The Interactive Fly
Genes involved in tissue and organ development
Fertilization typically involves membrane fusion between sperm and eggs. In Drosophila, however, sperm enter eggs with membranes intact. Consequently, sperm plasma membrane breakdown (PMBD) and subsequent events of sperm activation occur in the egg cytoplasm. It has been proposed that mutations in the sneaky (snky) gene result in male sterility due to failure in PMBD. This study supports this proposal by demonstrating persistence of a plasma membrane protein around the head of snky sperm after entry into the egg. It is further shown that snky is expressed in testes and encodes a predicted integral membrane protein with multiple transmembrane domains, a DC-STAMP-like domain, and a variant RING finger. Using a transgene that expresses an active Snky-Green fluorescent protein fusion (Snky-GFP), it is shown that the protein is localized to the acrosome, a membrane-bound vesicle located at the apical tip of sperm. Snky-GFP also allowed following the fate of the protein and the acrosome during fertilization. In many animals, the acrosome is a secretory vesicle with exocytosis essential for sperm penetration through the egg coats. Surprisingly, the Drosophila acrosome is a paternally inherited structure. Evidence is presented that the acrosome induces changes in sperm plasma membrane, exclusive of exocytosis and through the action of the acrosomal membrane protein Snky. Existence of testis-expressed Snky-like genes in many animals, including humans, suggests conserved protein function. The characteristics of Drosophila Snky, acrosome function and sperm PMBD is related to membrane fusion events that occur in other systems (Wilson, 2006).
Studies of fertilization have revealed the universal importance of membrane events to prepare and coordinate the gametes and ensure their successful union. These events have been most extensively studied in sperm of selected marine invertebrate and mammalian species and include dynamic changes in membrane proteins and lipids during sperm capacitation, acrosome exocytosis, binding, adhesion and membrane fusion with the egg. The majority of molecules with confirmed roles in these processes are cell adhesion molecules, ion channels or signal transduction components with molecular mechanisms of action that have been studied in multiple cell types. There is considerable interest in discovering proteins that act specifically during fertilization. The characterization of these molecules will contribute to a better understanding of the specialized properties of gametes and to the practical goal of identifying new targets for contraceptives (Wilson, 2006).
A genetic approach using Drosophila was employed to identify novel sperm molecules required for fertilization. Previous studies of male sterile mutants indicated that those affecting fertilization might be relatively rare. Therefore, a large number of male-sterile mutations were isolated and categorized to find a subset that disrupt sperm-egg interactions or induce paternal effect defects (Fitch, 1998; Wakimoto, 2004). Previously described were mutations in a gene called sneaky (snky), which met genetic criteria of specifically affecting fertilization (Fitch, 1998). Mutations of snky showed detectable effects only on male fertility. Sperm produced by mutant males were competent to enter the egg but arrested before sperm nuclear decondensation and aster formation. It was proposed that this sperm activation defect is due to a failure in breakdown of the sperm plasma membrane, a step that normally occurs immediately after sperm entry into the egg in Drosophila (Wilson, 2006).
This study provides phenotypic evidence of a role for Snky in affecting the integrity of the sperm plasma membrane during fertilization. The snky gene was molecularly identify and its transcript and predicted protein were characterized to investigate how the protein might function. The results indicate that Snky is an acrosomal membrane protein that is contributed to the early embryo. In addition to suggesting a possible molecular role for Snky, these studies have implications for the function and the fate of the acrosome during Drosophila fertilization (Wilson, 2006).
Fertilization in Drosophila is unorthodox in that it does not involve a membrane fusion event between the sperm and egg. The mechanism used by the sperm to penetrate through the egg plasma membrane remains an interesting mystery. In ultrastructural studies of fertilization in D. melanogaster and D. montana, Perotti (1975) observed that the entire sperm enters the egg, including the excessively long tail surrounded by its plasma membrane. There was no evidence for 'extra' membranes that would support uptake into the egg by endocytosis. While these studies did not capture the sperm before nuclear decondensation, Perotti described one case in which a decondensing sperm nucleus was surrounded by what appeared to be remnants of the sperm plasma membrane. These studies support the idea that the Drosophila sperm head must undergo PMBD in the egg cytoplasm. This step is required for the sperm nucleus to gain access to activating cytoplasmic factors that promote nuclear decondensation and replication, and for the centrosome to elaborate the sperm aster (Fitch, 1998). The current studies support this sequence of events and implicate Snky function in an event upstream of PMBD. The original proposal that snky mutant sperm fail in sperm activation because they arrest before PMBD was based on their poor accessibility to membrane impermeant chromatin dyes (Fitch, 1998). Further support was obtained by Ohsako (2003), who used an antibody to a proposed cell surface proteoglycan to demonstrate retention on the sperm head before and after insemination. In the current study, the rat CD2 protein was ectopically expressed on the sperm plasma membrane and it was showed that immunoreactivity to this specific integral membrane protein was retained on the head of snky1 sperm for at least 30 minutes after entry into the egg. These observations provide strong support for the proposal that snky sperm do not shed the plasma membrane surrounding the head. They also suggest that understanding the function of the Snky protein should provide clues about how PMBD is initiated or accomplished at the molecular level (Wilson, 2006).
Molecular studies show that Snky is a member of a family of related membrane proteins that are present in animals but are apparently absent in other lineages. Conservation among Snky family members points to three domains of potential functional significance. The most striking motif is the C4-C4 RING finger, which has been noted in at least four other proteins. In one of these, hNOT4, the C4-C4 RING has been shown to fold and bind zinc in a cross-brace fashion similar to the more common C3HC4 RING finger, suggesting structural and functional similarity between these variants. Although RING finger proteins have been implicated in a broad variety of cellular functions, recent studies suggest that their unifying role is in mediating protein-protein interactions in large multiprotein assemblages. Thus, Snky may play a role in organizing and holding together macromolecular complexes at the membrane via its C4-C4 RING. The second Cys-containing region is the patterned 6 Cys motif, the functional significance of which is indicated by conservation and by the recovery of a male sterile snky allele that mutates the second Cys to a Ser. Cysteines in this region may form disulfide bonds to create a binding pocket or otherwise allow interactions with additional proteins. The third region is the DC-STAMP-like region, named after Dendritic cell specific transmembrane protein, a plasma membrane protein that was first described for its expression in human dendritic cells. Thus far, the function of only a few DC-STAMP-domain-containing proteins have been examined through the analysis of mutations or other knockdown strategies. The general classification of these proteins has been as members of a new class of putative receptors. Mouse DC-STAMP is upregulated in differentiating osteoclasts, and required for osteoclast fusion to form multinucleate cells. The C. elegans SPE-42 is required in sperm and is proposed to act at a step in fertilization just before or during sperm-egg membrane fusion (Kroft, 2005). Snky is the third DC-STAMP domain-containing protein to have its function studied and, like Spe-42, it has a role in sperm function. Although the precise molecular function of the DC-STAMP domain remains unknown, these three examples support a role in mediating membrane-membrane interactions (Wilson, 2006).
The predicted structure of Snky, with its multiple transmembrane domains, and Snky-GFP localization studies provide evidence that Snky is an acrosomal membrane protein. This localization presents the intriguing question of how an acrosomal membrane protein is able to influence the integrity of the sperm plasma membrane. The findings also have broader implications for acrosome function. The acrosome is a Golgi-derived membrane-bound organelle found at the apical end of sperm, and its function has been extensively studied in marine invertebrates and mammals. In these organisms, the acrosome is best known as a specialized secretory vesicle that undergoes exocytosis. Like many secretory events, a rise in intracellular Ca2+ is required for acrosome exocytosis. This increase in Ca2+ requires influx into the sperm as well as efflux from internal stores. The acrosome has been identified as an internal source of Ca2+ and is believed to release Ca2+ to contribute to its own exocytosis (Walensky, 1995; Herrick, 2005). Ultrastructural studies show that exocytosis involves vesiculation of the outer acrosomal membrane and overlying plasma membrane. This results in the release of contents of the acrosome, which include hydrolytic enzymes and other components that facilitate binding and penetration through the egg coats. Exocytosis also exposes the inner acrosomal membrane, resulting in a new membrane patch and associated acrosomal molecules on the surface of the sperm. In marine invertebrates, this newly exposed region is the site of binding and membrane fusion with the egg, providing a direct physical link between the requirement for acrosome exocytosis and membrane fusion. In mammals, acrosome exocytosis is also a prerequisite for sperm to bind to and fuse with the egg. However, the plasma membrane that lies over the equatorial segment of the acrosome, and not the acrosome membrane itself, is believed to be the point of membrane fusion with the egg. Experimental studies of hamster sperm suggest that fusion competency of this specialized region of mammalian sperm requires changes that occur immediately before or during acrosome exocytosis, as well as the contents of the acrosome (Wilson, 2006 and references therein).
Considering these known acrosome functions, it is interesting that acrosomes are not universal features of animal sperm. Acrosomes are not present in the amoeboid sperm of nematodes, and they are occasionally absent or reduced in species that otherwise possess the flagellated sperm typical of animals. For instance, the absence or reduction of an acrosome in mature sperm of teleosts and certain insect species is well documented and is generally considered a derived condition. Hence the requirement for the acrosome during fertilization has been eliminated or bypassed in some lineages during the course of evolution. Ultrastructural studies show that an acrosome is typical of insect sperm. However, few studies have examined the role of the insect acrosome during fertilization. Studies of fertilization in the house fly Musca domestica suggested 'loosening or loss' of the sperm plasma membrane before entry into the micropyle of the egg, followed by exocytosis of acrosomal contents during passage of the sperm through the micropyle (Wilson, 2006).
These studies revealed a different fate for the Drosophila acrosome. The observation that Snky-GFP was a paternally contributed molecule to the early egg was a surprising finding. The persistence of a single prominent Snky-GFP structure, with an intensity and shape in eggs that appeared similar to those seen in mature sperm, suggests the possibility that the inherited structure was an intact acrosome. This possibility was further supported by studies tracking GFPsecr, a soluble protein that is secreted into the extracellular space when expressed in other Drosophila cells. Its robust and identical appearance to Snky-GFP argues against exocytosis, at least until after prometaphase of the first embryonic cell cycle (Wilson, 2006).
These cytological observations of the fate of the acrosome during normal fertilization, combined with the defect in PMBD observed in snky- mutant sperm, suggest that the acrosome may be acting primarily as a signaling vesicle to elicit changes in the overlying sperm plasma membrane. This activity requires the Snky acrosomal membrane protein, but occurs without acrosome exocytosis. Snky may be serving as a receptor that permits communication between the acrosome and plasma membrane. Alternatively, Snky or its associated proteins may serve to initiate or maintain contact between the membranes, or modify membrane lipids or proteins in preparation for PMBD. In this sense, Snky, DC-STAMP and SPE-42 may share a common mechanism in promoting membrane interactions (Yagi, 2005; Kroft, 2005). However, in the case of Snky, the pathway would lead to breakdown of the overlying plasma membrane, rather than fusion between two membranes (Wilson, 2006).
In Drosophila, Snky is required specifically for male fertility.
It will be interesting to determine whether Snky family members are required
for sperm function in zebrafish, which have sperm that lack acrosomes, and for
acrosome function in marine invertebrate and mammalian sperm. If human
Snky-like proteins are specifically required for sperm function, then they may
be potential targets for male contraceptives. More immediately, these studies of
Snky provide tools for further investigating how the membrane events that
occur during Drosophila fertilization compare to conventional views
of membrane dynamics during sperm activation and fertilization in animals.
Research on the acrosome has focused primarily on its function as a
specialized secretory or Ca2+ storage vesicle. These studies suggest
a primary role as signaling vesicle in Drosophila, with a newly
identified acrosomal membrane protein communicating directly or indirectly
with the plasma membrane to affect changes in membrane integrity. Comparisons
among species should continue to shed light on the intriguing ways in which
sperm structures and fertilization molecules, such as Snky, may be selected
for conservation or diversification during the course of evolution (Wilson, 2006).
Chromatin condensation is a typical feature of sperm cells. During mammalian
spermiogenesis, histones are first replaced by transition proteins and then by
protamines, while little of this process is known for Drosophila. This study characterizes
three genes in the fly genome, Mst35Ba, Mst35Bb, and
Mst77F. The results indicate that Mst35Ba and Mst35Bb
encode dProtA and dProtB, respectively. These are considerably larger than
mammalian protamines, but, as in mammals, both protamines contain typical
cysteine/arginine clusters. Mst77F encodes a linker histone-like protein
showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced
green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying
Drosophila lines show that these proteins become the important
chromosomal protein components of elongating spermatids, and His2AvDGFP
vanishes. Mst77F mutants [ms(3)nc3] are
characterized by small round nuclei and are sterile as males. These data suggest
the major features of chromatin condensation in Drosophila
spermatogenesis correspond to those in mammals. During early fertilization
steps, the paternal pronucleus still contains protamines and Mst77F but regains
a nucleosomal conformation before zygote formation. In eggs laid by
sesame-deficient females, the paternal pronucleus remains in a
protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the
sesame gene product is essential for removal of protamines while Mst77F
removal is independent of Sesame (Raja, 2005).
