InteractiveFly: GeneBrief

missing oocyte: Biological Overview | References


Gene name - missing oocyte

Synonyms -

Cytological map position - 22D4-22D4

Function - conserved novel protein

Keywords - regulates nuclear architecture and meiotic progression in early ovarian cysts, a novel interacting partner of the conserved nucleoporin Seh1, influences meiotic progression and oocyte fate

Symbol - mio

FlyBase ID: FBgn0031399

Genetic map position - chr2L:2,217,363-2,220,625

Classification - WD40 repeat protein

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1; see Drosophila Tor pathway) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. This study defined opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. As is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Missing oocyte (Mio) and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. These studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. The data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms (Wei, 2014b).

In yeast, the inhibition of the nutrient-sensitive target of rapamycin complex 1 (TORC1) in response to amino acid limitation is essential for cells to transit from the mitotic cycle to the meiotic cycle. In response to amino acid starvation, the Iml1 complex, comprising the Iml1, Nitrogen permease regulator-like 2 (Npr2), and Nitrogen permease regulator-like 3 (Npr3) proteins in yeast and the respective orthologs DEPDC5, Nprl2, and Nprl3 in mammals, inhibits TORC1 activity. The Iml1 complex, which has been renamed the 'GTPase-activating proteins toward Rags 1' (GATOR1) complex in higher eukaryotes, functions as a GTPase-activating protein complex that inactivates RagsA/B or Gtr1 in mammals and yeast, respectively, thus preventing the activation of TORC1. In the yeast Saccharomyces cerevisiae, mutations in the Iml1 complex components Npr2 and Npr3 result in a failure to down-regulate TORC1 activity in response to amino acid starvation and block meiosis and sporulation. As is observed in yeast, in Drosophila, Nprl2 and Nprl3 mediate a critical response to amino acid starvation (Wei, 2014a). However, their roles in meiosis and gametogenesis remain unexplored (Wei, 2014b).

Recent reports indicate that the Iml1, Npr2, and Npr3 proteins are components of a large multiprotein complex originally named the 'Seh1-associated' (SEA) complex in budding yeast and the 'GATOR' complex in higher eukaryotes. The SEA/GATOR complex contains eight highly conserved proteins. The three proteins described above, Iml1/DEPDC5, Npr2/Nprl2, and Npr3/Nprl3, form the Iml1/GATOR1 complex and inhibit TORC1. The five remaining proteins in the complex, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59, which have been designated the 'GATOR2' complex in multicellular organisms, oppose the activity of Iml1/GATOR1 and thus promote TORC1 activity (Wei, 2014b).

Little is known about the physiological and/or developmental requirements for the GATOR2 complex in multicellular organisms. However, in Drosophila the GATOR2 components Mio and Seh1 interact physically and genetically and exhibit strikingly similar ovarian phenotypes, with null mutations in both genes resulting in female sterility (Senger, 2011; Wei, 2014a). In Drosophila females, oocyte development takes place within the context of an interconnected germline syncytium, also referred to as an 'ovarian cyst'. Ovarian cyst formation begins at the tip of the germarium when a cystoblast, the daughter of a germline stem cell, undergoes four synchronous divisions with incomplete cytokinesis to produce 16 interconnected cells. Actin-stabilized cleavage furrows, called 'ring canals', connect cells within the cyst. Each 16-cell cyst develops with a single oocyte and 15 polyploid nurse cells which ultimately are encapsulated by a somatically derived layer of follicle cells to produce an egg chamber. Each ovary is comprised of ∼15 ovarioles that consist of a single germarium followed by a string of egg chambers in successively older stages of development. In mio- and seh1-mutant egg chambers, the oocyte enters the meiotic cycle, but as oogenesis proceeds, the oocyte fate and the meiotic cycle are not maintained stably (Senger, 2011; Wei, 2014a). Ultimately, a large fraction of mio and seh1 oocytes enter the endocycle and develop as polyploid nurse cells. A mechanistic understanding of how mio and seh1 influence meiotic progression and oocyte fate has remained elusive (Wei, 2014b).

This study demonstrates that the Iml1/GATOR1 complex down-regulates TORC1 activity to promote the mitotic/meiotic transition in Drosophila ovarian cysts. Depleting iml1 in the female germ line delays the mitotic/meiotic transition, so that ovarian cysts undergo an extra mitotic division. Conversely, mutations in Tor result in premature meiotic entry before the completion of the four mitotic divisions. Moreover, it was demonstrated that in the female germ line, the GATOR2 components Mio and Seh1 are required to oppose the TORC1 inhibitory activity of the Iml1/GATOR1 complex to prevent the constitutive down-regulation of TORC1 activity in later stages of oogenesis. These studies represent the first examination of the regulatory relationship between Iml1/GATOR1 and GATOR2 components within the context of a multicellular animal. Finally, these data reveal a surprising tissue-specific requirement for the GATOR2 complex in multicellular organisms and suggest a conserved role for the SEA/GATOR complex in the regulation of TORC1 activity during gametogenesis (Wei, 2014b).

