org Egalitarian

InteractiveFly: GeneBrief

egalitarian: Biological Overview | References

Gene name - egalitarian

Synonyms -

Cytological map position - 59F7-59F7

Function - RNA-binding protein

Keywords - oogenesis, microtubule-based transport of mRNAs

Symbol - egl

FlyBase ID: FBgn0000562

Genetic map position - 2R:19,622,427..19,634,348 [-

Classification - Egl_like_exo

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Sanghavi, P., Liu, G., Veeranan-Karmegam, R., Navarro, C. and Gonsalvez, G. B. (2016). Multiple roles for Egalitarian in polarization of the Drosophila egg chamber. Genetics [Epub ahead of print]. PubMed ID: 27017624
RNA-binding protein Egalitarian (Egl) is required for specification and maintenance of oocyte fate. Mutants in egl either completely fail to specify an oocyte, or if specified, the oocyte eventually reverts back to nurse cell fate. Due to this very early role for Egl in egg chamber maturation, it is unclear whether later stages of egg chamber development also require Egl function. In this study, Egl was depleted at specific stages of egg chamber development. In early stage egg chambers, Egl was shown to have an additional role in organization of oocyte microtubules. In the absence of Egl function, oocyte microtubules completely fail to reorganize. As such, the localization of microtubule motors and their cargo is disrupted. In addition, Egl also appears to function in regulating the translation of critical polarity determining mRNAs. Finally, in mid stage egg chambers, Egl does not appear to be required for microtubule organization, but rather for the correct spatial localization of oskar, bicoid and gurken mRNAs.
Vazquez-Pianzola, P., Schaller, B., Colombo, M., Beuchle, D., Neuenschwander, S., Marcil, A., Bruggmann, R. and Suter, B. (2016). The mRNA transportome of the Bicaudal D/Egalitarian transport machinery. RNA Biol [Epub ahead of print]. PubMed ID: 27801632
Messenger RNA (mRNA) transport focuses the expression of encoded proteins to specific regions within cells providing them with the means to assume specific functions and even identities. Bicaudal D and the mRNA binding protein Egalitarian interact with the microtubule motor dynein to localize mRNAs in Drosophila. Because relatively few mRNA cargos were known, Egl::GFP associated mRNAs were isolated and identified. The top candidates were validated by qPCR, in situ hybridization and genetically by showing that their localization requires BicD. In young embryos these Egl target mRNAs are preferentially localized apically, between the plasma membrane and the blastoderm nuclei, but also in the pole plasm at the posterior pole. Egl targets expressed in the ovary were mostly enriched in the oocyte and some were apically localized in follicle cells. The identification of a large group of novel mRNAs associated with BicD/Egl points to several novel developmental and physiological functions of this dynein dependent localization machinery. The verified dataset also allowed for development of a tool that predicts conserved A'-form-like stem loops that serve as localization elements in 3'UTRs.


Cytoplasmic sorting of mRNAs by microtubule-based transport is widespread, yet very little is known at the molecular level about how specific transcripts are linked to motor complexes. In Drosophila, minus-end-directed transport of developmentally important transcripts by the dynein motor is mediated by seemingly divergent mRNA elements. Evidence is provided that direct recognition of these mRNA localization signals is mediated by the Egalitarian (Egl) protein. Egl and the dynein cofactor Bicaudal-D (BicD) are the only proteins from embryonic extracts that are abundantly and specifically enriched on RNA localization signals from transcripts of gurken, hairy, K10, and the I factor retrotransposon. In vitro assays show that, despite lacking a canonical RNA-binding motif, Egl directly recognizes active localization elements. A physical interaction was revealed between Egl and a conserved domain for cargo recruitment in BicD and data is presented suggesting that Egl participates selectively in BicD-mediated transport of mRNA in vivo. This work leads to the first working model for a complete connection between minus-end-directed mRNA localization signals and microtubules and reveals molecular strategies that are likely to be of general relevance for cargo transport by dynein (Dienstbier, 2009).

Many proteins achieve an asymmetric localization within the cytoplasm through the transport of their mRNAs along the cytoskeleton by molecular motors. Despite the widespread occurrence of mRNA transport, the detailed mechanisms by which specific transcripts are recognized and recruited to motor complexes are poorly understood. One exception is during bud-specific enrichment of mRNAs along actin filaments in the yeast Saccharomyces cerevisiae, where proteins have been identified that can account for a complete link between localizing mRNAs and the cytoskeleton. However, many metazoans rely on microtubules to deliver mRNAs over the requisite longer distances, and mechanistic insights into how these transcripts are linked to motors are relatively sparse (Dienstbier, 2009).

