytoplasm, and hts phenotypes
were not rescued (Fig. 4D).
Expression of P{ShAdd::Ven} with either P{Cer::Ovhts} or
P{Ovhts::GFP} in a
htsG background showed the same
phenotype as the single rescue (Fig.
4E,F): only RCs were rescued, but not the fusome or any of the
phenotypes resulting from the loss of the fusome.
Recent work has shown that the fusome precursor, the spectrosome, first
begins to form during stage 11 of embryogenesis
(Wawersik and Van Doren,
2005). As the fusome develops from the spectrosome, it was
possible that the otu promoter was not providing Ovhts at an early
enough stage. However, earlier expression of Ovhts by driving
P{UASH-ovhts::GFP} with either P{nos-GAL4} or
P{tub-GAL4} produced the same result as the otu promoter
(data not shown).
To determine whether there is a somatic contribution to the hts
phenotype that results in our inability to rescue the fusome, we performed
clonal analysis with htsG.
Germline clones showed the same phenotype as homozygous mutants, whereas egg
chambers that had only follicle cell clones were normal (data not shown).
Therefore, the loss of the fusome and RCs is caused solely by the loss of
full-length Ovhts in the germline.
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G
(Fig. 5C) or
htsW532X (Fig.
5B) flies expressing P{Ovhts::GFP}, GFP was often absent
from germaria and only appeared later. Quantitation of this phenotype revealed
that in htsW532X there was a delay in 30% of germaria, and
in htsG there was a delay in
91% of germaria (Fig. 5D).
Thus, in hts mutants the recruitment of Ovhts-RC and actin, and
therefore the establishment of RCs, did not occur at the correct developmental
stage, suggesting that the fusome is necessary for the timing of RC
development.
Behavior of uncleavable Ovhts
We took advantage of the ability of S2 cells to cleave Ovhts to identify
amino acids necessary for its cleavage. We made a series of small in-frame
deletion mutations in the region of the predicted cleavage site, four of which
are shown in Fig. 6A, and
expressed them in S2 cells. The 1 deletion, which removes 20 amino
acids in the Tail domain, was cleaved at a wild-type level
(Fig. 6B). Deletion 2
removed the last 20 amino acids of the Tail domain, and 3 removed the
last nine amino acids of the Tail and the first 11 amino acids of the RC
domain. The 2 protein was cleaved, although less efficiently than
wild-type protein, whereas the 3 protein was not cleaved
(Fig. 6B). This result
demonstrates that the first 11 amino acids (ALVSQLAQKYA) of the RC domain are
required for cleavage.
To study the effect of uncleavable Ovhts in the ovary, we made a
P{Ovhts-3::GFP} transgene that was expressed from the
otu promoter. Except for the 20 amino acid deletion, this transgene
was identical to the P{Ovhts::GFP} transgene. Western analysis of
Ovhts-3::GFP from ovary extracts demonstrated that, as in S2 cells,
this protein is not cleaved (Fig.
6C). When expressed in wild-type flies, Ovhts-3::GFP was
present not only on RCs, but also on the fusome
(Fig. 6E,E').
Additionally, the 1B1 antibody, which normally only labels the fusome, now
also labeled RCs (Fig.
6E,E''). Therefore, the uncleaved protein localized to both
structures where the cleavage products are normally found.
Although wild-type flies expressing Ovhts-3::GFP were fertile and
produced apparently normal egg chambers, uncleaved Ovhts did cause a subtle,
but completely penetrant dominant defect in the disappearance of the fusome.
In wild-type germaria, the fusome begins to disappear where the RCs are
starting to form (Fig.
6D,D'',F). This results in unobstructed RCs with fragmented
fusome material between them, but not through them. In flies expressing
P{Ovhts-3::GFP}, fusome material was present within
the RCs as late as stage 2 and 3 egg chambers
(Fig. 6G). In some cases,
GFP-positive rings were occluded with GFP-negative fusome material that could
be visualized with 1B1 antibody (Fig.
6G',G'' arrowhead). Therefore, at least some of the
aberrant fusome contained only wild-type Ovhts-Fus protein and not the
N-terminal portion of P{Ovhts- 3::GFP}. Additionally,
RC rims were thicker than normal, less organized and misshapen
(Fig. 6, compare F with G).
These results suggest that the cleavage and proper maintenance of the Ovhts
domains may play a role in the transition from a fusome to RCs.
We also tested whether the P{Ovhts-3::GFP}
transgene could rescue hts mutants,
htsG and
htsW532X (data not shown). As with expression of
P{Ovhts::GFP}, P{Ovhts-3::GFP} rescued RCs but not
the fusome. The rescued RCs were also labeled with 1B1 antibody indicating
that the N-terminus of Ovhts was present.