Previous work demonstrated that in Drosophila the Iml1/GATOR1 complex mediates an adaptive response to amino acid starvation. This study tested the hypothesis that the Iml1/GATOR1 complex also has retained a role in the regulation of the early events of gametogenesis. Consistent with this model, this study found that in germline knockdowns of iml1, ovarian cysts delay meiotic entry and undergo a fifth mitotic division. This meiotic delay can be suppressed with the TORC1 inhibitor rapamycin. Thus, during Drosophila oogenesis the Iml1/GATOR1 complex promotes the transition from the mitotic cycle to the meiotic cycle through the down-regulation of the metabolic regulator TORC1. Increasing TORC1 activity by disabling its inhibitor delays meiotic progression, whereas germline clones of a Tor-null allele enter meiosis prematurely. Taken together, these data indicate that the level of TORC1 activity contributes to the timing of the mitotic/meiotic switch in Drosophila females and suggest that low TORC1 activity may be a conserved feature of early meiosis in many eukaryotes (Wei, 2014b).

However, in Drosophila, meiotic entry is not contingent on amino acid limitation at the organismal level. Indeed, the energy-intensive process of Drosophila oogenesis is curtailed dramatically when females do not have access to a protein source. Thus, to promote meiotic entry, Drosophila females must activate the Iml1/GATOR1 complex in a tissue-specific manner, using a mechanism that is independent of the overall nutrient status of the animal. At least two models can explain how Drosophila females might activate the Iml1/GATOR1 complex specifically in the germ line. In the first model, ovarian cysts locally experience low levels of amino acids during the mitotic cyst divisions and/or at the point of meiotic entry. These low levels of amino acids could reflect a non–cell-autonomous effect: The somatically derived escort cells that surround dividing ovarian cysts may function to create a low amino acid environment that triggers the activation of the Iml1/GATOR1 complex within developing ovarian cysts. Alternatively, the effect may be cell autonomous: The germ cells within dividing ovarian cysts may have a reduced ability to sense and/or import amino acids. In a second model, a developmental signaling pathway that is completely independent of local or whole-animal amino acid status directly activates the Iml1/GATOR1 complex. The identification of the upstream requirements for Iml1/GATOR1 activation in the female germ line will help distinguish between these two models (Wei, 2014b).

Although low TORC1 activity is required during early ovarian cyst development to promote the mitotic/meiotic switch, the dramatic growth of egg chambers later in oogenesis is a metabolically expensive process that is predicted to require high TORC1 activity. The current data indicate that the GATOR2 components Mio and Seh1 function to oppose the TORC1-inhibitory activity of the GATOR1 complex in the female germ line. In mio and seh1 mutants, TORC1 activity is constitutively repressed in the germ line of developing egg chambers, resulting in the activation of catabolic metabolism and the blocking of meiotic progression and oocyte development and growth (Wei, 2014b).

Previous data indicate that Mio and Seh1 act very early in oogenesis soon after the formation of the 16-cell cyst. The mio and seh1 ovarian phenotypes can be rescued by depleting the GATOR1 components nprl2, nprl3, or iml1 in the female germ line or by raising baseline levels of TORC1 activity by disabling an alternative TORC1 inhibitory complex, TSC1/2. These data are consistent with the model that the failure to maintain the meiotic cycle and the oocyte fate in mio and seh1 mutants is a direct result of inappropriately low TORC1 activity in the female germ line brought on by the deregulation of the Iml1/GATOR1 complex (Wei, 2014b).

Notably, null alleles of both mio and seh1 are viable, with many somatic tissues exhibiting no apparent developmental abnormalities and only limited reductions in cell growth. Thus, although Mio and Seh1 are critical for the activation of TORC1 and the development of the female gamete, these proteins play a relatively small role in the development and growth of many somatic tissues under nutrient-replete conditions. Whether this small role reflects the fact that components of the Iml1/GATOR1 complex are expressed at low levels in some somatic cell types or that the complex is present but needs to be activated by a signal, such as nutrient stress or a developmental signaling pathway, remains to be elucidated (Wei, 2014b).

In the future it will be important to gain a fuller understanding of the potential environmental and developmental inputs that regulate the activity of the Iml1/GATOR1 and GATOR2 complexes in multicellular organisms. These studies will provide much-needed insight into the basic mechanisms by which both environmental and developmental signaling pathways interface with the metabolic machinery to influence cell growth and differentiation (Wei, 2014b).

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. This study reports an unexpected tissue-specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. Seh1 associates with the product of the missing oocyte (mio) gene. In Drosophila, mio regulates nuclear architecture and meiotic progression in early ovarian cysts. Like mio, seh1 has a crucial germline function during oogenesis. In both mio and seh1 mutant ovaries, a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, the accumulation of Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, characterization of a seh1 null allele indicates that, although required in the female germline, seh1 is dispensable for the development of somatic tissues. This work represents the first examination of seh1 function within the context of a multicellular organism. Seh1 has been shown to play a role in the construction and/or maintenance of bipolar spindles in multiple organisms. In summary, these studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles (Senger, 2011).