One of the best prospects for elucidating microtubule-based mRNA transport is in the Drosophila syncytial blastoderm embryo, where a pathway for apical localization of a subset of endogenous mRNAs can be accessed by microinjection of in vitro synthesized, fluorescently labeled transcripts. Consistent with the nucleation of the minus ends of the microtubules in the apical cytoplasm, localization of these transcripts is driven by cytoplasmic dynein together with its accessory complex dynactin. Related machinery delivers mRNAs to the minus ends of microtubules in other Drosophila cell types, including oocytes and neuroblasts (Dienstbier, 2009).

The cis-acting RNA elements mediating asymmetric localization by dynein have been studied in detail for seven transcripts (the developmentally important mRNAs bicoid [bcd], fushi tarazu [ftz], gurken [grk], hairy [h], fs(1)K10 [K10], and wingless [wg], and the I Factor retrotransposon RNA) and contain one or more stem-loop structures. These 'localization signals' are necessary for minus-end-directed localization and also sufficient when inserted into heterologous transcripts (Dienstbier, 2009).

The localization signals in the different transcripts do not share significant primary sequence similarity and often have different lengths. This has led to two competing models: the first in which the RNA elements contain cryptic features that associate with a common recognition machinery, and the second in which they are recognized by different proteins, each able to independently provide a link to the dynein complex. It has not been possible to discriminate between these scenarios, because proteins that specifically bind any of these elements and are required for transport have not been identified (Dienstbier, 2009).

In addition to dynein/dynactin, the Egalitarian (Egl) and Bicaudal-D (BicD) proteins are also essential for targeting of mRNAs to the minus ends of microtubules (Mach, 1997; Bullock, 2001; Bullock, 2004; Hughes, 2004; Claussen, 2005). Egl and BicD are found in a complex with each other in vivo (together with other copies of themselves), although it is not known whether they interact directly (Mach, 1997; Oh, 2000; Navarro, 2004). Egl and BicD also associate with dynein light chain (Dlc) and the dynein/dynactin complex, respectively (Hoogenraad, 2001; Navarro, 2004), and are recruited to injected localizing mRNAs in embryos to bias the net movements of a bidirectional mRNA transport complex apically (Bullock, 2001; Bullock, 2006). Together, these observations have led to a model in which Egl and BicD associate with localization signals and increase the frequency of minus-end-directed dynein/dynactin movements (Tekotte, 2002; Bullock, 2006). Because neither Egl nor BicD has a known RNA-binding motif, it has been reasoned that they are recruited to localization signals by intermediary factors that directly contact the message (Pearson, 2004; Bullock, 2006; Dienstbier, 2009 and references therein).

Whether Egl has roles outside of mRNA transport has not been reported, but BicD functions in the transport of a subset of other cargoes for dynein. It has been proposed that the N-terminal two-thirds of mammalian BicD are sufficient for stimulating dynein transport and that the remaining C-terminal sequences (hereafter referred to as the CTD [C-terminal domain]) mediate a link between cargoes and the motor (Hoogenraad, 2003). This is based on the findings that the CTD can be functionally substituted by heterologous motifs for organelle recognition (Hoogenraad, 2003) and can bind Rab6, a membrane-linked GTPase that recruits dynein to Golgi vesicles (Dienstbier, 2009).

This study attempts to elucidate the mechanism of linkage of different mRNA localization signals to dynein. The surprising finding is reported that Egl is a selective RNA-binding protein that directly contacts active localization signals. Thus, seemingly divergent mRNA signals are recognized by the same factor. Egl associates with a conserved domain for cargo recruitment in BicD and is selectively required for mRNA transport in vivo. This work provides unique insights into the molecular links between localizing mRNAs and microtubule-based motors, and also sheds light on general mechanisms of cargo transport by dynein (Dienstbier, 2009).

Because of difficulties in finding shared features between dynein-dependent localization signals in different transcripts, it was not known whether dedicated factors are responsible for recognizing each of these elements. This uncertainty has severely restricted the ability to generalize conclusions from studies of localization mechanisms of individual transcripts. This work demonstrates that the same protein, Egl, is capable of specifically contacting minus-end-directed localization signals from multiple different transcripts. This conclusion is supported by the findings that (1) Egl and BicD are the only factors visibly enriched from embryonic extracts on all four localizing elements tested relative to a number of nonlocalizing controls, (2) Egl function in Drosophila is required for BicD-mediated transport of mRNAs and not other cargoes tested, (3) the majority of Egl, but not BicD, in cell extracts is found in a complex whose size is sensitive to Rnase treatment, and (4) recombinant Egl, but not BicD, binds RNA in vitro and is capable of discriminating between active apical localization signals and those containing subtle inactivating mutations (Dienstbier, 2009).

