SEGR | SHIM
| SIPT | SOB | UGRMT
| UCCR | UPB | Main
Page
CELL CYCLE REGULATION
DURING OOGENESIS
|
Mary A. Lilly, PhD, Head, Unit on Cell Cycle Regulation Amy
Hong, PhD, Visiting Fellow Takako
Iida, PhD, Visiting Fellow Stefania
Senger, PhD, Visiting Fellow Isamu
Sugimura, PhD, Visiting Fellow Mesha-Gay Brown, BA, Postbaccalaureate Fellow |
|
|
Highly
conserved nuclear protein required for the maintenance of the meiotic cycle
and the repair of double-stranded breaks during oogenesis and encoded by missing oocyte Iida, Senger To identify the pathways that direct entry into
and maintenance of the meiotic cycle in the single pro-oocyte, we screened
for mutants in which all 16 cells enter the endocycle and develop as nurse
cells. From this screen, we identified a new gene, missing oocyte (mio),
that is required for the maintenance of the meiotic cycle. In mio mutants, the oocyte enters the
meiotic cycle, forms mature synaptonemal complexes, and progresses to
pachytene. However, this meiotic state is not maintained. Ultimately, mio oocytes abandon the meiotic cycle,
enter the endocycle, and develop as nurse cells. We characterized the molecular structure of
the mio gene and determined that mio is predicted to encode a protein
of 867 amino acids that is highly conserved from yeast to humans. In higher
eukaryotes, all Mio family members share a similar domain structure. The
amino termini contain a series of four to six well-conserved WD40 repeats.
WD40 repeats often provide a surface for protein-protein interactions The
WD40 repeats found in mio family
members are most similar to those present in the chromatin-binding protein
CAF1p48/RbAp48, which is a component of numerous chromatin-remodeling
complexes. Specifically, CAF1p48/RbAp48 is found in complexes that modify
chromatin through the acetylation and deacetylation of histones. In addition
to the WD40 repeats, Mio family members contain a highly conserved 50–amino
acid domain near their C termini that shares structural similarities with two
well-characterized zinc binding domains, the RING finger and the PHD finger.
RING finger domains are present in a subclass of E3 ubiquitin ligases while
PHD fingers have been implicated in chromatin binding. While the “Mio domain”
does share structural similarities with these zinc-binding domains, it does
not fit the exact consensus of either a canonical RING finger or a canonical
PHD finger. Therefore, the biochemical function of this highly conserved
domain remains to be determined empirically. Mio accumulates to high levels in the oocyte
nucleus during early prophase of meiosis I. Double labeling with anti-Mio
antibodies and an antibody against the synaptonemal complex protein C(3)G
indicate that Mio specifically localizes to the nucleus of the oocyte soon
after the completion of premeiotic S phase, making Mio one of the earliest
nuclear markers for the oocyte that is not a known component of the
synaptonemal complex. Intriguingly, the mio ovarian phenotype is suppressed by inhibiting the formation
of the double-stranded breaks (DSBs) that initiate meiotic recombination
during meiosis. In mio single
mutants, the oocyte frequently enters the endocycle and becomes polyploid.
However, when placed in a genetic background in which DSB formation is
inhibited, the majority of mio egg
chambers retain an oocyte and develop to late stages of oogenesis. The
simplest interpretation of the data is that mio is required to repair the DSBs that initiate meiotic
recombination and that the inability to repair DSBs significantly contributes
to the mio phenotype. To obtain further insight into the pathway(s)
in which Mio functions, we undertook a screen to identify dosage-sensitive
modifiers of the mio phenotype. Our
initial analysis of the data from the screen indicates that mio is dominantly suppressed by
mutations in the Rad51 homolog spnA.
Rad51 is required for the repair of DSBs in both yeast and mammals. Further
studies of mio will help elucidate
the poorly characterized pathways that control meiotic progression and the
maintenance of oocyte identity. Iida T, Lilly MA. missing oocyte encodes a highly-conserved nuclear protein
required for the maintenance of the meiotic cycle and oocyte identity in
Drosophila. Development 2004;131:1029-1039. Regulation
of two variant cell cycles during the maturation of the Drosophila egg by the p27KIP1-like CDK inhibitor Dacapo
Hong, Brown; in collaboration with
Aladjem Animal oocytes undergo a highly conserved
developmental arrest in the prophase of meiosis I, often marking a
rapid-growth period for the oocyte, an arrest that is necessary to coordinate
meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst.
Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. In
contrast, its 15 mitotic sisters enter the endocycle and become polyploid in
preparation for their role as nurse cells. How germline cysts establish and
maintain these two independent cell cycles is unknown. We have shown that the
p21CIP/p27Kip1/p57Kip2-like cyclin-dependent
kinase inhibitor (CKI) Dacapo maintains the prophase I meiotic
arrest of the Drosophila oocyte. dacapo is a vital gene that
specifically inhibits the activity of CycE/Cdk2 complexes. CycE/Cdk2 activity
is required for S phase in Drosophila.