For mammals, the somatic set of histones are modified, as these
are in part replaced by specific variants during meiotic prophase. After
meiosis, histones are replaced by major transition proteins TP1 and TP2
and subsequently by highly basic protamines to ensure the
remodeling of chromatin to a typically highly condensed and transcriptionally
silent state of mature sperm. These replacements leads to a shift from
histone-based nucleosomal conformation to a radically different conformation,
resembling stacked doughnut structures containing protamines as major chromatin
condensing proteins and DNA. Some mammals have only one protamine gene,
while mice and humans have two genes encoding two
different protamines, both of which are essential for fertility and are
haploinsufficient. HILS1 (spermatid-specific
linker histone H1-like protein) has been proposed to participate in chromatin
remodeling in mouse and human spermiogenesis.
The transition between histone removal and its replacement
by protamines in mice and humans is characterized by small 6- to 10-kDa
transition proteins acting as a short-term chromosomal proteins.
In mice, the transition proteins TP1 and TP2 are redundant
in function. In fishes and birds, transition proteins are missing and protamines
directly reorganize the chromatin. In annelids and echinoderms, the nucleosomal
configuration is maintained in sperms,
while protamine-like proteins have been described for
mussels. These protamine-like proteins lack the typical
high cysteine content necessary for disulfide bridges.
Therefore, a doughnut-type chromatin structure as in mammals is unlikely to
occur in mussels. It has been proposed that
the protamine-like proteins in mussels belong to the histone H1 family. The
sperm chromatin of mussels contain core histones and thus a nucleosomal
configuration, but histone H1 is replaced by protamine-like molecules which
organize the higher order structure of the chromatin (Raja, 2005).
For Drosophila melanogaster, chromatin reorganization after meiosis has
not been studied at the molecular level. At the light microscopic level,
the Drosophila spermatid nucleus is initially round after meiosis and
then is shaped to a thin needle-like structure with highly condensed chromatin,
so that the volume of the nucleus is condensed over 200-fold.
In mammals, the volume of the nucleus is reduced over
20-fold. In the mature sperms of Drosophila, core
histones are not detectable by immunohistology. There is
histochemical evidence for the presence of very basic proteins in sperm,
but it still remains an open question whether histones are
replaced by protamine-like basic proteins in Drosophila. The analysis of
the Drosophila genome sequence
revealed that the proteins encoded by two genes show similarity to mammalian
protamines for which the male-specific transcripts Mst35Ba and
Mst35Bb have been found and have been proposed to
encode protamine-like proteins. Another male specifically
transcribed gene, Mst77F, is a distant relative of the histone H1/H5
(linker histone) family and has been proposed to play a role either as a
transition protein or as a replacement protein for compaction of the
Drosophila sperm chromatin. With enhanced green
fluorescent protein (eGFP) fusion for these abovementioned proteins, this study shows that Mst35Ba and Mst35Bb indeed encode protamines and
Mst77F encodes a linker histone-like protein. The expression pattern of
Mst77F overlaps the pattern of protamines as a chromatin component. Furthermore, during fertilization, the removal of protamines from the male
pronucleus requires the function of the maternal component, Sesame, but not for
the removal of Mst77F. It has been shown that sesame mutants cause
impairment of the entry of histones into the male pronucleus (Raja, 2005).
Mst35Ba and Mst35Bb are
present at cytological position 35B6 and 35B6-7, respectively, on the chromosome
arm 2L. These two genes are arranged in tandem, and both consist of three exons.
The 5'UTR, coding region, and the 3'UTR of these
genes are highly identical; they probably arose from a
recent gene duplication. The encoded protamines show over 94% identity to each
other (Raja, 2005).
A remarkable feature of protamines is their ability to form intermolecular
disulfide bridges, which is reflected by the conserved cysteine residues within
mammalian protamines. The dProtA and dProtB are of 146
amino acids (aa) and 144 aa, respectively, and thus longer than even the human
and mouse Protamine-2, which are 102 aa and 107 aa, respectively.
Both Drosophila protamines contain 10 cysteines each
and show significant similarity, particularly with respect to a high cysteine,
lysine, and arginine content to mammalian protamines.
Human and mouse Protamine-1 aligns to the
N-terminal half of the Drosophila protamines (from aa positions 27 to
82), and four cysteine residues are conserved and regularly spaced. In contrast,
Protamine-2 of human and mouse
shows relatively high similarity to the C-terminal half of the Drosophila
protamines, with four cysteines in this region that are conserved and regularly
spaced, whereas one cysteine is shared with the mouse and human Protamine-1 (Raja, 2005).
Mst77F is present at the cytological position 77F on the chromosome arm 3L and
lies within the large intron of PKA-R1. Mst77F is also male specifically
transcribed, and the encoded protein has been proposed to be a linker histone
H1/H5 type, which could also play the role of a transition protein or a
protamine. The Mst77F protein shares a
significant similarity to the HILS1 protein of mouse
and human HILS1, where the percentages of cysteine,
lysine, and arginine are similar to that of mHILS1 and hHILS1.
HILS1 protein has been recently described as a component of
the mammalian sperm nucleus. Drosophila Mst77F
encodes a protein of 215 aa with a molecular mass of 24.5 kDa and with a pI of
9.86. mHILS1 is of 170 aa and shows 39% similarity to Mst77F.
Mst77F contains 10 cystine residues as in Drosophila
protamines, and mHILS1 contains eight cystine
residues, of which four residues are conserved (Raja, 2005).
As there are considerable differences between the mammalian protamines as well
as between the mammalian HILS1 proteins and the presumptive Drosophila
homologue Mst77F, additional experiments are essential to clarify if these
proteins are indeed involved in the condensation of sperm chromatin (Raja,
2005).
Drosophila protamine mRNAs are transcribed at the primary spermatocyte
stage, whereas in mammals protamine mRNAs are synthesized at the round
spermatid stage and translationally repressed until the
elongated stage, which is mediated by 3'UTR. The Drosophila
ProtamineA-eGFP and ProtamineB-eGFP constructs do not contain the 3'UTR of the
respective protamine genes. Nevertheless, the transgenic flies carrying
these constructs still show repression of translation. So, in Drosophila,
the region responsible for the translational repression is most likely in the
5'UTR. Deletion constructs of Mst35Bb and Mst77F 5'UTRs fused to
the reporter lacZ show that the translation repression element is indeed
present in the 5'UTR. This holds true also for the mRNA of the
Mst77F-eGFP fusion gene, as is the case for all mRNAs investigated
concerning translational repression so far in male germ lines of
Drosophila. In contrast to mammalian spermatogenesis, in Drosophila transcription
ceases already with the entry into meiotic divisions.
Since the protamines are made in the elongated spermatids, the transcriptional
silencing in Drosophila spermatogenesis seems to be independent of
protamines (Raja, 2005).
When primary amino acid sequences of Drosophila protamines are compared
to mammalian protamines, it is quite evident that Drosophila protamines
are relatively large. dProtA and dProtB are over 94% identical to each other.
This could explain that both the protamines may be functionally redundant. Human
and mouse Protamine-1 aligns with the N terminus of both Drosophila
protamines, and Protamine-2 aligns more to the C
terminus. It is possible that the Drosophila
protamines undergo posttranslational cleavage at the N terminus, as is known for
mammals. The cytoplasmic eGFP fused at the C terminus
shows clear nuclear localization, indicating that the tagged protamine is
functionally intact. Drosophila protamines each contain 10 cysteine
residues at identical positions, while over 4 of 10 cysteines at the N terminus
and the C terminus are conserved with human and mouse Protamine-1 and
Protamine-2, respectively. With
nine cysteines, the content is highest in Protamine-1 of mice. Inter- or
intra-disulfide bridges can be formed between the cysteine-rich protamines to
condense the DNA. For mice it is shown that mutation in
protamine-1 or protamine-2 is haploinsufficient and causes male
sterility. A haploid situation was analyzed for the Mst35Ba and
Mst35Bb genes with the deficiency Df(2L)Exel8033/+;
these flies are fertile males and show normal
spermatogenesis. The large amount of identity that both dProtA
and dProtB exhibit can contribute to the functional redundancy (Raja, 2005).
Chromatin reorganization is an essential feature during spermiogenesis. The
functional significance of chromatin compaction during spermiogenesis is still
unknown. The main explanation seems to be that compaction of the sperm nucleus
is an essential factor for its mobility as well as for the penetration of sperm
into the egg and genomic stability. In mammals, somatic histones are in part
replaced by spermatid-specific variants during meiotic prophase,
later by major transition proteins TP1 and TP2,
and subsequently by highly basic protamines to ensure the
remodeling of chromatin to a typically highly condensed and transcriptionally
silent state of mature sperm. These replacements lead to a shift from
histone-based nucleosomal conformation to a radically different conformation,
resembling stacked doughnut structures containing major chromatin condensing
proteins and DNA in the nucleus (Raja, 2005).
In Drosophila, so far no proteins have been identified that are involved
in the packaging of the genome in the mature sperm nucleus. One observation,
that Histone3.3 variant and the somatic H3 isoform in Drosophila are
vanishing at the time of chromatin condensation, supports the view of histone
displacement, but it was still a
question of whether it is the real absence of histones at this stage in
Drosophila or whether the antibodies are not accessible to the mature
sperm due to the tight packaging of the chromatin. To
circumvent this problem, the GFP fusion approach was chosen, use was made of the
existing His2AvDGFP, and
Protamine-eGFP and Mst77F-eGFP fusion transgenic flies were generated
in order to
analyze the situation in Drosophila. The results clearly show that
histone His2AvD is lost from the spermatid nuclei at the time of appearance of
protamines and Mst77F during later stages of spermatid differentiation. The
exact molecular mechanisms underlying the histone displacement, degradation, and
incorporation of protamines onto the chromatin are poorly understood.
For mammals, evidence has been obtained that histone H2A is
ubiquitinated in mouse spermatids around the developmental time period when
histones are removed from the chromatin.
The mammalian HR6B ubiquitin-conjugating enzyme is the
homologue of yeast RAD6, and both can ubiquitinate histones in vitro.
Thus far, the mechanism of histone displacement and protamine
incorporation is unknown during spermiogenesis in Drosophila. In
flies as well as in mammals, many questions remain unanswered that need
to be addressed about these
underlying mechanisms of chromatin remodeling during spermiogenesis (Raja, 2005).
In mammals, transition proteins act as intermediates in the histone-to-protamine
transition. In mice, the onset of HILS1 and transition
proteins TP1 and TP2 (major forms) overlaps with the pattern of Protamine-1 and
later with Protamine-2 but HILS1 and the transition proteins are no longer present in the mature sperm.
Mice lacking both TP1 and TP2 show normal transcriptional
repression, histone displacement, nuclear shaping, and protamine deposition but
show the loss of genomic integrity with large numbers of DNA breaks leading to
male sterility. In Drosophila,
histones are displaced with synchronous accumulation of protamines and Mst77F.
Mst77F, a distant relative of the histone H1/H5 (linker histone) family, has
been proposed to play a role either as a transition protein or as a protamine
for compaction of the Drosophila sperm chromatin.