The pathways that control progression through the early meiotic cycle remain poorly understood in metazoans. Drosophila melanogaster provides a genetically tractable system with which to study the relationship between early meiotic progression and oocyte development. As in mammals and Xenopus, the Drosophila oocyte initiates meiosis within the context of a germline cyst. Drosophila ovarian cysts are produced through a series of four synchronous mitotic divisions during which cytokinesis is incomplete. Soon after the completion of the mitotic divisions, all 16 cells enter premeiotic S phase. However, only the true oocyte, which comprises one of the two cells at the center of the syncytium, remains in meiosis and goes on to produce a gamete. The other 15 cells lose their meiotic features, enter the endocycle, and develop as polyploid nurse cells. In contrast to the nurse cells, the single oocyte remains in prophase of meiosis I until it proceeds to the first meiotic metaphase late in oogenesis. The pathways that drive this complicated series of cell cycle transitions that are so critical to the development of the mature gamete remain a topic of great interest (Senger, 2011).

The missing oocyte (mio) gene was identified in a forward genetic screen for mutants affecting cell cycle regulation and oocyte differentiation in early ovarian cysts (Iida, 2004). In mio mutants, the oocyte enters the meiotic cycle, forms mature synaptonemal complexes and accumulates oocyte-specific markers. However, in the absence of Mio, the oocyte fate is not stably maintained. Soon after the nurse cells enter the endocycle in stage 1 of oogenesis, mio oocytes follow the nurse cells into the endocycle, lose the preferential accumulation of oocyte-specific markers and develop as pseudo-nurse cells. Thus, mio is required for the maintenance of the meiotic cycle and oocyte identity. The mio gene encodes a 975 amino acid protein that is highly conserved from yeast to humans (Iida, 2004). Yet, the molecular function of mio remains elusive. This study demonstrates that Mio associates with the conserved nucleoporin Seh1 (also known as Nup44A in Drosophila). Moreover, this study define a tissue-specific requirement for Seh1 during oogenesis (Senger, 2011).

Seh1 is a component of a nucleoporin subcomplex known as the Nup107-160 complex in higher eukaryotes and the Nup84 complex in yeast (Fahrenkrog, 2004; Hetzer, 2005; Wozniak, 2010). The Nup107-160 complex, which is the major structural component of the nuclear pore complex (NPC), consists of at least nine subunits in higher eukaryotes and functions in the regulation of mRNA export as well as in the assembly and distribution of NPCs within the nuclear envelope (Hetzer, 2005; Wozniak, 2010). Studies over the last five years have defined several physiological functions for the Nup107-160/Nup84 complex that appear to be independent of nucleocytoplasmic transport (Fahrenkrog, 2004; Wozniak, 2010). Most notably, in Xenopus egg extracts and HeLa cells, the Nup107-160 complex has a dynamic localization during the cell cycle (Hetzer, 2005). Although present on the nuclear envelope in interphase, the entire complex targets to kinetochores, spindles and spindle poles to varying extents during mitosis (Loiodice, 2004; Orjalo, 2006). Consistent with a mitotic function, depleting components of the Nup107-160 complex results in cell cycle abnormalities, including defects in mitotic spindle formation, chromosome segregation and cytokinesis (Orjalo, 2006; Platani, 2009). Moreover, recent evidence indicates that in HeLa cells and Xenopus egg extracts, the Nup107-160 complex mediates microtubule nucleation at kinetochores via its interaction with the γ-TuRC complex (Mishra, 2010). Unlike in other metazoans, in Drosophila Nup107 fails to localize to kinetochores at mitosis but is found concentrated in the spindle region (Katsani, 2008). In summary, the Nup107-160 complex is multifunctional, with roles in both nucleocytoplasmic transport and cell cycle regulation (Senger, 2011).

This study demonstrates that Mio, a protein that is required for maintenance of the meiotic cycle and oocyte fate during oogenesis, associates with the structural nucleoporin Seh1. Surprisingly, it was found that a seh1 deletion allele is viable but exhibits dramatically reduced female fertility. Closer examination reveals that, as is observed in mio mutants, in a fraction of seh1 ovarian cysts oocytes fail to maintain the meiotic cycle and oocyte fate into later stages of oogenesis. From these studies it is concluded that Seh1 has an essential germline function during oogenesis but is not required for the growth or development of somatic tissues (Senger, 2011).