Ovhts is expressed throughout all of oogenesis
Our ability to rescue RCs with Ovhts transgenes provided an opportunity to
investigate the function of Ovhts-RC. To determine when full-length Ovhts
needs to be expressed for RC localization, stage-specific induction of Ovhts
expression was done. Wild-type flies carrying a P{UASH-Ovhts::GFP}
transgene were crossed to two different Gal4 lines: MTD-Gal4 (see
Materials and methods) that induces strong expression throughout oogenesis or
P{bam-Gal4} that induces expression only in Region 1 of the
germarium. In ovaries from P{UASH-Ovhts::GFP}, MTD-Gal4 flies,
Ovhts::GFP was on RCs in all stages of oogenesis
(Fig. 7A). However, in ovaries
from P{UASH-Ovhts::GFP}; P{bam-Gal4}, Ovhts::GFP was only in the
germaria, and on rare occasions on RCs in stage 2 egg chambers
(Fig. 7B). Thus, continuous
expression of Ovhts-RC protein is needed to maintain localization to RCs.
|
G flies using the
P{nos-GAL4} driver whose expression is high in the germarium, low
during stages 2-6, and then high again starting approximately at stage 7. This
allowed rescue of RCs when they form in the germaria of mutants, followed by
about a day where little or no new Ovhts protein is produced. As expected
Ovhts::GFP was present on RCs in the germaria, absent in mid-stage egg
chambers, and present again in later egg chambers within the same ovariole
(Fig. 7C). Both the amount of
F-actin and its organization at RCs mirrored the presence of Ovhts::GFP
(Fig. 7C'). When
Ovhts::GFP was present, RCs appeared wild type. In egg chambers lacking
Ovhts::GFP, there were no clear F-actin-containing RC rims. There were,
however, actin-rich areas that may be disintegrating RC rims
(Fig. 7C',D). Thus,
continued expression of Ovhts is needed for the recruitment and/or maintenance
of F-actin on RCs. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yue and Spradling, 1992); yet
its exact functions have not been characterized completely. We provide
evidence that Ovhts is made as a full-length precursor protein that is
cleaved, allowing the N-terminal Adducin-like domain (Ovhts-Fus) to localize
to the fusome and the C-terminal unique domain (Ovhts-RC) to localize to RCs.
Ovhts-Fus is stable only in proliferating cells whereas Ovhts-RC is present
only after mitosis is complete, which further emphasizes the importance of
post-translational regulation for this protein. We created an uncleavable form
of the protein and show that it can dominantly delay early egg chamber
development. Finally, expression of transgenes at specific developmental time
points shows that Ovhts-RC is necessary for F-actin localization to RCs during
all stages of oogenesis.
Ovhts is a polyprotein whose products are differentially maintained
Our data demonstrate that Ovhts is made as a full-length precursor protein,
which is cleaved to form two mature proteins with separate functions. The
detection of cleavage products by western blotting and the differential
localization of the cleaved proteins in egg chambers provide strong support
for this conclusion. We have ruled out the alternative mechanism of producing
the downstream protein, Ovhts-RC, by the use of an internal ribosomal entry
site (IRES). There is no evidence of an IRES in the sequence of the
ovhts mRNA. Furthermore, both of the new hts alleles
described in this paper have point mutations 5' to any potential IRES
for Ovhts-RC translation, yet neither mutant produces Ovhts-RC.
|
). Viral
polyproteins are translation products of whole viral genomes, including both
replication enzymes and structural proteins
(Stanway et al., 2000). Viral
polyproteins undergo a number of different cleavages, some of which use
polyprotein-encoded proteases, and some of which occur through autoproteolysis
(Palmenberg, 1990). The
Vasopressin polyprotein includes vasopressin, neurophysin (NP) and a third
protein of unknown function. The products of the Vasopressin polyprotein are
released by at least two different uncharacterized cellular endoproteases
(Acher and Chauvet, 1988).
The Ovhts polyprotein is likely to be cleaved by an endoprotease rather
than undergoing autoproteolysis. The active site for autoproteolysis contains
one of three amino acids: Ser, Thr and Cys
(Perler et al., 1997). The
Ovhts cleavage region contains one Ser residue, which when mutated to either a
Cys or Ala did not affect Ovhts cleavage (L.N.P. and L.C., unpublished). This
suggests Ovhts is not cleaved autoproteolytically. Therefore, the more likely
mechanism of Ovhts cleavage is through a cellular endoprotease. Because
cleavage can occur in S2 cells as well as the ovary, the protease is likely to
be widely expressed; however, the cleavage region does not contain any known
protease cleavage sites.