In addition to the four elements tested in this study, Egl is also likely to associate directly with other mRNA localization signals because bcd, ftz, and wg recruit Egl in vivo and depend on its function for minus-end-directed transport (Dienstbier, 2009; Bullock; 2001; Bullock, 2004). Indeed, Egl binding may be the major, and perhaps only, specific determinant of the activity of an apical localization signal, as all three subtle inactivating mutations that were tested inhibit association of Egl from embryonic extracts (TLSδbub, TLSU6C, and hSL1C15G), and a fourth inactive point mutant (bcdSLV4496G-U) (Macdonald, 1997) prevents recruitment of Egl to bcd injected into embryos (Bullock, 2001). Presumably, despite differences in primary sequence composition, all of the characterized localization elements contain cryptic structural features that are recognized by Egl. Elucidating the structural basis of this recognition event will be the goal of future long-term studies (Dienstbier, 2009).

Interestingly, Egl exhibits some affinity for inactive localization elements when expressed recombinantly, as well as in embryonic extracts. Egl may well exhibit greater selectivity for active signals in the appropriate in vivo context. This could be because the composition of in vitro binding buffers is suboptimal. Alternatively, the incorporation of mRNAs into oligomeric particles within the cell may give rise to cooperative interactions between individual Egl and BicD complexes, thereby increasing cargo specificity. Nonetheless, an inherent degree of promiscuity by Egl in vivo would fit with a previous finding that its overexpression in embryos is sufficient to target a small amount of an endogenous nonlocalizing transcript population to the apical cytoplasm (Bullock, 2006) and could also be the basis of repeated emergence of apical localization signals during dipteran evolution (Dienstbier, 2009).

The mRNA elements that direct apical transport in the blastoderm embryo are also capable of mediating localization of transcripts toward the minus ends of microtubules during oogenesis (Bullock, 2001; Cohen, 2005; Van De Bor, 2005). It is therefore very likely that direct binding of Egl to these stem-loops is also functionally significant during these stages. Indeed, Egl and BicD have been shown to be components of motor complexes that transport grk from the nurse cells into the oocyte (Clark, 2007). Interestingly, within the oocyte the h and K10 elements are involved in localization to the anterior cortex, whereas those in grk and the I factor are also sufficient for translocation from the anterior to the dorso-anterior corner. Dorsalward movement is presumably due to the binding of the ILS and GLS to oocyte-specific factors in addition to Egl, either sequentially or simultaneously, or by modulating the mode of Egl binding (Dienstbier, 2009).

It has been shown that Egl and BicD are in a complex together in vivo (Mach, 1997). The current data shows for the first time that Egl, through its N-terminal 79 amino acids, directly interacts with BicD. In addition, Egl also binds Dlc through a consensus light chain-binding site between amino acids 963 and 969 (Navarro, 2004). BicD is able to recruit the dynein/dynactin complex (Hoogenraad, 2003) and Dlc associates with other dynein subunits. Thus, together with evidence for Egl RNA binding through amino acids 1-814, it is now possible to build a working model of a complete link between minus-end-directed mRNA signals and microtubules for the first time (Dienstbier, 2009).

Egl, BicD, and mRNA elements do not appear to be obligatory for particle assembly or bidirectional mRNA motility (Bullock, 2006). Instead, they are likely to be essential parts of a cassette that up-regulates minus-end-directed movement of a generic bidirectional mRNA transport complex. Other RNA-binding factors presumably package both localizing and nonlocalizing RNAs and provide additional links to motors (Dienstbier, 2009).

Within the minus-end regulatory cassette, the role of Egl is probably to recruit both BicD and Dlc to the mRNA to ensure efficient targeting of transcripts to the minus ends of microtubules. The presence of both Egl-interacting partners might be required for the stability of the motor complex. Alternatively, previous observations of the effects of altering protein concentrations on mRNA transport are consistent with Egl-Dlc and Egl-BicD interactions regulating different aspects of motility of the bidirectional motor complex: processivity and switching behavior, respectively (Bullock, 2006). Like Egl, Rab6 is able to associate with both BicD and a Dlc. Association with both BicD and Dlc may therefore be a common strategy used by cargo adaptors to ensure efficient minus-end-directed transport (Dienstbier, 2009).