To better understand how Mio influences the maintenance of the meiotic cycle and oocyte fate in Drosophila, proteins that co-purify with Mio were identified by tandem affinity purification. From these experiments, it was determined that Mio is present in a stable complex with the structural nucleoporin Seh1. Seh1 is a component of the Nup107 complex, which is the primary structural unit of the nuclear pore. Studies in multiple organisms indicate that, although Seh1 is a nucleoporin, it is not required for bulk nucleocytoplasmic transport and has a limited role in the localization of other nucleoporins to the NPC (Loiodice, 2004; Orjalo, 2006). In contrast to its limited role at the NPC during interphase, recent evidence indicates that Seh1 has an essential function during mitosis. In Xenopus egg extracts and mammalian tissue culture cells, a fraction of the Nup107 complex that includes Seh1 targets to kinetochores, spindles and/or spindle poles from early prometaphase through early anaphase (Belgareh, 2001; Orjalo, 2006). Moreover, reducing the levels of Nup107-160 components disrupts spindle assembly and cytokinesis (Aitchison, 1995; Bai, 2004; Mishra, 2010; Orjalo, 2006; Platani, 2009; Zuccolo, 2007). Importantly, the specific depletion of seh1 results in the failure of the Nup107 complex to target to kinetochores at mitosis and results in multiple mitotic defects (Zuccolo, 2007; Platani, 2009). Thus, Seh1 plays a role in the construction and/or maintenance of bipolar spindles in multiple organisms (Senger, 2011).

To define the role of Seh1 in Drosophila a seh1null deletion allele was generated. Considering the key role of Seh1 during mitosis in other organisms, it was surprising to find that seh1null homozygotes are viable. From this observation it is concluded that seh1 is dispensable in Drosophila for the mitotic cycle during the majority of somatic divisions. Consistent with a limited role for Seh1 during the mitotic cycle, the specific accumulation of Seh1 on kinetochores during mitosis was not observed. Although this might reflect a limitation of the reagents, these data are in agreement with previous work (Katsani, 2008) demonstrating that the core component of the Nup107-160 complex, the nucleoporin Nup107, fails to accumulate at kinetochores during mitosis in multiple Drosophila tissues (Senger, 2011).

These studies also suggest a limited and/or redundant role for Seh1 in supporting the general structure and/or function of the NPC in interphase cells. Specifically, it was found that multiple nucleoporins, including Nup107, target to the NPC in the absence of Seh1 in both germline and somatic tissues. The only nucleoporin mislocalization observed in seh1null mutants involved a limited displacement of Mtor from the nuclear envelope to the nucleoplasm in meiotic cysts in the germarium. Mtor is a component of the nuclear basket and is present on the cytoplasmic face of the NPC during interphase but relocates to the spindle matrix during mitosis (Lince-Faria, 2009; Qi, 2004). Whether the partial displacement of Mtor in the seh1 background reflects a direct role for Seh1 in recruiting and/or stabilizing Mtor at the NPC remains to be determined (Senger, 2011).

Although dispensable for somatic development, it was found that seh1 has an essential function in the female germline during oogenesis. seh1null females are nearly sterile, producing only a small number of adult progeny. Indeed, seh1null females lay fewer eggs and contain ovarioles with a diminished number of egg chambers relative to wild-type females. A possible contributory factor to this reduced egg production is the mitotic delay observed during the ovarian cyst divisions. It was found that in ovaries from seh1 females, stem cells and ovarian cysts in R1 of the germarium spend a greater proportion of their time in mitosis than those in wild-type females. This phenotype is consistent with the metaphase delay observed in mammalian cells and Xenopus egg extracts depleted of members of the Nup107-160 complex, including Seh1. Thus, mutations in seh1 alter the rate of egg chamber production, as well as the nature of the ovarian cyst divisions in the germarium (Senger, 2011).

In addition to affecting the overall rate of egg production, Seh1 influences the differentiation of the oocyte within the ovarian cyst. Similar to what is observed in mio mutants, in a fraction of seh1 ovarian cysts the oocyte enters the endocycle and develops as a pseudo-nurse cell. This does not reflect an inability ofseh1null oocytes to enter the meiotic cycle. On the contrary, seh1null ovarian cysts enter the meiotic cycle on schedule with the two pro-oocytes progressing to pachytene, as measured by the construction of a mature synaptonemal complex. However, soon after exiting the germarium, a fraction of seh1 mutant oocytes enter the endocycle and become polyploid. In Drosophila, oocyte differentiation, as well as the maintenance of the meiotic cycle, are contingent on the microtubule-based transport of mRNAs and proteins from the nurse cells to the oocyte (Huynh, 2004). The germline-specific RNA-binding protein Orb starts to accumulate in the oocyte in late R2a of the germarium. Defects that impair the microtubule-dependent accumulation of Orb in the oocyte correlate with the inability to maintain the meiotic cycle through later stages of oogenesis. This study found that in seh1 ovarian cysts, the specific accumulation of Orb in the oocyte, as well as the secondary migration of Orb protein from the anterior to the posterior of the oocyte, are often delayed and/or otherwise defective. Additionally, the microtubule-dependent translocation of centrioles from the anterior to the posterior of the oocyte nucleus in the stage 1 oocyte is defective in both mio and seh1 ovarian cysts. Thus, as is observed with mio, seh1 influences the ability of the oocyte to maintain the meiotic cycle and oocyte fate beyond the germarium (Senger, 2011).