Mst77F shows highest similarity to HILS1 with respect to the cysteines and basic
amino acid content but
not to mouse TP1, TP2, or H1t. Moreover, the results
show that the pattern of expression of Mst77F in the nucleus is similar to that
of mHILS1 in the nucleus, with the exception that Mst77F is also transiently
detected in the flagella and persists in mature sperm nuclei, unlike mHILS1. In
mammalian mature sperm nuclei, it is only the protamines that are the chromatin
condensing proteins which persist. This again raises the question of whether
Mst77F could also play the role of protamines. However, one additional copy of
dProtB (dProtA and dProtB showing 94% identity may be functionally redundant)
does not rescue the ms(3)nc3 phenotype, indicating that the
role of Mst77F may be completely or partially different from that of protamines
in the nucleus. However, a null mutation for Mst77F is required to answer
this question with respect to chromatin condensation. In
ms(3)nc3 mutants, the chromatin condensation with the
native protamines continues to take place. When a closer look was taken at the
deposition of ProtamineB-eGFP in
ms(3)nc3/Df(3L)ri-79c
trans-heterozygotes, it revealed that the condensed chromatin in the
tid-shaped nuclei is concentrated at the two
opposite ends, with a lightly stained chromatin spaced in the center. So the
chromatin condensation takes place but may not be complete with the
incorporation of the mutant Mst77F protein. The large amount of chromatin
compaction or condensation seen in Drosophila mature sperm when compared
to that of mouse and human sperm possibly could be the result of persistence of
Mst77F in the mature sperm nuclei. It remains to be clarified whether the sperm
nucleus contains further protamines that have not yet been properly annotated (Raja, 2005).
ms(3)nc3 is a second-site noncomplementation (nc) mutation
that was isolated in an ethylmethanesulfonate screen to identify interacting
proteins involved in microtubule function in Drosophila. This study shows
that ms(3)nc3 is a
single missense mutation from a T>A transition, causing the substitution of threonine instead of serine
at aa position 149. Mst77F shows a pattern of
expression similar to protamines in the nucleus and was also seen in the flagella until the
individualization stage. Since
ms(3)nc3 fails to complement class I alleles at the
ß2 tubulin locus, it is possible that Mst77F
has a dual role to play as a chromatin condensing protein in the nucleus and for
the normal nuclear shaping. Nuclear shaping is a microtubule-based event.
ms(3)nc3 leads to a tid-shaped nuclear
phenotype, where the nucleus fails to shape into a needle-like nuclei. Similar
defective nuclear shaping is seen with the few homozygous and heteroallelic
combinations of class I alleles of ß2 tubulin. The
incorporation of the defective subunit encoded by ms(3)nc3
may interfere with the function of the resulting complex. These data suggest the
involvement of an Mst77F (a linker histone variant) in the microtubule dynamics
during the nuclear shaping. This again complements the role of sea urchin
histone H1 in the stabilization of flagellar microtubules (Raja, 2005).
After the first steps in the fertilization process, the male gamete is still in
the highly compact protamine-based chromatin structure. In a wild-type egg, the
paternal pronucleus changes the shape from the needle-like to a spherical
structure. Furthermore, the male pronucleus acquires a nucleosome-based
structure before zygote formation and thus is transformed into a
replication-competent male pronucleus. sesame is a maternal effect
mutation in HIRA and had been mapped to 7C1.
HIRA family of genes (named after yeast HIR genes; HIR is an acronym for
'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene
transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. In
Drosophila, HIRA is expressed in the female germ line and a high level of
HIRA mRNA is deposited in the egg. Human HIRA is
shown to bind to histone H2B and H4. The WD repeats
present at the N-terminal part of HIRA could probably function as a part of a
multiprotein complex. Xenopus HIRA proteins are
also known in promoting chromatin assembly that is independent of DNA synthesis
in vitro. The corresponding maternal effect mutant sesame, in which the sperm fertilizes the egg but no zygote is formed, has been analyzed. Although the shape change of the nucleus to the spherical structure occurs in these mutants, maternal histones are not incorporated into the male pronucleus, which
strengthens the function of HIRA in binding to the core histones. This study shows
that neither Drosophila protamine is removed from the male pronucleus
in sesame mutants. This leads to the proposal that the transport and
incorporation of histones onto the chromatin in some manner is coupled to the
removal of protamines in which HIRA could play an important role in the
multiprotein complex required in this chromatin reconstitution process.
Mst77F removal from the male pronucleus in contrast to protamines
is independent of HIRA (Raja, 2005).
During spermiogenesis, chromatin reorganization of the complete genome is an
essential feature for male fertility. This process leads to an extremely
condensed state of the haploid genome in the sperm and requires a reorganization
of the paternal genome in the male pronucleus during fertilization and before
zygote formation. With the characterization of the chromatin condensing proteins
in Drosophila, it would be possible to gain more insight into the
mechanisms of sperm chromatin reorganization during spermiogenesis and
fertilization (Raja, 2005).
The Drosophila modulatory calcineurin-interacting protein (MCIP) sarah (sra) is essential for meiotic progression in oocytes. Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract; it is unknown whether calcium signaling is involved. MCIPs [also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down syndrome critical region 1)] are highly conserved regulators of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase 1 and 2. Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity, their in vivo functions remain unclear. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrestphenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. This is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process (Takeo, 2006; Horner, 2006).
sarah mutation disrupts several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. The conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation. Despite its independence from a sperm trigger, egg activation in Drosophila involves calcium-mediated pathways that are likely to be analogous to those in other animals. It is intriguing that among these downstream events is the acquisition of the egg's competence to remodel the sperm nucleus into the male pronucleus (Horner, 2006).
To explore the in vivo function of the Drosophila MCIP Sra, a null mutation in this locus was created by gene targeting. Homozygotes of the null allele sraKO are semilethal during larval or pupal stages. In addition, sraKO females were sterile, and their ovulation is abnormal (Ejima, 2004). These phenotypes were rescued by either sarah transgenes. Taken together, these results unambiguously demonstrate that sra is responsible for the phenotypes associated with sraKO, which include developmental defects in both sexes and ovulation and sterility in females (Takeo, 2006).
There is no apparent morphological abnormality in ovarian development in sraKO females, but eggs from sraKO mothers failed to hatch. Wild-type eggs at 2 hr after deposition already have completed meiosis and undergo synchronous mitotic nuclear division. In contrast, eggs—hereafter referred to as sra eggs—from sraKO mothers had a localized DAPI-stained signal in the cortical region near the anterior pole, indicating that sra eggs are arrested during meiosis. To analyze this phenotype in detail, the pattern of chromosome segregation and the spindle shape of sra eggs were examined. Spindle microtubules were visualized with tubulin antibody staining. In wild-type females, mature oocytes are arrested at metaphase of meiosis I, during which the chromosomes are seen as a large mass of chromatin. After the release of meiotic arrest during ovulation, individual chromosome arms become visible and migrate toward the poles; the chromosomes subsequently undergo meiosis II, after which nuclear fusion and the mitotic divisions of the zygote take place. In sra eggs, the meiotic chromosomes were seen in between the metaphase plate and the poles. It was confirmed that the oocytes taken from sra mutants including sraGS3080 and sraGS3168 are arrested at metaphase I as in the wild-type. Therefore, sra eggs are arrested at anaphase I shortly after the meiotic resumption from the metaphase I arrest (Takeo, 2006).
To determine whether the meiotic-arrest phenotype of sra eggs is caused by loss of function in the germline or somatic cells of sra mothers, mutant germline clones were generated by the flippase-dominant female sterile (FLP-DFS) technique. All eggs laid by wild-type females completed meiotic divisions, whereas most eggs (98%) from sra mothers arrested at anaphase of meiosis I. sra germline clones were also arrested at anaphase I, reproducing the phenotype of sraKO. A few sra eggs, including germline clones, were arrested at meiosis II. In these eggs, the two spindles were perpendicular to each other, rather than in tandem as in the wild-type. These results demonstrate that the meiotic defects in sra eggs are specifically attributable to the loss of function of sra in the female germline. Consistent with this conclusion, sra is highly expressed in the female germline during oogenesis (Ejima, 2004) and in early embryos. Furthermore, the meiotic-arrest phenotype caused by sra mutations was almost fully rescued by nos-GAL4/UASp-sra transgenes. These results establish that sra is required in the germline for meiotic progression in Drosophila females (Takeo, 2006).
Sra is a Drosophila member of the modulatory calcineurin-interacting protein (MCIP) family of proteins, which are known to function as endogenous regulators of calcineurin. Calcineurin consists of two subunits, CnA and CnB. The Drosophila genome contains three genes encoding CnA subunits (CanA1, Pp2B-14D, and CanA-14F) and two genes encoding CnB subunits (CanB and CanB2). The functions of calcineurin have been poorly analyzed in Drosophila. It is hypothesized that sra functions as an endogenous regulator of calcineurin in Drosophila. Analyses of the expression pattern of Drosophila calcineurin genes by RT-PCR revealed that all three CnA and both CnB genes were expressed in larvae and adult females, but among these, only Pp2B-14D, CanA-14F, and CanB2 are expressed in early embryos and ovaries. Therefore, these three calcineurin subunits are candidates for interacting with Sra in the female germline (Takeo, 2006).
A constitutively active form of calcineurin can be created by truncating the C-terminal part of CnA (Sullivan, 2002; Gajewski, 2003). Misexpression of the active form of Pp2B-14D (Pp2B-14Dact) causes morphological abnormalities in eyes and wings (Sullivan, 2002). To determine the effects of sra on calcineurin signaling, whether activated calcineurin-dependent phenotypes can be modified by coexpression of sra was tested. Overexpression of sra alone in developing eyes by using GMR-GAL4 did not induce any phenotypic change. Flies misexpressing Pp2B-14Dact showed a mild rough-eye phenotype, which was completely suppressed by coexpression of sra. Similarly, misexpression of Pp2B-14Dact in the posterior compartment of developing imaginal discs by using en-GAL4 resulted in loss of wing veins and reduction of wing size. These wing phenotypes were also completely suppressed by coexpression of sra, whereas overexpression of sra alone had no effect on wing morphology. Furthermore, overexpression of sra rescued the lethality induced by the muscle-specific expression of Pp2B-14Dact by using 24B-GAL4. All these results clearly show that sra has an inhibitory effect on calcineurin signaling (Takeo, 2006).
If Sra acts as an inhibitor of calcineurin in vivo, it was speculated that calcineurin signaling is hyperactivated in sra mutants; that is, hyperactivation of calcineurin signaling might also affect the meiotic phenotype as in sra mutants. It was found that females carrying nos-GAL4 and UASp-Pp2B-14Dact had fully developed ovaries, but were sterile or semisterile, depending on the transgenic lines. The sterility or semisterility caused by nos>Pp2B-14Dact was effectively rescued by co-overexpression of sra, demonstrating that sra counteracts activated calcineurin (Takeo, 2006).
To characterize the meiotic phenotype caused by Pp2B-14Dact, eggs were stained from semisterile females expressing nos>Pp2B-14Dact with DAPI and tubulin antibody to visualize chromosomes and spindles, respectively. A total of 63 eggs were examined. Of these, 10% (6/63) developed normally, whereas 14% (9/63) had neither DAPI signaling nor tubulin antibody staining. The remaining 76% showed complex abnormalities that could be classified into three types: (1) dispersed chromatins with no obvious spindle (33%); (2) apparently normal chromosomes with an abnormal spindle (38%), and (3) a mass of chromatin with an apparently normal spindle (5%). Also the nuclei of mature oocytes taken from nos>Pp2B-14Dact females were observed to see whether meiotic arrest at metaphase I is normal. It was found that the majority had abnormal nuclei containing dispersed chromatins, and that the remaining were arrested at metaphase I or anaphase I. These results suggest that calcineurin signaling was activated to a greater extent in the germline of females constructed in this way than in sra mutants. Taken together, these results demonstrate that the regulation of calcineurin signaling is critical for female meiosis, and its regulator Sra/MCIP is essential for meiotic progression at the time of egg activation in the Drosophila female (Takeo, 2006).
In vertebrates whose meiotic arrest occurs at metaphase II, arrest is released at the time of fertilization. The mechanisms of meiotic arrest and resumption have been extensively studied in mice and frogs, and several key components have been identified, including Cdc2/Cyclin B (MPF) and MAP kinase. In addition, the so-called “Ca2+ transient” mediated by IP3 signaling has been linked to egg activation; this transient promotes completion of meiosis, ion-channel opening, and cortical granule exocytosis. Recent studies have revealed that Ca2+/calmoduin-dependent protein kinase II (CaMKII) is physiologically activated in mouse oocytes in response to fertilizing sperm. CaMKII is implicated in the regulation of the timing of re-entry into mitosis through the phosphorylation of Cdc25C, a phosphatase mediating G1/M transition by dephosphorylating MPF in Xenopus. More recently, CaMKII was shown to phosphorylate an anaphase-promoting complex/cyclosome (APC/C) inhibitor, Emi1-related protein (Erp1), resulting in its degradation and thereby releasing the brakes on the cell cycle from metaphase II in Xenopus eggs (Takeo, 2006).
Less is known about the mechanism of egg activation in Drosophila. Genetic screens for female-sterile mutations have identified several genes involved in female meiosis. For example, twine, a homolog of Drosophila cdc25, is required for arrest at metaphase I in mature oocytes. cortex (cort) and grauzone (grau) mutant eggs exhibit meiotic arrest at meiosis II with defects in cytoplasmic polyadenylation and translation of maternal bicoid mRNAs. cort encodes an APC/C activator protein Cdc20, suggesting that APC/C-Cdc20Cort-mediated inactivation of MPF is required for the translational control of poly(A)-dependent maternal mRNA. grau encodes a member of the C2H2-type zinc-finger protein family and activates transcription of cort to induce the completion of female meiosis. In addition, a recent study reported that a small cell-cycle regulator, Cks30A, plays an essential role in meiotic progression by associating with Cdk1 (Cdc2)/cyclin complexes and mediating Cyclin A degradation in the female germline. Therefore, cell-cycle regulators involved in meiotic progression are likely to be conserved between vertebrates and Drosophila (Takeo, 2006).