The linkage of the RC domain to Adducin may facilitate Drosophila ring canal development
The arrangement of the Ovhts polyprotein is conserved in
Drosophila. Twelve Drosophila species have been sequenced to
date (Grumbling and Strelets,
2006). We annotated the hts homolog from each species,
and found that the overall gene organization was highly conserved (L.N.P. and
L.C., unpublished). In all cases, an exon encoding the novel RC domain was
similarly positioned near the 3' end of the gene. Whereas the Adducin
domains are greater than 90% identical, the RC domain is only 43% conserved,
indicating that it is diverging much more quickly. The RC domain is not
present as an independent gene or as part of the Adducin gene of
other sequenced insect genomes. Thus, it appears that the Ovhts polyprotein
was acquired after the divergence of Drosophila.
To explain the existence of an Ovhts polyprotein, we propose a model in which the Ovhts polyprotein functions to provide Ovhts-RC at a critical time and place and in sufficient abundance through its linkage to Ovhts-Fus. At all stages of oogenesis the Ovhts full-length precursor is translated and cleaved. In Region 1 of germaria, the Ovhts-Fus product is maintained and localizes to the fusome. However, the Ovhts-RC product cannot be detected in Region 1, indicating that it is rapidly degraded. Starting in Region 2, the Ovhts-RC domain begins to be maintained and localizes to RCs. This coincides with the completion of mitotic cell cycles, and perhaps also the expression of an Ovhts-RC binding partner required for its stability. Ovhts-RC first appears as puncta around the fusome, suggesting that the cleavage of the polyprotein may take place on or near the fusome. Another possibility is that the Ovhts-RC stabilizing factor is itself associated with the fusome. In either case the Ovhts-RC protein is in close proximity to the arrested cleavage furrows where it is needed to initiate RC development.
An open question is why the transgenes we tested do not restore the fusome in hts mutants. We ruled out the effects of secondary mutations by using several combinations of hts alleles from different backgrounds (i.e. P-element insertions, point mutations and deficiencies), and we deem the existence of another protein isoform unlikely as we performed 3' RACE and found only the known transcripts. The most likely possibility is that expression of ovhts is required at a specific time early in development for the formation and stability of spectrosomes in germline progenitor or stem cells, and we did not recapitulate this expression pattern in the rescue experiments.
Ovhts cleavage facilitates a smooth transition of fusome to ring canals
The presence of the uncleavable Ovhts-3::GFP protein affects the
transition from a fusome to an RC, even though it localizes to both
structures. Therefore, cleavage is not required for localization but is
necessary for the full function of the Ovhts polyprotein products. The
expression of uncleavable Ovhts-3::GFP causes the aberrant persistence
of fusome material in RCs. Strikingly, the fusome material includes endogenous
wild-type Ovhts-Fus protein that has failed to degrade. As it is known that
mammalian Adducins function as heterotetramers, a direct interaction of
wild-type Ovhts-Fus or ShAdd with Ovhts-3::GFP may occur and result in
pulling fusome material into the lumen of the RCs. This hypothesis is
supported by the fact that RCs in hts mutants that lack wild-type
Ovhts-Fus are rescued by Ovhts-3::GFP and do not have fusome plugs or
misshapen rims. The dominant-negative effect of Ovhts-3::GFP on
wild-type RCs disappears by stage 3, suggesting that uncleaved Ovhts is
tolerated in egg chambers that have degraded the fusome. Therefore, it is the
transition from fusomes to RCs that is particularly sensitive to the presence
of uncleaved Ovhts.
Ovhts-RC is required throughout oogenesis
RC formation has been described as a multi-step process beginning with the
arrested cleavage furrow followed by the recruitment of Filamin, Ovhts-RC and
F-actin, and completed with the addition of other actin binding proteins
(Hudson and Cooley, 2002). We
found that the recruitment of Ovhts-GFP to RCs is delayed in hts
mutants lacking a fusome, supporting a role for the fusome (and possibly
prelocalization of Ovhts-RC) in the initiation of RC development. Furthermore,
without continual expression of Ovhts-RC
(Fig. 7), F-actin was lost from
the inner rim of RCs. As the F-actin cytoskeleton of RCs is highly dynamic
(Kelso et al., 2002), the
absence of Ovhts-RC in hts mutant RCs may tip the balance of actin
dynamics toward depolymerization, resulting in the depletion of RC F-actin.
Thus, Ovhts-RC is needed continuously either to stabilize polymerized actin or
to promote actin polymerization.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/4/703/DC1
|
ACKNOWLEDGMENTS |
|---|
G allele. We also thank Vann
Bennett for mammalian MARCKS domain antibody. We are grateful to all members
of the Cooley lab for help with editing and insightful conversation. This work
was supported by a NSF predoctoral fellowship to L.N.P., a Burroughs Wellcome
Fund Postdoctoral Fellowship of the Life Sciences Research Foundation to T.S.,
and a National Institutes of Health grant (GM52702) to L.C. |
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2, P<0.001. Scale bars: 10
µm.