As Seh1 has been implicated in a variety of cellular functions, there are several possibilities as to how it might influence oocyte development and meiotic progression. First, Seh1 might act at the NPC to regulate the nucleocytoplasmic transport of specific molecules required for oocyte differentiation and growth. Second, Seh1 might regulate the activity of Mtor and/or other nucleoporins that have recently been implicated in transcriptional regulation (Vaquerizas, 2010). Finally, consistent with the alterations in mitotic cyst division and Orb localization, Seh1 might directly influence the organization and/or function of microtubules within ovarian cysts. Currently, the third model is favored because it is the most congruent with previous observations on the role of Seh1 in other organisms as well as with the current data (Senger, 2011).

This study has shown that Mio and Seh1 are present in a stable complex and that both proteins are dispensable for somatic development but are required for the development of the mature egg. Additionally, it was found that Seh1 is required for Mio protein stability. In the seh1 mutant background, Mio protein levels are reduced dramatically. Furthermore, depleting seh1 via RNAi in S2 tissue culture cells results in a rapid reduction in Mio protein levels. These results suggest the following simple model. Seh1 influences oocyte growth and the maintenance of the oocyte fate through its ability to promote the stability of the Mio protein. In the absence of Seh1, Mio protein levels fall, resulting in a mio-like phenotype. However, two lines of evidence suggest that Mio and Seh1 have a more complex interaction. First, overexpressing mio in the seh1 mutant background fails to rescue the seh1 phenotype. This failure to rescue is observed even though the seh1 15; UAS-mio ovaries have high levels of Mio protein in the germline. This strongly suggests that the seh1 ovarian phenotype is not due solely to the instability of the Mio protein in the absence of Seh1. Second, seh1 acts as a strong dominant suppressor of the mio 16-nurse-cell phenotype. A possible model to explain this counterintuitive result is that mio and seh1 act in opposing directions to regulate a common pathway that is crucial for the maintenance of the oocyte fate. Misregulation of this common pathway by either mio or seh1 could result in the reversion of the oocyte to the default state of nurse cell (Senger, 2011).

In the future, studies of Mio and Seh1 will help elucidate the pathways that drive oocyte development and meiotic progression and contribute to understanding of how individual NPC components drive tissue-specific differentiation (Senger, 2011).

The GATOR2 component Wdr24 regulates TORC1 activity and lysosome function

TORC1 (see Drosophila Tor) is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. The genome of Drosophila contains a single Sea2/Wdr24 homolog encoded by the gene CG7609 that shares 25% identity and 44% similarity to yeast Sea2 and 37% identity and 54% similarity to the human homolog WDR24. This study defines the in vivo role of the GATOR2 component Wdr24 in Drosophila. Wdr24 was shown to have both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, it was shown that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, it was found that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. Wdr24 was also shown to promotes lysosome acidification and autophagic flux in mammalian cells. Taken together these data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes (Cai, 2016).

In metazoans multiple conserved signaling pathways control the integration of metabolic and developmental processes. TORC1 is an evolutionarily conserved multi-protein complex that regulates metabolism and cell growth in response to an array of upstream inputs including nutrient availability, growth factors and intracellular energy levels. The catalytic component of TORC1 is the serine/threonine kinase Target of Rapamycin (TOR). When nutrients are abundant, TORC1 activity promotes translation, ribosome biogenesis as well as other pathways associated with anabolic metabolism and cell growth. However, when nutrients or other upstream activators are limiting, TORC1 activity is inhibited triggering catabolic metabolism and autophagy (Cai, 2016).

The Seh1 associated/GTPase-activating protein toward Rags (SEA/GATOR) complex is a newly identified upstream regulator of TORC1 that can be divided into two putative sub-complexes GATOR1 and GATOR2 (Bar-Peled, 2013; Dokudovskaya, 2011; Panchaud, 2013). The GATOR1 complex, known as the Iml1 complex or the Seh1 Associated Complex Inhibits TORC1 (SEACIT) in yeast, inhibits TORC1 activity in response to amino acid limitation. SEACIT/GATOR1 contains three proteins Npr2/Nprl2, Npr3/Nprl3 and Iml1/DEPDC5. Recent evidence, from yeast and mammals, indicates that the components of the SEACIT/GATOR1 complex function through the Rag GTPases to inhibit TORC1 activity. Notably, Nprl2 and DEPDC5 are tumor suppressor genes while mutations in DEPDC5 are a leading cause of hereditary focal epilepsies (Cai, 2016).

The GATOR2 complex, which is referred to as Sevh1 Associated Complex Activates TORC1 (SEACAT) in yeast, activates TORC1 by opposing the activity of GATOR1. The SEACAT/GATOR2 complex is comprised of five proteins, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59. Computational analysis indicates that multiple components of the GATOR2 complex have structural features characteristic of coatomer proteins and membrane tethering complexes. In line with the structural similarity to proteins that influence membrane dynamics, in Drosophila the GATOR2 subunits Mio and Seh1 localize to multiple endomembrane compartments including lysosomes, the site of TORC1 regulation, and autolysosomes. In metazoans, members of the Sestrin and Castor family of proteins bind to and inhibit the GATOR2 complex in response to leucine and arginine starvation respectively. This interaction is proposed to inhibit TORC1 activity through the derepression of the GATOR1 complex. However, how GATOR2 opposes GATOR1 activity, thus allowing for the robust activation of TORC1, remains unknown. Additionally, the role of the GATOR2 complex in the regulation of both the development and physiology of multicellular animals remains poorly defined (Cai, 2016).