The involvement of calcineurin signaling in female meiosis has not previously been described in any organism. These studies on sra are the first to demonstrate that regulation of a Ca2+-dependent phosphatase is critical for the progression of female meiosis. Analyses of mutations in calcineurin genes and identification of the substrates in germline cells should facilitate further understanding of the role of calcineurin signaling in female meiosis (Takeo, 2006).
In the Drosophila oocyte, meiosis is arrested in the first division of metaphase: at this stage a tapered spindle forms, aligned parallel to the egg surface. The chromosomes are therefore located in the cortical region near the anterior pole, whereas fusion of parental complements occurs in the inner ooplasm. How does the female pronucleus reach the interior of the egg? The second meiotic spindles are arranged in tandem, end to end, and disposed perpendicular to the longitudinal axis of the egg with the innermost spindle carrying the female pronucleus. This pattern of spindle organization is probably involved in the migration of the female pronucleus deeper into the egg near the cytoplasmic domain of the male pronucleus. The precise time at which the mitotic spindle of Drosophila changes orientation is unknown. However, spindle rotation from a position parallel to the egg surface to a radial orientation presumably occurs during or shortly after the oocyte passes through the oviduct. How spindle orientation is achieved and maintained during meiosis is an intriguing question. Microtubules linking spindle poles to the oocyte surface have been implicted in the rotation and anchoring of the meiotic apparatus in Xenopus oocytes and in other organisms, but this does not seem to be the case in the Drosophila oocyte, since the meiotic spindles lack astral microtubels. However, the observation that a transient array of microtubules links the meiotic apparatus to discrete subcortical foci suggests that in Drosophila the orientation of the spindle also requires a functional interaction between the spindle and the oocyte cortex (Riparbelli, 1996 and references).
The microtubule array of mitosis II observed between the twin spindles at metaphase, anaphase and telophase might be an intermediate between the anastral poles of the meiotic I spindles and the astral poles of the mitotic spindles in early embryos. A complex pathway of spindle assembly takes place during resumption of meiosis at fertilization, consisting of a transient array of microtubules radiating from the equatorial region of the spindle toward discrete foci in the egg cortex. A monastral array of microtubules is observed between twin metaphase II spindles in fertilized eggs. These microtubules originating from disc-shaped material stain with Rb188 antibody specific for an antigen asssociated with the centrosome of Drosophila embryos (DMAP190 or CP190). Therefore, the Drosophila egg contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubues in a monastral array after activation, but are unable to organize bipolar spindles (Riparbelli, 1996).
The meiosis II spindle of Drosophila oocytes is distinctive in structure, consisting of two tandem spindles with anastral distal poles and an aster-associated spindle pole body between the central poles. Assembly of the
anastral:astral meiosis II spindle occurs by reorganization of the meiosis I spindle, without breakdown of the meiosis I
spindle. The unusual disc- or ring-shaped central spindle pole body forms de novo in the center of the elongated
meiosis I spindle, followed by formation of the central spindle poles. gamma-Tubulin transiently localizes to the
central spindle pole body, implying that the body acts as a microtubule nucleating center for assembly of the central
poles. The first step in formation of the central pole body is the appearance of puckers in the center of the the meiosis I spindle, followed by the pinching out from the spindle of a disc or ring of microtubules that becomes the central pole body. The manner in which the central spindle pole body forms suggests the involvement of a microtubule motor. If so, the motor involved is likely to be different from Ncd (Nonclaret disjunctional), since loss of Ncd function does not seem to prevent its formation. Following the formation of the central spindle pole body, the microtubules arrayed to either side of the central body narrow into poles, forming the mature meiosis II spindle. The central poles become more tapered during progression through meiosis II, and the central spindle pole body also changes in morphology: the disc or ring becomes asterlike, then enlarges into a ring that lies between the two central telophase II nuclei (Endow, 1998).
Localization of gamma-tubulin to the meiosis II spindle is dependent on the microtubule motor protein,
Ncd. Absence of Ncd results in loss of gamma-tubulin localization to the spindle and
destabilization of microtubules in the central region of the spindle. Likewise, during meiosis I, the minus-end motility of Ncd and its crosslinking activity are probably needed to focus microtubules into spindle poles for the correct functioning of meiosis I. Assembly of the anastral:astral meiosis II spindle
probably involves rapid reassortment of microtubule plus and minus ends in the center of the meiosis I spindle. This
can be accounted for by a model that also accounts for the loss of gamma-tubulin localization to the spindle and
destabilization of microtubules in the absence of Ncd (Endow, 1998).
A model for assembly of the Drosophila oocyte meiosis II spindle is suggested: gamma-Tubulin is first recruited or relocalized, possibly as gamma-TuRC, to the midbody of the meiosis I spindle, where it functions to nucleate microtubules for formation of the meiosis II central spondle poles. The loss of gamma-tubulin localization to the spindle in the absence of Ncd suggests that the Ncd motor serves to recruit or anchor gamma-tubulin to the center of the spindle. The Ncd motor would then stabilize newly nucleated microtubule minus ends and focus the microtubules into poles. The unstabilized plus ends of the microtubules in the center of the spindle (remaining from meiosis I) would undergo rapid depolymerization as a consequence of dynamic instability. Stabilization of the newly nucleated microtubule minus ends and depolymerization of the plus ends would cause a rapid sorting out of the microtubules in the center of the meiosis I spindle, replacing microtubule plus ends with minus ends. The distal poles of the meiosis II spindle would be retained from the meiosis I spindle and maintained by the same forces that originally formed them: the crosslinking activity and minus-end movement of Ncd along spindle microtubules (Endow, 1998).
Evidence is provided that a distinct midzone is present in the Drosophila melanogaster female meiosis I spindle. This region has the ability to bind the Pavarotti kinesin-like (PAV-KLP) and Abnormal spindle (Asp) proteins, indicating a correct organization of the central spindle microtubules. The core component centrosomal protein centrosomin (CNN) has been identified at an unexpected site within the anaphase I spindle, indicating a role for CNN during the biogenesis of the female meiotic apparatus. However, there are no apparent defects in the midzone organization of cnn oocytes, whereas defects occur later when the central aster forms. The primary mutant phenotype of cnn oocytes is the failure to form a developed central microtubule organizing center (MTOC), although twin meiosis II spindles usually do form. Thus the central MTOC may not be essential for the formation of the inner poles of twin meiosis II spindles, as generally proposed, but it might be involved in maintaining their proper spacing. The proposal is discussed that, in the presence of a central MTOC, a chromatin-driven mechanism of spindle assembly like that described during meiosis I may control the morphogenesis of the twin meiosis II spindles (Riparbelli, 2005).
Eighty years ago, the pioneering studies of Huettner (Huettner, 1924) on the maturation of the Drosophila melanogaster oocyte revealed that chromosome segregation during the first meiosis is supported by a peculiar spindle apparatus that transforms during the second meiosis into twin spindles arranged in tandem and disposed perpendicularly to the longitudinal axis of the egg. These spindles ensure the reductional divisions and the formation of the haploid complements, the innermost of which is the female pronucleus. Huettner first observed that the meiotic spindles are anastral and lack centrioles at their poles, though they are tapered. Despite the potential of these observations for clarifying acentrosomal pathways of microtubule (MT) organization, the female meiotic apparatus of Drosophila has received little attention for many years. During the last decade some researches have concentrated their attention on the morphogenesis and organization of the spindle during metaphase of the first meiotic division. Immunohistochemical analysis failed to reveal centrosomal components, such as CP60, CP190 and gamma-tubulin, at the spindle poles, confirming, in its essential aspects, the general description of Huettner. The role of gamma-tubulin during meiosis is, however, controversial since mutants for this gene have abnormal first meiotic spindles, but meiosis is not terminally arrested and appears mostly normal. Several lines of evidence suggest that meiotic spindle assembly in the Drosophila female begins with a chromosome-driven mechanism of MT organization. Mutational analysis indicates that, in the absence of centrosomes, MT bundling and bipolarity of anastral meiotic spindles requires kinesin-like proteins with minus-end-directed MT motor activity. Defects in the genes nonclaret disjunctional (ncd) and subito (sub), which encode kinesin-like proteins, lead to the formation of abnormal meiotic I spindles with frayed or undefined poles. The cooperative interaction between motor proteins and the products of the genes mini spindles (msps) and transforming acidic coiled-coil protein (tacc), localized at the acentrosomal poles of the first meiotic spindle, might be crucial for the bipolarity of the meiotic spindle. The MT minus-end-associated Abnormal spindle (Asp) protein, localized at the extremities of the meiotic spindle, could be also involved in the stabilization of focused poles (Riparbelli, 2005 and references therein).
Oocytes are activated to resume meiosis by passage through the oviduct. The spindle, positioned parallel to the cortex during metaphase arrest, reorients perpendicular to the oocyte surface during completion of meiosis I. The spindle elongates during anaphase-telophase of the first meiosis and transforms during meiosis II into two tandemly arranged spindles (Huettner, 1924). Although the meiotic II spindles are anastral, an unusual structure, from which an extensive array of MTs nucleates, forms between the two internal spindle poles (Riparbelli, 1996). It has been shown that this structure, in contrast to the poles of the meiotic spindles, contains several centrosomal proteins usually found at the mitotic spindle poles, such as gamma-tubulin, CP60 and CP190 and centrosomin (CNN) (Llamazares, 1999). However, although this central MTOC contains centrosomal components and is able to nucleate an astral array of MTs, it lacks centrioles, is unable to duplicate and disappears after the completion of meiosis (Riparbelli, 2005).
Whereas previous analyses provided insight into the mechanisms of spindle assembly and chromosome segregation during metaphase of the first meiosis, they provided no clear information on the kinetics of spindle morphogenesis during transition from meiosis I to II. This aspect was investigated by time-lapse analysis of ncd-gfp oocytes (Endow, 1998). The main event of the transforming of meiotic I spindle in twin tandem arranged meiotic II spindles is the formation in the elongated meiotic I spindle of lateral puckers, which are correlated with the organization of the central MTOC (Endow, 1998). However, despite the usefulness of this elegant in vivo approach, some aspects, such as the individualization of the twin spindles and the origin and function of the central MTOC, remain unclear. The findings presented in this report both expand and clarify earlier studies on the structural organization and dynamics of the female meiotic spindle, suggesting an alternative model of meiosis II spindle assembly (Riparbelli, 2005).
After oocyte activation, meiosis resumes and the metaphase-arrested spindle elongates and undergoes a pivoting movement to reorient perpendicularly to the surface (Endow, 1997). Two distinct populations of interpolar MTs, interior and peripheral, form a top-like anaphase spindle. The interior MTs run parallel to the main axis of the spindle to form longitudinal thick bundles, whereas the peripheral ones spread out from the poles to form opposite cone-shaped half spindles. The peripheral MTs, which form a cage around the interior bundles, intersect at the equatorial plane of the spindle and extend both deeply into and above this plane until they reach the cell cortex. Thus, the spindle appears to be anchored to the oocyte surface through the peripheral MT set that forms the inner half spindle. Some clusters of MTs appear through the equatorial region of the spindle where opposite MTs overlapped and/or interacted tangentially. As anaphase progresses the clustering of MTs is more evident through the spindle equator. Most of the interior MT bundles terminate in a small non-fluorescing area at the opposite poles. Co-labeling with propidium iodide indicated that these regions of lower MT density correspond to the chromosomes and that the bundles might therefore be kinetochore fibers. The kinetochore MTs of different chromosomes form separated bundles and do not focus on a common pole. Thus the spindle poles appear broad and frayed. Although the density of the MTs made it difficult to resolve the structure of the central region of the spindle, suitable optical sections showed that some MT bundles overlap in this region. During telophase a diffuse tubulin accumulation was observed at the middle of the spindle at the sites where the interior MTs overlap (Riparbelli, 2005).
Transition from meiosis I to meiosis II is marked by the expansion of the cage of MTs that surround the interior interpolar MTs to form an extensive network. Some of the peripheral MTs also contact the oocyte cortex. Astral arrays of MTs of various size become apparent at this time within the equator of the spindle and outside its boundaries. Intermediate to the formation of these structures might be the clusters of overlapping MTs observed during the first meiosis. These clusters become increasingly compact at the onset of second meiosis and could become centers from which MTs spread. A distinct tubulin aggregate was seen to bulge out at the middle of the interior MT bundles. Kinetochore MTs run almost parallel in each half spindle both outward and inwards to each chromosome set they hold. Thus, kinetochore fibers form distinct parallel 'minispindles' and the outer poles appear frayed. The inner poles cannot be distinguished at this time. Optical sections midway along the spindle show an irregular belt-shaped equatorial tubulin aggregate that could be derived from the tubulin accumulation observed during telophase of the first meiosis. Several MTs that span from this aggregate end at the cytoplasmic asters or mingle within the peripheral network (Riparbelli, 2005).