Binding of both Egl and Rab6 to BicD is sensitive to the same amino acid substitution in the CTD. Egl and Rab6 recognize localizing mRNAs and Golgi vesicles, respectively, raising the possibility that BicD functions in the transport of different cargoes through mutually exclusive association of the CTD with cargo-specific adaptors. It was found that relatively subtle overexpression of Egl not only augments BicD-dependent apical mRNA transport (Bullock, 2006), but also antagonizes BicD function in lipid droplet motility. This implies that, through competition for the BicD CTD, the pathways for microtubule-based transport of different cargoes can be finely balanced. Alteration of the availability of adaptors for BicD is therefore a potentially effective strategy for regulating net sorting of cargoes (Dienstbier, 2009).

Experiments involving the tethering of cargoes to BicD domains also shed light on potential general mechanisms of dynein-based cargo transport. As is the case for mammalian BicD (Hoogenraad, 2003), removal of the CTD of the Drosophila protein stimulates transport by dynein. This situation presumably mimics a version of the full-length protein bound to a cargo adaptor in which an autoinhibitory effect of the C terminus is negated (Hoogenraad, 2003). The N terminus of BicD can efficiently capture dynein/dynactin components from cell extracts (Hoogenraad, 2003), suggesting that this interaction could be entirely sufficient for productive transport. However, the results indicate that, at least in Drosophila, the capacity of BicDδC to mediate net movement of tethered cargoes is dependent on its association with endogenous BicD transport complexes. Such a scenario was not directly tested in the previous mammalian cell assays (Dienstbier, 2009).

In the case of minus-end-directed mRNA transport in flies, the CTD appears to provide an essential link, through Egl, to Dlc. In addition, the CTD can associate with the dynamitin subunit of dynactin (Hoogenraad, 2001). The significance of this interaction was not clear in light of a model in which only the N-terminal sequences of BicD are important for transport by dynein. The finding that the CTD is needed in trans for the activity of BicDδC revives the possibility that the dynamitin interaction is functionally important (Dienstbier, 2009).

The ability of BicDδC::CP, but not BicD::CP, to target heterologous cargoes apically is likely to reflect a role for the CTD in inhibiting association with other copies of BicD. Consistent with this notion, BicDδC::CP accumulates in large, apically enriched puncta, whereas the full-length protein fused to the coat protein fails to form discrete particles and has a uniform distribution. Together with the observation that BicD is able to associate with other copies of itself in vivo, these results imply that dimerization or oligomerization of BicD could be an important step in the activation of transport by cargo binding. Future experiments will be aimed at determining the copy number of components of the transport complex in the presence and absence of a bound consignment (Dienstbier, 2009).

A targeted gain-of-function screen identifies genes affecting salivary gland morphogenesis/tubulogenesis in Drosophila

During development individual cells in tissues undergo complex cell-shape changes to drive the morphogenetic movements required to form tissues. Cell shape is determined by the cytoskeleton and cell-shape changes critically depend on a tight spatial and temporal control of cytoskeletal behavior. The formation of the salivary glands in the Drosophila embryo, a process of tubulogenesis, was used as an assay for identifying factors that impinge on cell shape and the cytoskeleton. To this end a gain-of-function screen was performed in the salivary glands, using a collection of fly lines carrying EP-element insertions that allow the overexpression of downstream-located genes using the UAS-Gal4 system. A salivary-gland-specific fork head-Gal4 line was used to restrict expression to the salivary glands, in combination with reporters of cell shape and the cytoskeleton. A number of genes known to affect salivary gland formation was identified, confirming the effectiveness of the screen. In addition, many genes not implicated previously in this process were found, some having known functions in other tissues. This paper reports the initial characterization of a subset of genes, including chickadee, rhomboid1, egalitarian, bitesize, and capricious, through comparison of gain- and loss-of-function phenotypes (Maybeck, 2009. Full text of article).

Egalitarian (Egl) and BicaudalD (BicD) are two proteins that act together with cytoplasmic Dynein in the localization of mRNAs in Drosophila embryos and the oocyte, with Egl interacting directly with Dynein light chain. Overexpression of egl using EP(2)938 led to salivary glands that were C-shaped or shortened. Shortened glands could also be observed when egl was expressed in the glands using a UAS-egl construct. GFP-positive cells that appeared to lose contact with the gland could be observed. Because both BicD and Egl have essential functions during oogenesis, an analysis of egl or BicD null embryos is not possible. Therefore, embryos from mothers carrying two hypomorphic alleles of egl that were mated to heterozygous fathers were examined. Embryos with reduced Egl function often showed a disrupted epidermis, with large patches that appeared to completely lack apical Crumbs labeling. This phenotype was variable, however. Also, during later stages of invagination at stage 13, the placode area was often disrupted and lacked apical Crumbs. Salivary gland morphogenesis was disrupted in egl mutant embryos in that the cells of the placode often did not change their apices in a coordinated way (although Crumbs still accumulated apically in the placode), the invagination hole appeared too large and extended, and the invaginated portion of the glands often had an irregular shape. In the invaginated portion of a gland, Crumbs was not concentrated near the apical cell junctions as in the wild type, where this accumulation appears as a honeycomb lattice. Instead, Crumbs was delocalized all over the apical surface and large accumulations could also be found intracellularly (Maybeck, 2009).