Recent evidence from Drosophila indicates that the requirement for the GATOR2 complex may be context specific in multicellular animals. In Drosophila, null alleles of the GATOR2 components mio and seh1 are viable but female sterile. Surprisingly, somatic tissues from mio and seh1 mutants exhibit little if any reductions in cell size and have nearly normal levels of TORC1 activity. In contrast, TORC1 activity is dramatically decreased in ovaries from mio and seh1 mutant females. This decrease in TORC1 activity is accompanied by the activation of catabolic metabolism in the female germ line, a dramatic reduction in egg chamber growth and difficulties maintaining the meiotic cycle. Thus, there is a surprising tissue specific requirement for the GATOR2 components Mio and Seh1 during oogenesis. However, the in vivo role of the other members of the GATOR2 complex in the regulation of cellular metabolism remains undefined (Cai, 2016).

This study defines the in vivo requirement for the GATOR2 component Wdr24 in Drosophila. Wdr24 was found to have two distinct functions. First, Wdr24 is a critical effector of the GATOR2 complex that promotes TORC1 activity and cellular growth in a broad array of tissues. Second, Wdr24 is required for the TORC1 independent regulation of lysosome function and autophagic flux. Notably, two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. Taken together these data support the model that multiple components of the GATOR2 complex have both TORC1 dependent and independent roles in the regulation of cellular metabolism (Cai, 2016).

This study describes a dual role for the GATOR2 component Wdr24 in the regulation of TORC1 activity and lysosome dynamics. Wdr24 is a critical effector of the GATOR2 complex that promotes TORC1 activity in both germline and somatic tissues. This lies in contrast to the GATOR2 components Mio and Seh1, which have a limited role in the regulation of TORC1 activity in many cell types. Surprisingly, a second function of Wdr24 was identified that is independent of TORC1 status, the regulation of lysosome acidification and autophagic flux. Taken together these data support the model that the GATOR2 complex regulates both the response to amino acid starvation and lysosome function (Cai, 2016).

Whole animal studies often reveal tissue-specific and/or metabolic requirements for genes that are not readily observed in cell culture. In mammalian and Drosophila tissue culture cells, RNAi based depletions of the GATOR2 components Mio, Seh1, Wdr59, and Wdr24 result in decreased TORC1 activity in return to growth assays (Bar-Peled, 2013, Wei, 2014). These data have resulted in the model that all components of the GATOR2 complex are generally required for TORC1 activation (Wei, 2014). However, the characterization of mio and seh1 null mutants in Drosophila, demonstrated that Mio and Seh1 are critical for the activation of TORC1 and inhibition of autophagy in the female germ line, but play a relatively small role in the regulation of TORC1 activity and autophagy in somatic tissues under standard culture conditions. Thus, the requirement for at least a subset of GATOR2 complex components is tissue and/or context specific (Cai, 2016).

This study reports that the GATOR2 component Wdr24 is required for the full activation of TORC1 in both germline and somatic cells of Drosophila. Consistent with the global down-regulation of TORC1 activity in the absence of Wdr24, wdr24 mutant adults are notably smaller than controls and are female sterile. Depleting the GATOR1 components nprl2 and nprl3 in the wdr24 mutant background rescued the low TORC1 activity, growth defects, and female sterility of wdr24 mutants. Thus, the GATOR2 component Wdr24 is required to oppose GATOR1 activity in both germline and somatic cells of Drosophila. From these results it is proposed that Wdr24 is a key effector of the GATOR2 complex required for the full activation of TORC1 in most cell types (Cai, 2016).

There are several potential models to explain the differential requirement for individual GATOR2 proteins in Drosophila. First, there may be tissue specific requirements for individual GATOR2 subunits. In this model the different phenotypes observed in the seh1 and mio versus wdr24mutants reflects a qualitative difference in the requirement for these proteins in different tissues. However, an alternative model is favored in which Wdr24 is the core effector of GATOR2 activity, with Mio and Seh1 functioning primarily as positive regulators of GATOR2 activity. In this second model, the differential phenotypes observed in the seh1 and mio versus wdr24 mutants reflects a quantitative difference in the requirement for GATOR2 activity in different tissues. The distinction between these two models awaits the identification of the molecular mechanism of Wdr24 and GATOR2 action (Cai, 2016).