As meiosis progresses the peripheral MT array gradually disassembles and the MT asters become free in the cytoplasm or remain connected to the remnant of the peripheral MT framework or to the spindle by thin threads and the equatorial tubulin cluster is more evident and protruding. Free asters are transient structures that disappear over time and are no longer visible from anaphase II on. The outward extremities of the minispindles formed by the kinetochore fibers focus on common outer poles, whereas the inner kinetochore fibers still run almost parallel. Thus, the individualization of the meiosis II twin spindles occurs gradually and requires the formation of the internal poles that start to be organized during late prophase/early prometaphase when the inwards extremities of the minispindles have partially coalesced midway along the spindle. Short MTs form a loose cloud at the spindle equator. During prometaphase/early metaphase, the twin spindles begin to be distinguishable, though their inner poles are formed by three to four distinct bundles and appear broad. The MTs at the spindle equator are more prominent and form an extensive astral array, the 'central aster', which increases in size during subsequent metaphase and anaphase. At telophase the MTs of the central aster reach their maximum length, although they have decreased in density (Riparbelli, 2005).
Although female meiosis in Drosophila differs from male meiosis and mitosis in that spindle poles lack centrosomes and cytokinesis does not occur, this study demonstrates that the central region of the spindle has a common organization in these systems and that a structured midzone exists in the female meiotic spindle. At least two main events are involved in the formation of the spindle midzone in Drosophila cells (Inoue, 2004): the overlay of antiparallel MTs and the release of a subpopulation of MTs from the spindle poles. The central region of the female meiosis spindle during late anaphase I has the ability to bind PAV-KLP, the Drosophila ortholog of MKLP. This protein has been found in association with the putative MT plus ends at the midzone of anaphase spindles of mitotic cells and spermatocytes where it might function in regulating the dynamics and organization of the overlapping MTs. Thus the female meiotic spindle could have a structured midzone formed by overlapping antiparallel MTs. This is consistent with the finding, from sagittal optical sections, that opposite MT bundles overlap at the middle of the central spindle. The increased density of MTs in the central region of the spindle at telophase of the first meiosis might be consistent with the release of a subset of MTs from the opposite spindle poles. When this process occurs in mitotic cells and spermatocytes (Riparbelli, 2002), the Asp protein accumulates at the minus ends of the central spindle MTs. The Asp staining observed in the central region of the spindle during telophase of the first meiosis could be such a protein localization, at the predicted MT minus ends, suggesting that MTs of the central spindle undergo similar dynamics during female meiosis and in both mitosis and male meiosis (Riparbelli, 2005).
Morphological evidence indicates that several differences exist between the meiosis I and II spindles: the meiosis I spindle is anastral, whereas the twin meiosis II spindles have outer anastral poles and an unusual central aster between the inner poles (Riparbelli, 1996; Endow, 1997). These observations suggest that spindle assembly could require different mechanisms during meiosis I and II. Whereas the organization of the meiosis I spindle involves a chromosomal-dependent pathway of MT organization, the individualization of the twin spindles during meiosis II requires the formation of new poles in the center of the prophase II spindle. However, the mechanism by which the inner poles form is unclear. This issue has been difficult to address because the formation of the inner spindle poles occurs in a region of very high MT density, and thus any stage of this process is obscured from view. It has been proposed that the formation of the inner poles might require the reorganization of the central spindle in which the polarity of MT ends reverse (Endow, 1998). This hypothesis is supported by the presence of an unusual MTOC that has been postulated to play a major role in the process of MT nucleation for the formation of the inner half spindles during meiosis II (reviewed by Megraw, 2000). However, normal-looking twin bipolar spindles can form during meiosis II, both when the central aster is defective as in polo mutants (Riparbelli, 2000) and when it is very reduced as in wispy, KLP3A and cnn mutants (Riparbelli, 2005).
A new model of spindle assembly during meiosis II is suggested. Spindle elongation during anaphase/telophase of the first meiosis moves the homologous chromosomes to opposite poles that appear broad and frayed because the kinetochore fibers of different chromosomes do not focus on a common pole during later meiosis I stages. Starting from late prophase II, two opposite half spindles become evident, with outer focused poles where Asp accumulates. By contrast, inner poles are not evident at this point. However, the accumulation of Asp midway along the spindle suggests that the minus ends of opposite interpolar MTs are facing into the central region of the spindle. These observations are consistent with the finding, in each prophase II half spindle, of two opposite MT populations, with their plus ends near the chromatin and their minus ends outwards. The outward minus ends are focused on distinct poles, whereas the minus ends facing the spindle equator remain unbound. Control of spindle assembly could be achieved during meiosis II by the same chromosome-driven mechanisms of MT organization working in meiosis I. MTs in the vicinity of the condensed chromatin could be sorted into bundles and focused in stable poles by the cooperative interaction of molecular motors and cross-linking proteins. Loss of ncd function results, indeed, in the destabilization and fragmentation of both inner and outer spindle poles (Endow, 1998). Accordingly, the inner half spindles are formed during late prophase by parallel MTs that are first bundled at the spindle equator in distinct foci, and that then coalesce into a single pole. This model of meiotic spindle assembly is consistent with the formation of twin spindles in oocytes lacking normal-shaped central asters. It is proposed, therefore, that the acentrosomal pathway makes an essential contribution to spindle formation during meiosis II, even when a functionally active MTOC is present in the oocyte. An alternative mechanism might be that the inner half meiosis II spindles could be formed by a mixture of two MT populations: MTs nucleated in the vicinity of the chromosomes and MTs of the central aster. The gradual tethering together of these two MT populations could give rise to the tapered inner spindle poles, as proposed during the assembly of the mitotic spindle in cultured vertebrate cells. To what extent inner half spindles might be formed by two MT populations is difficult to determine, since individual MTs and their origin cannot be resolved within the assembling spindle. However, the Asp staining observed during transition from prophase to metaphase of the second meiosis suggests that the MTs radiating from the central aster contribute minimally, if at all, to the formation of the internal half spindles. The Asp staining is strong in the central region of the spindle, whereas it is weaker in the central aster, pointing to a lower MT density. This suggests that the contribution of the central MTOC is not essential for the higher MT density within the region where the inner poles will organize. Consistently, optical sections at the level of the spindle equator during prophase of the second meiosis reveal a continuity between the peripheral MT network and the ring-like tubulin aggregate, but no link is observed between this structure and the MTs of the central spindle. Finally, staining of Asp indicates that the minus ends of the inner half meiosis II spindle MTs start to coalesce into a single pole and, therefore, are completed before a distinct central aster is apparent. On the basis of these considerations the first hypothesis is favored in which the function of the central aster is redundant for the meiosis spindle organization (Riparbelli, 2005).
However, if the meiosis II spindles are assembled by an acentrosomal pathway, what is the role of the central aster? In the absence of a developed central aster, the twin spindles can be improperly spaced or oriented with respect to the long axis of the oocyte, resulting in the failure to correctly position the female pronucleus. The central aster could, therefore, be needed to keep the correct spacing between neighboring twin spindles. Accordingly, twin spindles are misaligned and at variable distances from each other when the central aster is defective as in polo mutants (Riparbelli, 2000). Mutation in cnn has been shown (Vaizel-Ohayan, 1999) to impair the spatial organization of mitotic spindles in the early Drosophila embryo (Riparbelli, 2005).
In vivo observations revealed that the precursor of the central spindle could be identified by a tubulin pucker midway along the central spindle at the end of meiosis I (Endow, 1998). This structure first appears as a tubulin aggregate to the spindle midzone, where antiparallel MTs overlap and recruit the PAV-KLP motor and CNN proteins. There are two possible mechanisms in the formation of the central aster. One possibility is that the MTs are nucleated by a discrete MTOC. The other possibility is that motor proteins may cross-link and organize randomly nucleated MTs into aster-like structures. The finding of several centrosomal proteins at the focus of the central aster during meiosis II (Megraw, 2000), strongly points to the first possibility. Since gamma-tubulin at the centrosome seems to be dependent on cnn function (Terada, 2003), the finding of CNN at the spindle midzone during anaphase I could indicate the prerequisite for the recruitment of gamma-tubulin at the central spindle and, therefore, for the nucleation of the central aster MTs. However, in cnn mutants that lack this protein at the spindle midzone in anaphase I and fail to accumulate gamma-tubulin, a faint astral array of MTs forms at the middle of the spindle during prometaphase/early metaphase II. In the absence of gamma-tubulin the central aster becomes poorly organized during subsequent meiotic stages and then disappears. It is therefore proposed (see Fig. 8 of Riparbelli, 2005 ) that the assembly of this structure might first rely on the cooperative interaction of motor proteins and acentrosomal MTs to form a polarized array of MTs that spread out from the equatorial region of the spindle. This process does not require ncd function, since mutants for the Ncd motor protein assemble an astral array of MTs at the beginning of meiosis II (Endow, 1998). Recruitment of material along these MTs might then contribute to the accumulation of centrosomal proteins, thus leading to the formation of a true MTOC that in turn could lead to further growth of the central aster. According to this model the presence of centrosomal material and other components, such as PAV-KLP, at its focus might be due to a fortuitous recruitment along its MTs. This is consistent with the finding that when the integrity of the central aster is affected in cnn mutant oocytes, there is little or no accumulation of PAV-KLP between twin spindles. By contrast, the motor protein is always found at the midzone of mutant spindles during both anaphase I and anaphase II, where it is needed for spindle dynamics. This is consistent with the observation that the accumulation of pericentrin and gamma-tubulin at the vertebrate centrosome is inhibited in the absence of tubulin or by microinjection of antibodies against cytoplasmic dynein. This two step mechanism could explain why gammaTub37CD mutants show an astral array of MTs at the beginning of metaphase II in the absence of detectable gamma-tubulin. The sperm aster is usually retained to be assembled by a true centrosome derived from both paternal and maternal sources. In particular the male gamete provides the centriole around which maternal components accumulate to form a mature centrosome able to nucleate MTs and to reproduce. Although rigorous conclusions are difficult to draw from negative results, the observation that cnn mutant oocytes have a developed sperm aster lacking CNN and gamma-tubulin, points to alternative mechanisms of sperm aster assembly. However, the possibility cannot be excluded that the amount of these proteins could be so much lower in mutant oocytes that they have escaped immunofluorescence analysis (Riparbelli, 2005).
In C. elegans, genome-wide screens have identified just five essential centriole-duplication factors: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. These proteins are widely believed to comprise a conserved core duplication module. In worm embryos, SPD-2 is the most upstream component of this module, and it is also essential for pericentriolar material (PCM) recruitment to the centrioles. Drosophila Spd-2 is a component of both the centrioles and the PCM and has a role in recruiting PCM to the centrioles. Spd-2 appears not, however, to be essential for centriole duplication in somatic cells. Moreover, PCM recruitment in Spd-2 mutant somatic cells is only partially compromised, and mitosis appears unperturbed. In contrast, Spd-2 is essential for proper PCM recruitment to the fertilizing sperm centriole, and hence for microtubule nucleation and pronuclear fusion. Spd-2 therefore appears to have a particularly important role in recruiting PCM to the sperm centriole. It is speculated that the SPD-2 family of proteins might only be absolutely essential for the recruitment of centriole duplication factors and PCM to the centriole(s) that enter the egg with the fertilizing sperm (Dix, 2007).
The predicted Drosophila ortholog of C. elegans SPD-2 is encoded by the gene CG17286, which is referred to as Drosophila Spd-2 (Pelletier, 2004). Rabbit Spd-2 antibodies were generated and affinity-purified; these antibodies recognized centrosomes at all stages of the cell cycle in embryos and specifically in mitosis in brain. A Spd-2-GFP (green fluorescent protein) fusion protein was generated, that localizes to centrosomes throughout the cell cycle in both embryos and larval brain cells (Dix, 2007).
In brain cells, Spd-2-GFP was more strongly recruited to mitotic centrosomes, but it also localizes to interphase centrosomes. Because larval brain cells recruit very little pericentriolar material (PCM) during interphase, this localization suggests that Spd-2-GFP is associated with centrioles in interphase and is also recruited to the PCM in mitosis. In addition, Spd-2-GFP localizes to the large centrioles present in the primary spermatocytes. Taken together, these observations indicate that, like its C. elegans counterpart, Spd-2 localizes both to the centrioles and to the PCM (Dix, 2007).