What could be the mechanism leading to a loss of apical surface identity or constituents? Egl and BicD together with Dynein act as minus-end-directed microtubule motors, and as in most epithelial cells, the minus ends of microtubules are located near the apical surface in the salivary glands. hairy mRNA is one of the best-understood cargoes of Egl- and BicD-mediated transport (Bullock 2003; Bullock, 2006), and Hairy has been shown to be important for the regulation of apical membrane growth during salivary gland formation, in part through modulation of Crumbs. Thus, affecting hairy transcript localization through lowered levels of Egl and BicD could in turn affect the maintenance of apical membrane identity in the secretory cells. Alternatively, recent reports have shown that crumbs mRNA itself, and also the RNA of the Crumbs-associated protein Stardust (Std), are apically localized and this apical localization is important for function. Thus, if crumbs mRNA localization were dependent upon Egl and BicD, then reduction of Egl and BicD would result in a loss of functional Crumbs at the apical surface, leading to a loss of epithelial characteristics. The apical localization at least of stardust mRNA appears developmentally regulated in the embryo. Thus, it is possible to envision that salivary gland cell apical maintenance is Egl and BicD dependent and especially sensitive to levels of Egl and thus to Crumbs in comparison to other epithelial tissues at the same stage (Maybeck, 2009).

A Dynein-dependent shortcut rapidly delivers axis determination transcripts into the Drosophila oocyte

The primary axes of Drosophila are set up by the localization of transcripts within the oocyte. These mRNAs originate in the nurse cells, but how they move into the oocyte remains poorly understood. This study investigates the path and mechanism of movement of gurken RNA within the nurse cells and towards and through ring canals connecting them to the oocyte. gurken transcripts, but not control transcripts, recruit the cytoplasmic Dynein-associated co-factors Bicaudal D (BicD) and Egalitarian in the nurse cells. gurken RNA requires BicD and Dynein for its transport towards the ring canals, where it accumulates before moving into the oocyte. The results suggest that bicoid and oskar transcripts are also delivered to the oocyte by the same mechanism, which is distinct from cytoplasmic flow. It is proposed that Dynein-mediated transport of specific RNAs along specialized networks of microtubules increases the efficiency of their delivery, over the flow of general cytoplasmic components, into the oocyte (Clark, 2007).

Within nurse cells, a new path of Dynein-dependent transport to the ring canals has been identified that links the nurse cells to the oocyte. grk RNA moves along this route, and the data suggest that bcd and osk transcripts also follow the same path. This intracellular shortcut requires BicD and is distinct from the route taken by general cytoplasmic components and control RNAs, which move into the oocyte less effectively. The data suggest that the distinction between RNA components that follow this direct path and those that do not is the ability to recruit the Dynein-associated co-factors BicD and Egl. It is proposed that Dynein-dependent transport of grk, bcd and osk transcripts towards the ring canals follows a MT network, which is distinct from other networks in the nurse cells (Clark, 2007).

It was not possible to determine the proportion of grk RNA particles that move compared with ones that were stationary because it is hard to distinguish stationary particles from autofluorescence. By contrast, rapidly moving RNA particles of the same intensity are easy to distinguish from background. By showing directly that Dynein is required for the transport of axis specification transcripts from the nurse cells to the oocyte, this work explains previous work on this topic that did not directly address the mechanism of transport from the nurse cells into the oocyte. The results also explain why the movement of bcd and osk mRNA into the oocyte is MT dependent, and why pair-rule transcripts, which are transported in the blastoderm embryo in a MT-dependent manner, by Dynein, are also transported into the oocyte when exogenously expressed in the nurse cells. It is suggested that nurse cell-to-oocyte transport is likely to be a fairly promiscuous transport system that can deliver any transcript that has the capacity for transport by the Dynein motor complex along MTs to their minus ends. It is therefore likely that the Dynein-dependent shortcut is deployed by many other transcripts that are localized in the oocyte during mid-oogenesis, such as orb, K10 and nanos (nos). In fact, given that the oocyte nucleus is largely transcriptionally inactive, it is possible that up to 10% of all transcripts thought to be localized in the oocyte could first be transported by the same Dynein-dependent mechanism into the oocyte (Clark, 2007).