A novel TORC1 independent role has been identified for Wdr24 in the regulation of lysosome dynamics and function. In wdr24 mutants, the down-regulation of TORC1 activity and the accumulation of autolysosomes occur independent of nutrient status. It was initially hypothesized that in the absence of the GATOR2 component Wdr24, the deregulation of the GATOR1 complex results in low TORC1 activity, triggering the constitutive activation of autophagy and the accumulation of autolysosomes. Surprisingly, however, epistasis analysis determined that the accumulation of lysosomes could be decoupled from both the chronic inhibition of TORC1 activity and the activation of autophagy. Raising TORC1 activity in the wrd24 mutant background, by depleting either components of the GATOR1 or TSC complex, failed to rescue the accumulation of abnormal lysosomal structures. Notably, it was determined that two additional members of the GATOR2 complex, Mio and Seh1, also regulate lysosomal behavior independent of both GATOR1 and the down-regulation of TORC1 activity. From these data it is inferred that multiple components of the GATOR2 complex have a TORC1 independent role in the regulation of lysosomes (Cai, 2016).

An increased number of autolysosomes is often associated with reduced autophagic flux due to diminished lysosomal degradation. Consistent with reduced autophagic flux, in Drosophila wrd24-/- mutants accumulated enlarged autolysosomes filled with undegraded material. Moreover, lysosomes in the wrd24-/- mutants failed to quench the GFP fluorescence of a GFP-mCherry-Atg8a protein. These phenotypes are consistent with decreased lysosomal pH and degradative capacity. In order to examine in detail the role of Wdr24 in the regulation of lysosome function a wrd24-/- knockout HeLa cell line was generated that recapitulated the phenotypes observed in Drosophila wrd24-/- mutants. Specifically, wrd24-/- HeLa cells had have decreased TORC1 activity and accumulate a large number of autolysosomes. Using multiple assays it was determined that wrd24-/- lysosomes had reduced degradative capacity and autophagic flux and thus accumulate proteins that are normally degraded by lysosomal enzymes such as p62, LC3II and Cathepsin D. Additionally, it was determined that wrd24-/-lysosomes have increased pH relative to wild-type cells, again consistent with reduced lysosomal function. Taken together these data confirm that Wdr24 plays a key role in the regulation of lysosomal activity (Cai, 2016).

This study shows that components of the GATOR2 complex function in the regulation of TORC1 activity and in the TORC1 independent regulation of lysosomal dynamics and autophagic flux. These two functions suggest that the GATOR2 complex may regulate cellular homeostasis by coordinating TORC1 activity with the dynamic regulation of lysosomes during periods of nutrient stress. Intriguingly, several recent reports describe a very similar dual function for the RagA/B GTPases in both mice and zebrafish (Kim, 2014; Shen,2016). RagA/B play a critical role in the activation of TORC1 in the presence of amino acids (Kim, 2008; Sancak, 2008). Surprisingly, however, TORC1 activity was not found to be significantly decreased in cardiomyocytes of RagA/B knockout mice (Cai, 2016).

Nevertheless, the RagA/B mutant cardiomyocytes have decreased autophagic flux and reduced lysosome acidification. From published data, it was concluded that the RagA/B GTPases regulate lysosomal function independent of their role in the regulation of TORC1 activation in some cell types. Similarly, RagA is required for proper lysosome function and phagocytic flux in microglia. Notably, Mio, a component of the GATOR2 complex is found associated with RagA (Bar-Peled, 2013). Thus, in the future it will be important to determine if components of the GATOR2 complex function in a common pathway with the Rag GTPases to regulate lysosomal function (Cai, 2016).

In Saccharomyces cerevisiae single mutants of wrd24/sea2/ and wdr59/sea3 do not exhibit defects in TORC1 regulation but do have defects in vacuolar structure. Moreover, several recently identified genes that regulate the GATOR2-GATOR1-TORC1 pathway in response to amino acid limitation are restricted to metazoans. These data make it tempting to speculate that the ancestral function of the GATOR2 complex maybe the regulation of lysosome/vacuole function and autophagic flux. Indeed, the finding that GATOR2 components regulate lysosome dynamics is particularly intriguing in light of the observation that GATOR2 complex is comprised of proteins with characteristics of coatomer proteins and membrane tethering complexes. Notably, the GATOR2 complex components Mio, Seh1 and Wdr24 localize to lysosomes and autolysosomes. Similarly, these proteins associate with the vacuolar membrane in budding yeast. Thus, going forward it will be important to examine if the GATOR2 complex acts directly on lysosomal membranes to regulate their structure and/or function. More broadly, future studies on the diverse roles of the SEACAT/GATOR2 complex will further understanding of the complex relationship between cellular metabolism and the regulation of endomembrane dynamics in both development and disease (Cai, 2016).

missing oocyte encodes a highly conserved nuclear protein required for the maintenance of the meiotic cycle and oocyte identity in Drosophila

In Drosophila, a single oocyte develops within a 16-cell germline cyst. Although all 16 cells initiate meiosis and undergo premeiotic S phase, only the oocyte retains its meiotic chromosome configuration and remains in the meiotic cycle. The other 15 cells in the cyst enter the endocycle and develop as polyploid nurse cells. A longstanding goal in the field has been to identify factors that are concentrated or activated in the oocyte, that promote meiotic progression and/or the establishment of the oocyte identity. This study presents the characterization of the missing oocyte gene, an excellent candidate for a gene directly involved in the differentiation of the oocyte nucleus. The missing oocyte gene encodes a highly conserved protein that preferentially accumulates in pro-oocyte nuclei in early prophase of meiosis I. In missing oocyte mutants, the oocyte enters the endocycle and develops as a polyploid nurse cell. Genetic interaction studies indicate that missing oocyte influences meiotic progression prior to pachytene and may interact with pathways that control DNA metabolism. These data strongly suggest that the product of the missing oocyte gene acts in the oocyte nucleus to facilitate the execution of the unique cell cycle and developmental programs that produce the mature haploid gamete (Iida, 2004; PubMed).