Interestingly, Drosophila Sas-4, Sas-6, and Sak/Plk4 specifically localize to the proximal and distal ends of the centrioles in primary spermatocytes, and it has been proposed that this localization might be common to proteins involved in the duplication process. In contrast, Spd-2 equally distributes along the length of the centrioles in these cells as detected using both the GFP fusion protein and antibodies, suggesting that Spd-2 might not contribute to centriole duplication in the same manner as do the other proteins (Dix, 2007).
To test whether Spd-2 is essential for centriole duplication, a stock was obtained carrying a mutation in the Spd-2 gene. The G20143 mutant stock is an enhanced and promoter (EP) line carrying a P element insertion directly after the initiating ATG codon of the Spd-2 gene. On a Western blot, Spd-2 protein was not detected in Spd-2 mutant brains, and quantitative Western-blot analysis revealed that Spd-2 protein levels were reduced by more than 90%. No Spd-2 was detected at centrosomes or centrioles in Spd-2 homozygous mutant brain cells by immunofluorescence. Thus, this P element insertion severely reduces the expression of the Spd-2 protein. All of the phenotypes described below were rescued by the Spd-2-GFP transgene, demonstrating that the lack of Spd-2 is responsible for these defects (Dix, 2007).
Previous studies have shown that flies lacking centrioles are viable but are severely uncoordinated because of a lack of cilia in their mechanosensory neurons. However, Spd-2 mutant flies are viable and are not uncoordinated, suggesting that they possess both centrioles and cilia. Consistent with this observation, centrioles were detectable in both wild-type (WT) and Spd-2 mutant brains expressing the centriolar markers GFP-PACT, Sas-4-GFP, and Asl-GFP. Quantification of centriole numbers in fixed brain preparations revealed that, in contrast to mutants lacking the other centriole-duplication proteins Sas-4, Sas-6, and Sak/Plk-4, there was no dramatic decrease in centriole numbers in Spd-2 mutant cells compared to the WT. These results are consistent with the recent finding that depletion of Spd-2 in a genome-wide screen in Drosophila cultured cells did not affect centriole duplication (Dix, 2007).
To determine whether Spd-2 is essential for centriole duplication in other tissues, the large centrioles were examined in the primary spermatocytes of the male germline. Strikingly, centriole numbers actually increased in the primary spermatocytes lacking Spd-2, suggesting that centrioles can over duplicate in the absence of Spd-2 in some tissues. The presence of extra centrioles leads to the formation of multipolar spindles, resulting in severe meiotic defects and male sterility. Taken together, these results suggest that Spd-2 is not essential for centriole duplication in Drosophila somatic cells. Thus, SPD-2 is the first centriole-duplication factor from the C. elegans pathway that does not have an essential role in this process in Drosophila (Dix, 2007).
In the C. elegans embryo, SPD-2 is essential for PCM recruitment to the centrioles, and it appears to be one of the earliest effectors in the recruitment pathway (Pelletier, 2004; Kemp, 2004). To test whether Spd-2 is also required for this process in flies, the recruitment of the PCM markers Cnn and γ-tubulin to mitotic centrosomes was assessed in WT and Spd-2 mutant third-instar larval brain cells. Both Cnn and γ-tubulin were still detectable on the majority of mitotic centrosomes in Spd-2 brains, but they were present at significantly decreased levels compared to the WT. Thus, Spd-2 appears not to be essential for PCM recruitment in mitotic larval brain cells, but is required to ensure the efficiency of this process (Dix, 2007).
It was asked whether the reduced levels of PCM at Spd-2 mutant centrosomes would affect the ability of the centrosomes to nucleate MTs and hence drive spindle formation. It was found that the mitotic index in WT and Spd-2 mutant third-instar larval brain cells was very similar, suggesting that mitosis occurs with normal timing in mutant cells. Moreover, a live analysis of the mitotic spindles in larval neuroblasts expressing GFP-α-tubulin revealed that the centrosomes in Spd-2 mutant neuroblasts nucleated robust astral MT arrays that participated in spindle formation in a manner indistinguishable from those in WT cells. In addition, Spd-2 mutant neuroblasts invariably divided asymmetrically, as was the case in WT neuroblasts. Thus, in Spd-2 mutant third-instar larval brain cells, the reduced efficiency of PCM recruitment does not detectably perturb mitosis. This is in contrast to C. elegans embryos lacking SPD-2, in which PCM recruitment, MT nucleation, and mitosis are dramatically impaired (Dix, 2007).
The data suggest that Spd-2 is not essential for centriole duplication and has only a minor role in PCM recruitment in Drosophila somatic cells. Despite this fact, Spd-2 mutant females produced embryos that did not hatch as larvae. In fixed 0-4 hr collections of Spd-2 embryos mated with WT males (hereafter Spd-2 embryos), most embryos had been fertilized but were arrested very early in development (Dix, 2007).
To determine the nature of the defect in Spd-2 embryos, 0-15 min collections of embryos were fixed and stained to visualize MTs, PCM (Cnn), and centrioles (Asl-GFP). The first mitotic spindle was frequently observed in 0-15 min collections of WT embryos, but it was never observed in Spd-2 embryos, suggesting that mutant embryos arrest before the first mitosis. In many mutant embryos, it was clear that pronuclear migration had failed, and male and female pronuclei were often observed that remained spatially separated within the embryo. This was never observed in WT embryos of a similar stage (as determined by the morphology of the polar bodies; data not shown). Thus, it appears that Spd-2 mutant embryos are unable to develop because of a failure in pronuclear fusion (Dix, 2007).
Next, whether there were defects in female meiosis that might explain the early arrest of the Spd-2 embryos was analyzed. It was found that female meiosis proceeded normally in Spd-2 embryos, generating a female pronucleus that was positioned appropriately for subsequent capture by the sperm aster. Defects, however, were observed in the recruitment of Cnn to the shared central pole of the meiosis II spindles in Spd-2 embryos. This observation suggests that Spd-2 can have a role in recruiting PCM proteins to MT organizing centers (MTOCs) that do not contain centrioles (Dix, 2007).
To test whether the mutant embryos fail in pronuclear fusion because of a defect in sperm aster assembly, the distribution was analyzed of PCM and MTs around the sperm centriole that enters the egg at fertilization. In the majority of WT embryos in metaphase or later stages of meiosis II, large amounts of Cnn and MTs were recruited around the sperm centriole. In contrast, in Spd-2 embryos at the same stage, sperm centrioles were rarely associated with Cnn or MTs. Thus, in contrast to somatic cells, Spd-2 has an essential role in recruiting the PCM to the sperm centriole: In its absence, the sperm centrosome does not organize a robust array of astral MTs, pronuclear fusion fails, and mutant embryos arrest prior to the first mitotic division (Dix, 2007).
These results suggest that Spd-2 has a particularly important role in PCM recruitment to the sperm centriole. It was therefore wondered whether Spd-2 might also be essential for centriole duplication specifically during the first centriole-duplication event after fertilization. It was observed, however, that in all late meiosis II embryos in which the centrioles could be detected, a newly duplicated centriole could be distinguished in both WT and Spd-2 embryos. This suggests that the single sperm-derived centriole is capable of undergoing the first duplication event in the absence of maternally supplied Spd-2 protein, although the interpretation of this result is not straightforward (Dix, 2007).
These data indicate that the Drosophila ortholog of C. elegans SPD-2 is not essential for centriole duplication and has only a minor role in PCM recruitment in somatic cells. This might suggest that the function of SPD-2 has diverged between worms and flies. It is noted, however, that the essential role of C. elegans SPD-2 in centriole duplication and PCM recruitment has, to date, only been demonstrated in the embryo, during the first events after. The data show that Spd-2 plays a particularly important role in recruiting the PCM to the centriole that enters the fly embryo at fertilization. Thus, it is possible that this family of proteins has a conserved function, which is only absolutely essential when the sperm centriole(s) first enter the fertilized egg (Dix, 2007).
Why would SPD-2 proteins be essential for PCM recruitment to the sperm centriole but not to the centrioles in somatic cells? One possibility is that SPD-2 proteins have a specialized role in facilitating the de novo recruitment of PCM proteins from maternal stores to the “naked” centriole(s) that enter the oocyte at fertilisation. This is a unique situation, because in all subsequent rounds of mitotic PCM recruitment, the centrioles are already associated with at least a small amount of PCM that was present on the centriole during interphase. Hence, SPD-2 proteins might not be essential for PCM recruitment once the PCM has initially been loaded onto the sperm centrioles. It remains possible, however, that SPD-2 proteins are also essential for PCM recruitment to the centrioles during subsequent embryonic cycles. Unfortunately, this possibility cannot be tested because Spd-2 embryos arrest prior to the first mitotic division (Dix, 2007).
As is the case for PCM recruitment, there are currently no data suggesting that SPD-2 is essential for centriole duplication in worm somatic cells. Indeed, the results of a recent candidate-based siRNAi screen in human tissue culture cells suggested that human SPD-2 (Cep192) might not be essential for centriole duplication in somatic cells (Kleylein-Sohn, 2007). It therefore remains possible that SPD-2 proteins could be specifically required for the initial duplication of the fertilizing sperm centriole. At a first glance, these data appear to contradict this hypothesis because it was shown that the first round of centriole duplication can occur in Drosophila embryos lacking Spd-2. It remains possible, however, that the initial events of centriole duplication have already occurred in the sperm prior to fertilization, and so are not dependent on the maternally supplied pool of Spd-2. It is not possible to test this hypothesis directly by the fertilization of Spd-2 embryos with Spd-2 mutant sperm because these sperm are immotile (Dix, 2007).
How could SPD-2 proteins coordinate these two important centrosome-cycle events—centriole duplication and centrosome maturation? It is proposed that SPD-2 could act as a general protein-recruitment factor, which is only absolutely essential for the recruitment of centriole-duplication factors and PCM proteins to the sperm centriole(s) after fertilization. More work will elucidate whether SPD-2 proteins are also dispensable for these processes in the somatic cells of other species, or whether the functions of worm and fly SPD-2 proteins have diverged. In either case, these data highlight the importance of studying the roles of proteins implicated in the centrosome cycle in a range of organisms and cellular contexts (Dix, 2007).
Conventional centrosomes are absent from a female meiotic spindle in many animals. Instead, chromosomes drive spindle assembly, but the molecular mechanism of this acentrosomal spindle formation is not well understood. This study screened female sterile mutations for defects in acentrosomal spindle formation in Drosophila female meiosis. One of them, remnants (rem), disrupted bipolar spindle morphology and chromosome alignment in non-activated oocytes. It was found that rem encodes a conserved subunit of Cdc2 (Cks30A). Since Drosophila oocytes arrest in metaphase I, the defect represents a new Cks function before metaphase-anaphase transition. In addition, it was found that the essential pole components, Msps and D-TACC, are often mislocalized to the equator, which may explain part of the spindle defect. The second cks gene cks85A, in contrast, has an important role in mitosis. In conclusion, this study describes a new pre-anaphase role for a Cks in acentrosomal meiotic spindle formation (Pearson, 2005).
Spindle formation in female meiosis is unique in terms of the absence of conventional centrosomes. Instead, chromosomes have a central role in the assembly of spindle microtubules. This acentrosomal (also called acentriolar or anastral) spindle formation is common in female meiosis for many animals including mammals, insects and worms. Despite potential medical implications, this spindle formation is much less studied than centrosome-mediated spindle formation in mitosis (Pearson, 2005).
Drosophila provides a valuable tool to study the acentrosomal spindle formation in vivo. Unlike many other species, mature non-activated Drosophila oocytes arrest in metaphase of meiosis I until ovulation, which coincides with fertilization. This provides a unique opportunity to study spindle formation, without interference from chromosome segregation or meiotic exit (Pearson, 2005).
Two components of acentrosomal spindle poles, Msps and D-TACC, physically interact and are crucial for spindle bipolarity. Other studies have identified essential components for spindle formation, such as kinesin-like proteins (Ncd and Sub, γ-tubulin, and a membrane protein surrounding the spindle (Axs). Some of these spindle components are probably modulated by cell-cycle regulators, but knowledge of the regulation is limited. To identify essential components and regulators, a cytological screen was performed for mutants defective in acentrosomal spindle formation of non-activated oocytes (Pearson, 2005).
Through the screen, remnants was identified and identified as a mutant of a Drosophila Cks/Suc1 homologue, Cks30A. Cks is the third subunit of the Cdc2 (Cdk1)-cyclin B complex, but the role of Cks is less clearcut than that of other subunits of the complex. It is implicated in entry into mitosis/meiosis, metaphase-anaphase transition, exit from mitosis/meiosis and inactivation of Cdk inhibitors. This study shows that Cks30A is required for spindle morphogenesis and chromosome alignment in the metaphase I spindle in arrested mature oocytes. This requirement of a Cks before metaphase-anaphase transition represents a new function that has not previously been identified. Furthermore, it was found that essential spindle pole components Msps and D-TACC mislocalize in the mutant, which may be partly responsible for the spindle defects (Pearson, 2005).