The Dynein-dependent transport route uncovered within the nurse cells is likely to allow transcripts encoding axis specification determinants to be delivered rapidly at key times in oogenesis. In particular, cytoplasmic transport during stages 5-8 is likely to be relatively slow and non-specific, so delivery of transcripts from the nurse cell nuclei to the oocyte cytoplasm is likely to be very slow, if it involves an undirected diffusion-based process. Certainly, osk and bcd mRNA and other transcripts are thought to form large multimeric complexes in the nurse cells, so are unlikely to be easily dispersed within the cytoplasm by free diffusion. osk and grk are transported into the oocyte at the same stages of oogenesis, and both require Bruno and Hrp48 (also known as Hrb27C - FlyBase); however, it is unclear whether they are transported within the same complexes into the oocyte. At stage 10B, the mechanism this paper has described is not required, because the rapid dumping of all of the cytoplasmic contents of the nurse cells into the oocyte occurs. However, by stage 10B, most of the major patterning transcripts have probably been localized in the oocyte (Clark, 2007).

This work does not address directly the speed of passive diffusion of RNA into the oocyte or the mechanism of cytoplasmic flow and dumping in stage 10B. Although Kinesin 1 is required for cytoplasmic movements within the oocyte, it is not required for the general growth of the oocyte or for the presence of mRNAs in the oocyte. These observations suggest that Kinesin 1 is not important for cytoplasmic transport or for specific mRNA transport into the oocyte (Clark, 2007).

The existence of a specific intracellular route for the transport of transcripts in nurse cells adds to existing evidence that there are various minus-end destinations to which different cargos are delivered by Dynein within the same cell. For example, within the oocyte, bcd RNA is transported to the cortex if injected into the oocyte, but to the anterior, after transport into the oocyte, following injection into the nurse cells. grk RNA is transported in two steps, both of which depend on Dynein. The second step is towards the oocyte nucleus, and is unique to grk and I factor RNA, but is not shared with bcd and K10 transcripts, despite the fact that all of these transcripts are probably being transported by Dynein. There are, therefore, likely to be several distinct MT routes along which Dynein can transport cargos within egg chambers. In neurons, choices between distinct MT routes are made by Kinesin-dependent vesicle transport depending on the presence of a specific neurotransmitter-receptor-interacting protein, GRIP1. How Dynein chooses between distinct MT networks is less clear, but could be based on distinct isoforms of the motor complex, on distinct kinds of MTs with different tubulin isoforms, or on their decoration with different MT-associated proteins. In addition, there is evidence that cargos can influence the behaviour of their motor, raising the interesting possibility that cargos could also influence the choice of MT route adopted by their motors. This work suggests that the presence of BicD and Egl could also influence the choice of MT route adopted by motors. Future work, including new approaches for co-visualizing MTs and RNAs in living cells, will be required to distinguish between all of these possible ways of selecting intracellular routes. Whatever the basis of such distinct routes, they are likely to exist for various kinds of molecular motors and to be functionally important for a wide range of tissues and cargos (Clark, 2007).

Guidance of bidirectional motor complexes by mRNA cargoes through control of dynein number and activity

During asymmetric cytoplasmic mRNA transport, cis-acting localization signals are widely assumed to tether a specific subset of transcripts to motor complexes that have intrinsic directionality. This study provides evidence that mRNA transcripts control their sorting by regulating the relative activities of opposing motors on microtubules. In Drosophila embryos it is shown that all mRNAs undergo bidirectional transport on microtubules and that cis-acting elements produce a range of polarized transcript distributions by regulating the frequency, velocity, and duration of minus-end-directed runs. Increased minus-end motility is dependent on the dosage of RNA elements and the proteins Egalitarian (Egl) and Bicaudal-D (BicD). These proteins, together with the dynein motor, are recruited differentially to different RNA signals. Cytoplasmic transfer experiments reveal that, once assembled, cargo/motor complexes are insensitive to reduced cytoplasmic levels of transport proteins. Thus, the concentration of these proteins is only critical at the onset of transport. This work suggests that the architecture of RNA elements, through Egl and BicD, regulates directional transport by controlling the relative numbers of opposite polarity motors assembled. The data raise the possibility that recruitment of different numbers of motors and regulatory proteins is a general strategy by which microtubule-based cargoes control their sorting (Bullock, 2006).

mRNA localization signals modulate the kinetics of microtubule-based transcript movements. In mammalian cells, uniformly distributed mRNAs undergo short, unidirectional transport events that are augmented in duration and frequency by the RNA signal from the localizing β-actin transcript. In Drosophila syncytial blastoderm embryos, individual nucleotide changes in the hairy (h) transcript slow the rate of delivery of injected fluorescently labeled mRNAs to the apical cytoplasm. However, it is unclear whether localization elements simply limit dissociation of mRNAs from their RNA binding protein(s), and hence the microtubule, or actively regulate motor movement (Bullock, 2006).