REFERENCES

Search PubMed for articles about Drosophila Missing oocyte

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Bar-Peled, L., Chantranupong, L., Cherniack, A. D., Chen, W. W., Ottina, K. A., Grabiner, B. C., Spear, E. D., Carter, S. L., Meyerson, M. and Sabatini, D. M. (2013). A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340: 1100-1106. PubMed ID: 23723238

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Dokudovskaya, S., Waharte, F., Schlessinger, A., Pieper, U., Devos, D. P., Cristea, I. M., Williams, R., Salamero, J., Chait, B. T., Sali, A., Field, M. C., Rout, M. P. and Dargemont, C. (2011). A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae. Mol Cell Proteomics 10: M110 006478. PubMed ID: 21454883

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Huynh, J. R. and St Johnston, D. (2004). The origin of asymmetry: early polarisation of the Drosophila germline cyst and oocyte. Curr Biol 14: R438-449. PubMed ID: 15182695

Iida, T. and Lilly, M. A. (2004). missing oocyte encodes a highly conserved nuclear protein required for the maintenance of the meiotic cycle and oocyte identity in Drosophila. Development 131: 1029-1039. PubMed ID: 14973288

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Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. and Guan, K. L. (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 10: 935-945. PubMed ID: 18604198

Kim, Y. C., Park, H. W., Sciarretta, S., Mo, J. S., Jewell, J. L., Russell, R. C., Wu, X., Sadoshima, J. and Guan, K. L. (2014). Rag GTPases are cardioprotective by regulating lysosomal function. Nat Commun 5: 4241. PubMed ID: 24980141

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Mishra, R. K., et al. (2010). The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores. Nat. Cell Biol. 12: 164-169. PubMed ID: 20081840

Orjalo, A. V., et al. (2006). The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly. Mol. Biol. Cell 17: 3806-3818. PubMed ID: 16807356

Panchaud, N., Peli-Gulli, M. P. and De Virgilio, C. (2013). Amino acid deprivation inhibits TORC1 through a GTPase-activating protein complex for the Rag family GTPase Gtr1. Sci Signal 6: ra42. PubMed ID: 23716719

Platani, M., et al. (2009). The Nup107-160 nucleoporin complex promotes mitotic events via control of the localization state of the chromosome passenger complex. Mol. Biol. Cell 20, 5260-5275. PubMed ID: 16807356

Qi, H., et al. (2004). Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila. Mol. Biol. Cell 15: 4854-4865. PubMed ID: 15356261

Sancak, Y., Peterson, T. R., Shaul, Y. D., Lindquist, R. A., Thoreen, C. C., Bar-Peled, L. and Sabatini, D. M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320: 1496-1501. PubMed ID: 18497260

Senger, S., Csokmay, J., Akbar, T., Jones, T. I., Sengupta, P. and Lilly, M. A. (2011).The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis. Development 138(10): 2133-42. PubMed ID: 21521741

Shen, K., Sidik, H. and Talbot, W. S. (2016). The Rag-Ragulator complex regulates lysosome function and phagocytic flux in microglia. Cell Rep 14: 547-559. PubMed ID: 26774477

Vaquerizas, J. M., et al. (2010). Nuclear pore proteins nup153 and megator define transcriptionally active regions in the Drosophila genome. PLoS Genet. 6: e1000846. PubMed ID: 20174442

Wei, Y. and Lilly, M. A. (2014a). The TORC1 inhibitors Nprl2 and Nprl3 mediate an adaptive response to amino-acid starvation in Drosophila. Cell Death Differ 21: 1460-1468. PubMed ID: 24786828

Wei, Y., Reveal, B., Reich, J., Laursen, W. J., Senger, S., Akbar, T., Iida-Jones, T., Cai, W., Jarnik, M. and Lilly, M. A. (2014b). TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila. Proc Natl Acad Sci U S A 111(52):E5670-7. PubMed ID: 25512509

Wozniak, R., Burke, B. and Doye, V. (2010). Nuclear transport and the mitotic apparatus: an evolving relationship. Cell. Mol. Life Sci. 67: 2215-2230. PubMed ID: 20372967

Zuccolo, M., et al. (2007). The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions. EMBO J. 26: 1853-1864. PubMed ID: 17363900


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date revised: 20 January 2015

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