For molecular analysis of the acentrosomal spindle in Drosophila female meiosis, female sterile mutants were screened for spindle defects in non-activated oocytes. Female sterile mutants on the second chromosome have previously been isolated. This study focused on classes of mutants that lay eggs that do not develop beyond the blastoderm stage. This category of mutants includes known meiotic mutants affecting spindle formation, such as fs(2)TW1 (γ-tubulin 37C) and subito (a kinesin-like protein (Pearson, 2005).
The identity of the remnants (rem) gene was identified by positional cloneing. The rem gene was previously mapped to 30A-C using a deficiency Df(2L)30AC (Schupbach, 1989). One missense mutation was identified in the gene CG3738 (cks, hereafter called cks30A; Finley, 1994). There were no other mutations within coding sequences and splicing junctions in the region. In addition, the amount and size of the transcripts that are known to be expressed in adult females was tested, and no differences were found between rem and wild type (Pearson, 2005).
Cks30A is one of two Drosophila homologues of Saccharomyces cerevisiae Cks1/Schizosaccharomyces pombe Suc1, a conserved subunit of the Cdc2 (Cdk1)/cyclin B complex, and has been shown to interact with Cdc2 (Finley, 1994). The mutation in rem1 results in a conversion of the 61st amino acid from proline to leucine. This proline is completely conserved among all Cks homologues, further confirming that the mutation is not a polymorphism. Crystal structure analysis has indicated that this residue forms part of the interaction surface with Cdc2 (Bourne, 1996). Immunoblots using an anti-human Cks1 antibody indicated that this mutation disrupts the stability of the Cks30A protein (Pearson, 2005).
To explain the role of Cks30A, focus was placed on the rem1 mutant in non-activated oocytes, which arrest in metaphase I. Non-activated oocytes were dissected from wild type and the rem1 mutant, and chromosomes and spindles were visualized by immunostaining (Pearson, 2005).
In wild type, non-activated mature oocytes contain a single bipolar spindle around chromosomes. Bivalent chromosomes align symmetrically with chiasmatic chromosomes at the equator and achiasmatic chromosomes that are located nearer the poles. The rem1 mutant was able to enter meiosis, condense chromosomes and assemble microtubules around chromosomes. However, only a minority of spindles showed normal spindle morphology and chromosome alignment (Pearson, 2005).
The most prominent defect in the rem1 mutant was chromosome misalignment. This defect was observed in about a half of the spindles. Even in the cases in which the spindle remained well organized, chiasmatic chromosomes often moved away from the equator and lost overall symmetrical distribution. The second class of defect in the rem1 mutant was abnormal spindle morphology. Although the abnormality varied from spindle to spindle in the rem1 mutant, the most typical defect was the formation of ectopic poles near the spindle equator. The focusing of spindle poles seemed to be unaffected (Pearson, 2005).
Further quantitative analysis showed no significant difference between the phenotypes of rem1 homozygotes (rem1/rem1) and hemizygotes (rem1/Df). This indicates that the rem1 mutation is genetically amorphic. A recent independent study (Swan, 2005a) has indicated that another weaker allele remHG24 shows similar abnormalities at a lower frequency. These results indicate that Cks30A is required before the metaphase-anaphase transition for spindle morphology and chromosome alignment (Pearson, 2005).
To gain an insight into the spindle defects in female meiosis, the localization of Msps was examined. Msps protein belongs to a conserved family of microtubule regulators, including XMAP215, and is the first protein identified at the acentrosomal poles in Drosophila. An msps mutation often leads to the formation of a tripolar spindle in female meiosis I (Pearson, 2005).
In wild type, Msps protein is accumulated at the acentrosomal poles of the metaphase I spindle in female meiosis, although the localization sometimes spreads to the spindle microtubules. In the rem1 mutant, although the Msps protein is still concentrated at the poles, it is often accumulated around the equator of the spindle. Mislocalization of this important pole protein to the equator in the rem1 mutant may sometimes lead to the formation of ectopic spindle poles near the equator (Pearson, 2005).
Msps localization is dependent on another pole protein D-TACC, which binds to Msps. To test whether D-TACC also mislocalizes, the localization of D-TACC was examined in the rem1 mutant. In wild type, D-TACC is highly concentrated at the acentrosomal pole. In the rem1 mutant, D-TACC often accumulates at the spindle equator, although it is still concentrated around the poles to some degree. In summary, Cks30A is required for correct localization of the essential pole proteins, Msps and D-TACC (Pearson, 2005).
To gain an insight into how the defect in the Cdc2 complex leads to Msps or D-TACC mislocalization to the spindle equator, the localization of cyclin B was examined. Cyclin B is considered to be the main determinant of the activity and cellular localization of the Cdc2 complex. Immunostaining in non-activated oocytes showed that cyclin B is localized to the metaphase I spindle, with a concentration around the spindle equator. This cyclin B localization could suggest a possible regulatory role of the Cdc2 complex in the transport of Msps and D-TACC from the spindle equator to the poles. The cyclin B localization is not affected in the rem mutant, suggesting that Cks30A mainly affects the substrate specificity of the Cdc2 complex, as shown in other systems (Pearson, 2005).
The Drosophila genome contains one more predicted cks homologue (CG9790), which is called cks85A. Although mammalian genomes also have two Cks genes, they are more similar in sequence to each other than to either of the two cks genes in Drosophila (Pearson, 2005).
The gene expression pattern of the two cks genes was examined during Drosophila development. RNAs were isolated from various stages of development and analysed by reverse transcription-PCR (RT-PCR) using primers that correspond to each of the cks genes. cks30A gave strong signals in adult females and embryos, whereas it gave only weak signals in adult males, larvae and pupae. This maternal expression pattern is consistent with the observed female sterile phenotype of the cks30A (rem1) mutant. In contrast, cks85A signals were obtained more uniformly throughout the development without sex specificity in adults. In S2 cultured cells, which originated from embryos, both genes were well expressed (Pearson, 2005).
To identify the Cks proteins, an anti-human Cks1 antibody was used for immunoblots of protein extracts from embryos and S2 cells. Although the antibody recognized many proteins, two bands were detected within a range of molecular weights consistent with the Cks proteins. In embryos laid by the rem1 mutant, the amount of the smaller band was greatly reduced. To further confirm their identity, S2 cells were subjected to RNA interference (RNAi) using doublestranded RNAs (dsRNAs) corresponding to the cks genes. It was found that both of the bands disappeared when both genes were simultaneously knocked down by RNAi. It indicated that, consistent with RT-PCR results, S2 cells produced both the Cks proteins and that RNAi effectively depletes them (Pearson, 2005).
Cytological analysis showed that cks85A RNAi results in a significant increase in chromosome misalignment/missegregation and spindle abnormality in mitosis after an extended time, whereas cks30A RNAi has a lesser impact on mitotic progression. About a half of anaphase or telophase cells had lagging chromosomes or chromosome bridges after cks85A RNAi. In some cases, spindles contained scattered chromosomes the sister chromatids of which were either attached or detached. The frequency of multipolar spindles was also increased. The genetic and RNAi results indicated that cks85A has an important function in mitotic progression, whereas cks30A mainly functions in female meiosis (Pearson, 2005).
This study has shown a new pre-anaphase function of a Cks protein in acentrosomal spindle formation during Drosophila female meiosis. Through a cytological screen, spindle defects in remnants among female sterile mutants. Cytological analysis showed that Cks30A is required for correct formation of the acentrosomal spindle and chromosome alignment in female meiosis I. The observation on mislocalization of the essential pole components, Msps and D-TACC, in the mutant provides a molecular insight into a role of Cks30A in spindle morphogenesis (Pearson, 2005).
Cks/Suc1 protein is the third subunit of the Cdc2-cyclin B complex, which is conserved across eukaryotes. Although it has been known to be essential for the cell cycle, the function seems to be less straightforward than that of the other subunits of the Cdc2 complex. One reason is that Cks also interacts with other Cdks and has Cdk-independent functions. Even if Cks is limited to roles in mitosis/meiosis, Cks proteins are implicated in entry into mitosis/meiosis, metaphase-anaphase transition and also exit from mitosis/meiosis. Furthermore, the roles of Cks were further complicated by the fact that animal genomes encode two Cks homologues (Pearson, 2005).
Studies in Caenorhabditis elegans and mice showed that one of two cks genes is required for female fertility. Similarly, the results indicated that one of two Drosophila cks homologues, cks30A, is expressed maternally and is required for female meiosis. Further analysis indicated that Cks30A is required for proper bipolar spindle formation and chromosome alignment in mature oocytes arrested in metaphase I. In C. elegans, depletion of one of the Cks proteins by RNAi results in a failure to complete meiosis I. Similarly, in mice, oocytes from a Cks2 knockout cannot progress past metaphase I and a small percentage of oocytes show chromosome congression failure. In both cases, the defects were interpreted mainly as post-metaphase defects. Since Drosophila non-activated oocytes are arrested in metaphase I until ovulation, pre-anaphase function of Cks30A can be distinguised from possible post-metaphase function. This study clearly showed that Drosophila Cks30A has a function in establishing metaphase I, in addition to later functions that have reported recently (Swan, 2005a; Pearson, 2005).
At the moment, it is not known how the cks30A mutation disrupts spindle formation and chromosome alignment in female meiosis. It has been thought that a loss of Cks function affects the Cdc2 activity towards certain substrates. It was found that the essential pole components, Msps and D-TACC, mislocalize to the spindle equator in the mutant. Previously, it was hypothesized that Msps is transported by the Ncd motor and anchored to the poles by D-TACC. D-TACC localizes to the poles independently from Ncd, but may also be transported from the spindle equator along microtubules by other motors. Cks30A-dependent Cdc2 activity may be required for activating the transport system at the onset of spindle formation in female meiosis. Consistently, it was found that cyclin B is concentrated around the equator of the metaphase I spindle. Msps is the XMAP215 homologue and belongs to a family of conserved microtubule-associated proteins. It is a major microtubule regulator, both in mitosis/meiosis and interphase. The mislocalization of this microtubule-regulating activity could lead to the disruption of spindle organization in the mutant (Pearson, 2005).
Meiosis is a highly specialized cell division that requires significant
reorganization of the canonical cell-cycle machinery and the use of
meiosis-specific cell-cycle regulators. The anaphase-promoting complex (APC, a machine for degrading proteins; see APC subunits Cdc27 and morula; for review see Acquaviva, 2006)
and a conserved APC adaptor/activator, Cdc20 (also known as Fizzy), are required for
anaphase progression in mitotic cells. The APC has also been implicated in
meiosis, although it is not yet understood how it mediates these non-canonical divisions. Cortex (Cort) is a diverged Fzy homologue that is expressed in the female germline of Drosophila, where it functions with the Cdk1-interacting protein Cks30A to drive anaphase in meiosis II. This study shows
that Cort functions together with the canonical mitotic APC adaptor Fzy to
target the three mitotic cyclins (A, B and B3) for destruction in the egg and
drive anaphase progression in both meiotic divisions. In addition to
controlling cyclin destruction globally in the egg, Cort and Fzy appear to
both be required for the local destruction of cyclin B on spindles. Cyclin B associates with spindle microtubules throughout meiosis I and
meiosis II, and dissociates from the meiotic spindle in anaphase II. Fzy and
Cort are required for this loss of cyclin B from the meiotic spindle. These
results lead to a model in which the germline-specific APCCort
cooperates with the more general APCFzy, both locally on the
meiotic spindle and globally in the egg cytoplasm, to target cyclins for
destruction and drive progression through the two meiotic divisions (Swan, 2007).
The cell divisions of female meiosis and the ensuing mitotic cycles of
early embryogenesis represent two examples of non-canonical cell cycles.
Meiosis differs from the typical mitotic cycle in several respects. Most
notably, two divisions occur in sequence without an intervening S-phase,
resulting in the production of four haploid gametes. Additionally, the first
meiotic division involves the segregation of homologous chromosomes and occurs
without sister chromatid segregation, whereas the second meiotic division
involves the segregation of sister chromatids, as occurs in mitosis. The
regulation of meiosis requires a significant reorganization of the canonical
cell-cycle machinery and the use of a number of meiosis-specific cell-cycle
regulators. One example is in the regulation of anaphase - the
coordinated series of events that results in the segregation of chromosomes to
produce two daughter nuclei. In mitotically dividing cells, anaphase
progression crucially depends on the inactivation of the mitotic kinase Cdk1
(also known as Cdc2) and on the subsequent release of sister chromatid
cohesion through the destruction of cohesin complexes. These events are
controlled by an E3 ubiquitin ligase -- the anaphase-promoting complex (APC) --
in association with an adaptor protein, Fzy, and this complex targets mitotic
cyclins and securin (potential Drosophila homolog; Pimples) for destruction (reviewed in
Peters, 2002). The role of the
APC in meiosis appears to be more complex than in mitotic cells. For example,
the APC only partially inhibits Cdk1 activity between meiotic divisions
and sister chromatid cohesion persists at centromeres through anaphase I. It is not yet clear how the activity of the APC is modified in these specialized
cell divisions (Swan, 2007).