To investigate whether mRNA cargoes regulate the movement of motors on microtubules, improved microscopy and automatic tracking software has been used to analyze the detailed movements of mRNAs in the Drosophila blastoderm. Here, the microtubules have a stereotypical arrangement, with minus ends nucleated apically and plus ends extending basally (Bullock, 2006).

Particles of injected, fluorescently labeled h mRNA are transported bidirectionally, undergoing relatively long runs in the minus-end direction interspersed with shorter reversals (plus-end runs), as well as periods of little or no persistent movement (pauses). The net rate of apical transport of the h mRNA particles is ~150 nm/s, with active transport in both directions reaching velocities of up to ~1-1.5 μm/s (Bullock, 2006).

The characteristics of mRNA motion are reminiscent of those of other bidirectional cargoes. Firstly, the distances of runs in both directions approximate a decaying exponential distribution, as if each opposing motor activity ceases productive cargo transport due to a constant-probability event. Secondly, particles frequently undergo rapid switching between minus- and plus-end motility. This suggests that the mRNA binds opposite polarity motors at all times, with the net distribution determined by differences in their relative activities (Bullock, 2006).

It is not clear how control of net transport of other bidirectional cargoes is achieved, although opposing motor activities appear to be mutually dependent. Indeed, inhibition of the minus-end-directed motor dynein, which is required for apical mRNA localization, by preinjection of anti-dynein intermediate chain (Dic) antibodies or the dynein inhibitor vanadate, inhibits motility of h mRNA in both directions. The identity of the motor engaged during plus-end movement is not known, and it could even be dynein; recent work suggests that the motor with its accessory complex dynactin can undergo bidirectional motion on a single microtubule in vitro (Bullock, 2006).

To address the mechanistic role of apical localization signals, the behavior of h transcripts was contrasted to those of Krüppel (Kr), which are distributed evenly in the cytoplasm, or several heterologous mRNAs such as those derived from transcription of a plasmid backbone. Neither the Kr nor heterologous mRNA populations become enriched apically following injection, but each of these mRNAs rapidly assembles into particles with a spectrum of sizes similar to those of h. Surprisingly, the nonlocalizing transcripts undergo short bidirectional movements that initiate with similar kinetics to localizing mRNAs (Bullock, 2006).

The movements of the nonlocalizing transcript populations are indistinguishable from one another and are clearly motor driven; like those of h, persistent movements in both directions frequently reach velocities of ~1-1.5 μm/s and are sensitive to anti-Dic (anti-dynein intermediate chain) injection, as well as to hypomorphic mutations in the gene encoding dynein heavy chain (dhc64C). Consistent with a physiological function of bidirectional transport in achieving uniform spreading of mRNAs, endogenous Kr transcripts are retained in the perinuclear region upon injection of anti-Dic antibodies. Transport of uniform mRNAs presumably facilitates encounters with other posttranscriptional machinery (Bullock, 2006).

The ability of localizing and nonlocalizing mRNAs to undergo bidirectional transport led to an examination of which aspects of motion are significant for determining their different net distributions. Compared to nonlocalizing transcripts, apically localizing mRNAs spend more time undergoing minus-end transport and less time moving in a plus-end direction or pausing. There is a ~2.5-fold increase in the mean distance of minus-end runs of h compared to nonlocalizing mRNAs. This does not reflect increased dissociation of nonlocalizing mRNAs from recognition factors; for all RNAs tested, the distribution of plus-end run lengths is indistinguishable. Furthermore, minus-end runs are often immediately followed by reversals, which implies that mRNAs remain associated with a microtubule (Bullock, 2006).

Localizing mRNAs also have a subtle, but significant, increase in mean velocity of minus-end transport (10%-15%) compared to nonlocalizing RNAs, whereas plus-end velocity is not significantly different for all mRNAs tested. Localizing mRNAs are also ~1.4 times more likely than nonlocalizing mRNAs to undergo a minus-end run instead of a plus-end run following a pause. In contrast, the likelihood of a minus-end run or pause following a plus-end run is similar for localizing and nonlocalizing mRNAs (Bullock, 2006).

Overall, these data indicate that the h RNA localization signal is not obligatory for linking mRNAs to molecular motors. Instead, it gives rise to net apical localization by increasing the probability of initiation and maintenance of rapid minus-end-directed excursions of a bidirectional motor complex (Bullock, 2006).