Throughout much of the growth phase of Drosophila
oogenesis, the levels of cki Dacapo oscillate in the 15-polyploid nurse cells
but remain persistently high in the single oocyte. We have shown that both
modes of Dacapo regulation are functionally important. In the oocyte,
prophase I arrest is lost or not properly established in germline cysts that
lack Dacapo. This is the first demonstration of a cip/kip family member
functioning in a normal meiotic cycle. In addition, our data indicate that
Dacapo is part of the biochemical oscillator that drives the nurse cell
endocycle. Specifically, we find that, in polyploid nurse cells, the
oscillations of Dacapo facilitate the relicensing of DNA replication origins
during endoreplication by inhibiting CycE/Cdk2 activity at the end of each
endocycle S phase. Our data are consistent with recently proposed models
suggesting that the periodic expression of members of the cip/kip family of
Cdk inhibitors direct entry into the Gap phase during endo-replicative
cycles. We propose that it is through the differential regulation of the cki
Dacapo that two dramatically different cell cycles, the meiotic cycle and the
endocycle, are independently maintained within the common cytoplasm of the
ovarian cyst. Currently, we are performing genetic screens to identify
additional genes that regulate CycE/Cdk2 during oogenesis. Recent evidence suggests that during the
mitotic cycle inappropriately high G1 cyclin activity leads to
genomic instability due to the inefficient formation of prereplication
complexes. In this model, an inappropriately low density of DNA replication
origins leads to the production of persistently stalled forks that have the
potential to become recombinogenic. We find that mutations in dap lead to a dramatic increase in the
presence of stalled replication forks in endocycling nurse cells. In
addition, dap nurse cells have
extremely low levels of the prereplication complex component Dup/Cdt1. Our
data suggest a model in which Dap inhibits CycE/Cdk2 activity during the Gap
phase and thus promotes the efficient relicensing of DNA replication origins.
Intriguingly, a similar role has been proposed for the CKI Sic1 in promoting
replication origin licensing in late G1 in S. cerevisiae. Hong A, Lee-Kong S, Iida T, Sugimura I, Lilly
MA. The p27cip/kip ortholog dacapo
maintains the Drosophila oocyte in prophase of Mediation
of intercellular ER connectivity in Drosophila
ovarian cysts by the fusome Iida; in collaboration with Lippincott-Schwartz,
Snapp Gametogenesis in diverse organisms involves
the formation of germline cysts containing interconnected germ cells. Drosophila ovarian cysts arise through
a series of four synchronous incomplete mitotic divisions. After each round of
mitosis, a membranous organelle, the fusome, grows along the cleavage furrow
and the remnants of the mitotic spindle to connect all cystocytes in a cyst.
The fusome is essential for the pattern and synchrony of the mitotic cyst
divisions as well as for oocyte differentiation. Using live cell imaging,
GFP-tagged proteins, and photobleaching techniques, we have demonstrated that
fusomal endomembranes are part of a single continuous endoplasmic reticulum
(ER) that is shared by all cystocytes in dividing ovarian cysts. Membrane and
lumenal proteins of the common ER freely and rapidly diffuse between
cystocytes. The fusomal ER mediates intercellular ER connectivity by linking
the cytoplasmic ER membranes of all cystocytes within a cyst. Before entry
into meiosis and onset of oocyte differentiation (between region 1 and region
2A), ER continuity between cystocytes is lost. Furthermore, analyses of hts and Dhc64c mutants indicate that intercellular ER continuity within
dividing ovarian cysts requires the fusome cytoskeletal component and suggest
a possible role for the common ER in synchronizing mitotic cyst divisions. Our results have implications for
communication in other syncitial systems as well, such as spermatozoa in
mammals and the ectoderm of hydra cnidoblasts. In both of these examples,
groups of synchronously developing cells contain evidence of intercellular ER
connections between dynamic ring canals. Thus, a shared ER may be a general
mechanism for coordinated cyst development. Snapp EL, Iida T, Frescas D,
Lippincott-Schwartz J, Lilly MA. The fusome mediates intercellular ER
connectivity in Drosophila ovarian
cysts. Mol Biol Cell
2004;15:4512-4521. COLLABORATORS Mirit Aladjem, PhD, Laboratory of Molecular Pharmacology,
NCI, Ruth Lehmann, PhD, Howard Hughes Medical Institute,
Department of Cell Biology, Skirball Institute of BioMolecular Medicine, New
York University School of Medicine, New York, NY Jennifer Lippincott-Schwartz,
PhD, Cell Biology and Metabolism
Branch, NICHD, For further information, contact mary_lilly@nih.gov |
|