In most eukaryotes, the meiotic cell cycle is followed by another atypical
cell cycle -- the cleavage divisions of early embryogenesis. In
Drosophila, these cleavage cycles occur as a series of synchronized,
rapid nuclear divisions and are referred to as syncytial divisions. The female
meiotic cell cycle is not only closely linked to the syncytial mitotic cell
cycle in time, but it also occurs within a shared cytoplasm -- that of the egg.
Therefore, these two distinct cell cycles share a common pool of cell-cycle
regulators, and may share common strategies for spatially and temporally
regulating cell-cycle progression within a syncytium (Swan, 2007).
One way in which the syncytial cell cycle is modified appears to be in the
limited destruction of mitotic cyclins in each cell cycle, apparently by
restricting their destruction to the area of the mitotic nuclei. Although
there is evidence that cyclin destruction is spatially regulated in somatic
cells, this strategy appears to be of particular importance in the syncytial embryo of Drosophila as a means to conserve mitotic cyclins for the duration of the rapid syncytial divisions. Several lines of evidence suggest that at least one cyclin, cyclin B, undergoes limited local destruction on mitotic spindles in the syncytial embryo. It is not yet known what mediates this local cyclin B destruction, and it is also not known whether this is unique to the syncytial mitotic cell cycle or if it occurs in the preceding meiotic divisions (Swan, 2007).
Drosophila represents an excellent model system for understanding
how the canonical cell-cycle machinery is developmentally modified, and how
novel cell-cycle regulators are used to control meiosis and syncytial
divisions. cortex (cort) encodes a Cdc20/Cdh1 (Cdh1 is also
known as Fzr and Rap)-related protein, that appears to be required
specifically in female meiosis (Chu, 2001; Lieberfarb, 1996; Page, 1996) and functions with a germline-specific Cks gene, Cks30A, to mediate the destruction of cyclin A (Swan, 2005a; Swan, 2005b). This study shows that the canonical APC adaptor Fzy functions together with Cort to target mitotic cyclins for destruction, and to drive anaphase in both meiosis I and meiosis II. Female meiosis, like the subsequent syncytial mitotic cell cycles, appears to involve the local destruction of cyclin B, and both Cort and Fzy were found to be required for this process (Swan, 2007).
In most cell types, in both Drosophila and in other metazoans, the
APCFzy drives anaphase progression by targeting mitotic cyclins and other mitotic proteins for destruction. This study shows that the female germline is an exception in that the APCFzy is not sufficient. A germline-specific APC adaptor, Cort, cooperates with Fzy to mediate cyclin destruction in meiosis (Swan, 2007).
The cort gene encodes a diverged member of the Fzy/Cdh1 family
(Chu, 2001). Fzy/Cdh1
homologues interact with the APC and with specific sequences (D-box, KEN box
or A-box) found on cyclins and on other APC targets. As such, Fzy/Cdh1
proteins act as specificity factors to target proteins for ubiquitination and
eventual destruction. Cort protein, like all Fzy/Cdh1-family proteins,
contains seven WD domains in the C-terminal-half of the protein, implicated in substrate recognition (Pfleger, 2001). Cort has an N-terminal C-box (amino
acids 482, 483) and a C-terminal IR tail (amino acids 54-60), both implicated
in binding to the APC. In addition to containing these conserved functional
domains, Cort displays a conserved ability to mediate cyclin destruction.
cort mutations result in the overaccumulation of cyclin A, cyclin B
and cyclin B3 in the egg (Swan, 2005a), whereas the ectopic expression of Cort in the wing disc leads to a reduction in the levels of these mitotic cyclins. Taken together, these results indicate that Cortex encodes a functional member of the Fzy/Cdh1 family (Swan, 2007).
Although the Drosophila genome has four genes that encode Fzy/Cdh1
proteins, only two of these proteins, Fzy and Cort, are expressed at
detectable levels in the female germline (Raff, 2002;
Jacobs et al., 2002; Chu, 2001). The role of these two APC adaptors has been studied both individually and in double
mutants, and it was found that they function together to promote anaphase in
both the first and second meiotic divisions of female meiosis. In most cell
types in Drosophila and other eukaryotes, a single APC complex,
APCFzy, is responsible for cyclin destruction and anaphase
progression. It is therefore surprising that, in the female germline of
Drosophila, two APC adaptors are necessary for meiotic progression.
In the case of meiosis I, Cort and Fzy appear to play largely redundant roles, since only removing both genes results in a significant block in meiosis I. The
two APC complexes may also be functionally redundant with respect to global
cyclin levels. Mutations in either fzy or cort result in an
increase in the levels of cyclin A, cyclin B and cyclin B3, whereas mutation
in both genes results in even-further increases in cyclin levels (Swan, 2007).
Although Cort and Fzy have overlapping roles in promoting anaphase I, both
are essential for meiosis II. This could simply reflect a greater quantitative
requirement for APC activity in meiosis II. Alternatively, the two APC
complexes could have distinct roles in the second meiotic division. Consistent
with this latter possibility, mutations in either cort or
fzy both result in arrest at different stages of meiosis II:
cort mutants arrest with the sister chromatids associated, and
therefore in metaphase, whereas fzy mutants almost invariably arrest
with separated sister chromatids, and are therefore in anaphase. cort
and fzy also result in different patterns of cyclin B stabilization
on the arrested spindles, suggesting roles in metaphase and anaphase,
respectively. Therefore, Cort may function to initiate sister chromatid
separation at the onset of anaphase II and Fzy may primarily function later,
in anaphase II. Alternatively, the later arrest observed in fzy could
simply reflect the fact that the fzy alleles that have been used are
not nulls, and it is possible that a complete loss of Fzy activity would also
result in a metaphase arrest, as seen in cort. However, comparing the
meiosis II phenotypes of fzy with Cks30A-null mutants
suggests that the later arrest in fzy is not simply due to residual
activity. Cks30A-null mutants have a weaker meiotic arrest than
fzy; they complete meiosis at high frequency
(Swan, 2005a), but they
display a higher frequency of metaphase arrest or delay. The fact that
fzy does not similarly cause a delay in metaphase of meiosis II
suggests that it is only required at anaphase. Therefore, it is possible that
Fzy is crucial at anaphase, whereas Cort is necessary for the metaphase to
anaphase transition (Swan, 2007).
The different temporal requirements for Cort and Fzy prior to and after
sister chromatid separation, respectively, could be related to their apparent
differences in substrate specificity. Western analysis
reveals that Cort is
more important for the destruction of cyclin A and cyclin B3, whereas Fzy
appears to play a greater role in cyclin B destruction in the egg. In mitotic
cells, cyclin destruction occurs sequentially. Cyclin A is destroyed first, in
prometaphase, and this is a prerequisite for sister chromatid separation.
Cyclin B destruction occurs at anaphase onset and is necessary for later
anaphase events, subsequent to sister chromatid separation.
Therefore, it is possible that Cort promotes the early stages of meiotic
anaphase by targeting cyclin A for destruction, whereas Fzy is more crucial
later, through its targeting of cyclin B for destruction (Swan, 2007).
The meiotic cell cycle differs in many respects from the standard mitotic
cycle. Whereas APC-mediated destruction of mitotic regulators appears to be
required for anaphase progression in most or all mitotic cells, the role of
the APC and cyclin destruction in meiosis is not as well-understood. This
analysis of the two APC adaptors Cort and Fzy has permitted an evaluation of
the role of the APC complex in female meiosis in Drosophila. The APC is required for anaphase progression in both meiotic divisions.
Correlating with its requirement for the completion of meiosis, the APC is
required for the destruction of mitotic cyclins. At least one of these
cyclins, cyclin B, is a crucial substrate in meiosis, because the expression
of a stabilized form of cyclin B disrupts this process. Therefore, APC
activity and cyclin destruction are required for anaphase progression in both
meiotic divisions, in addition to in mitosis. APC activity has been implicated
in both meiotic divisions in C. elegans and in
the mouse, and in the second, but not the first, meiotic division in
Xenopus. In yeast, two APC complexes, the mitotic APCFzy
and a meiosis-specific complex (APCAma1 in S. cerevisiae
and APCMfr1 in S. pombe) function together to mediate
protein destruction in meiosis. It now appears that Drosophila also uses two APC complexes in female meiosis, and this may turn out to be a common strategy in other eukaryotes (Swan, 2007).
Cks30A belongs to a highly conserved family of proteins that bind to and
stimulate the activity of the mitotic kinase Cdk1. In Xenopus, the
Cks30A homologue Xep9 stimulates the Cdk-dependent phosphorylation of APC
subunits, and thereby promotes the activation of the APCFzy complex. The current
results suggest that Cks30A may have a similar role in stimulating both the
APCFzy and APCCort in female meiosis in
Drosophila. (1) Cks30A, like cort and
fzy, is required for the completion of meiosis II and, like
fzy, it is required for the completion of the first mitotic division
of embryogenesis (Lieberfarb, 1996; Page, 1996; Swan,
2005). (2) Cks30A, as are Cort and Fzy, is necessary for
global cyclin destruction in the Drosophila egg and for local cyclin
B destruction on the meiotic spindle. Global levels of cyclin A
and cyclin B3 are elevated to a greater extent in Cks30A mutants than
in single mutants for cort or fzy, consistent with the idea
of Cks30A activating both Cort and Fzy. (3) Cks30A is
necessary for the activity of ectopically expressed Cort in the adult wing. Cks30A may also play a role in activating APCFzy in mitotic cells.
the temperature-sensitive fzy6 allele is lethal at all
temperatures in a Cks30A-null background, suggesting that the Cks30A-dependent activation of APCFzy becomes essential when Fzy activity is compromised (Swan, 2007).
Although Cks30A appears to promote the activity of the APCCort
and the APCFzy, these complexes seems to retain some activity in
the absence of Cks30A. Whereas cort and fzy cause an arrest
in meiosis II, Cks30A-null mutants are typically delayed only in
meiosis II (Swan,
2005a). Also, although cyclin A and cyclin B3 levels are elevated
more in Cks30A eggs than in either fzy or cort, their levels
are still not as high as in fzy; cort double mutants, indicating that
Fzy and Cort can destroy cyclin A and cyclin B3 to some degree in the absence
of Cks30A. Cyclin B destruction is even less dependent on Cks30A, because
cyclin B levels are affected less in Cks30A mutants than in either
cort or fzy single mutants. Therefore, Cks30A may be more
crucial for the activity of APCCort and APCFzy complexes
on cyclin A and cyclin B3, and less crucial for their activity on cyclin B.
The relatively weaker meiotic arrest in Cks30A mutants compared to
fzy; cort double mutants may also indicate that the APC has other
meiotic targets that can be destroyed in the absence of Cks30A (Swan, 2007).
Cyclin B undergoes local oscillations in its association with mitotic
spindles in syncytial embryos, appearing transiently along the full length of
the mitotic spindle in early metaphase and gradually disappearing from the
spindle starting at the centrosomes and ending at the kinetochores. The
timing of this loss of cyclin B from the spindle, at the onset of anaphase,
corresponds with the timing of cyclin B destruction in other cell types,
suggesting the possibility that cyclin B is locally destroyed on the spindle
in anaphase. This study shows that cyclin B is subject to similar local
oscillations in the female meiotic cycles, and that cyclin B
destruction is necessary for the completion of female meiosis. Importantly, the local loss of cyclin B from the spindle in meiosis is
dependent on the APC adaptors Cort and Fzy, and that the local loss of cyclin
B from the spindle in mitosis depends on Fzy. These results
strongly suggest that the local loss of cyclin B from the spindle in anaphase
of meiosis II and anaphase of mitosis is actually due to its local
destruction (Swan, 2007).
The pattern of accumulation and loss of cyclin B from the spindle in
meiosis differs in some respects compared to syncytial mitotic cycles. (1) In metaphase of mitosis, cyclin B initially accumulates throughout the spindle microtubules, whereas, in metaphase of the meiotic divisions, cyclin B first appears exclusively at the spindle mid-zone. This difference may reflect the fact that the meiotic spindle does not contain centrosomes and cyclin B may, therefore, not load onto spindles from centrosomes and progress along the spindles to the kinetochores, as has been proposed for mitosis.
(2) The timing of cyclin B destruction appears to be different between the meiotic and mitotic cycles. Most strikingly, there is no loss of cycli