To address whether localization signals are binary switches, the effects were tested of altering the sequence of the h element upon mRNA movement. The weak localizing h transcript 1328A→U [which has a mutated base in the first of two stem loops (SL1 and SL2a) that comprise the localization signal] undergoes slightly longer runs in the minus-end direction than nonlocalizing transcripts, whereas plus-end run lengths are the same as those of nonlocalizing and wild-type h transcripts. This mode of localization is probably employed by certain endogenous mRNAs. For instance, endogenous ken transcripts are only partially enriched apically, and injected ken localizes with kinetics indistinguishable from those of h1328A→U. Conversely, replacing the h localization signal with three copies of SL1 (hSL1x3) leads to apical accumulation ~2-fold faster than h due to significant increases in the initiation, velocity, and maintenance of minus-end runs. Plus-end motility of hSL1x3 is indistinguishable from that of the other transcripts tested. These findings indicate that mRNA sequences can generate a range of motile behaviors of bidirectional transport complexes. (Bullock, 2006).

To investigate how mRNA signals regulate minus-end transport, the potential roles of Egalitarian (Egl) and Bicaudal-D (BicD) were investigated. These proteins are components of a complex required for accumulation of several mRNAs at the minus end of microtubules in Drosophila, and their recruitment to an injected localizing transcript population can be observed above normal cytoplasmic levels. Egl binds directly to Dynein light chain (Dlc), and mammalian BicD associates with components of the dynein and dynactin complexes and recruits membranous vesicles for transport (Bullock, 2006).

It is not possible to characterize transport in egl or BicD null embryos because both factors have earlier essential functions. However, hypomorphic mutations in egl or BicD strongly reduce duration and velocity of minus-end, but not plus-end, runs of both wild-type and h1328A→U mutant particles. Weak apical accumulation of endogenous ken transcripts is also abolished by these mutations (Bullock, 2006).

In order to test the full requirements for Egl and BicD in transport of mRNAs, embryos were preinjected with blocking antibodies specific to each protein. Antibodies to either protein block net asymmetric movement of mRNAs. However, whereas anti-Dic or vanadate injection results in very little movement of mRNAs, anti-Egl- or anti-BicD-injected embryos frequently display short runs of localizing and nonlocalizing mRNAs in both directions. This limited motility does not result from residual protein activity because it cannot be significantly reduced by injecting the antibodies into partial loss-of-function embryos or by coinjecting the two antibodies. Thus, Egl or BicD is not obligatory for linking mRNAs to a motor, which is compatible with the impairment of apical mRNA anchorage upon inhibition of dynein, but not Egl or BicD (Bullock, 2006).

Inhibition of Egl and BicD modulates the transitions between pauses, minus-end runs, and plus-end runs similarly to perturbation of localization signals. Thus, like RNA localization signals, Egl and BicD promote the initiation and maintenance of rapid minus-end-directed movement of mRNAs along microtubules (Bullock, 2006).

Nonlocalizing transcripts can, however, make use of the Egl/BicD machinery very occasionally; a small subset of Kr and plasmid mRNA particles undergo relatively long minus-end-directed runs that are sensitive to inhibition of either protein (Bullock, 2006).

The data demonstrate that Egl and BicD augment minus-end-directed movements of mRNAs on microtubules. Interestingly, the frequency, speed, and duration of minus-end runs are significantly reduced when the level of wild-type BicD protein is reduced to ~6% of normal (BicDHA40/r5), indicating that BicD has concentration-dependent roles in regulating motility. Indeed, overexpression of BicD results in more efficient apical transport of injected h. Egl and its binding protein Dlc also function dose-dependently; minus-end motility of h on microtubules is significantly increased upon overexpression of either protein and reduced by halving egl gene dosage (Bullock, 2006).

Interestingly, elevating Egl, BicD, or Dlc levels augments minus-end motility in different ways. Very similar to increasing the number of localization elements, overexpression of Egl increases minus-end run length and minus-end velocity and modulates the transitions between travel states. In contrast, overexpressing BicD only modulates transitions between travel states, and increasing Dlc levels only increases minus-end run length and minus-end velocity. Egl might therefore function to independently recruit the activities of BicD and Dlc to mRNA signals. Indeed, different domains of Egl mediate association with these two proteins. Consistent with a concentration-dependent role of transport proteins during localization of endogenous mRNAs, there is also a subtle increase in the apical enrichment of endogenous uniform mRNAs upon Egl or Dlc overexpression. Strikingly, there is only a 2- to 2.5-fold increase in levels of Egl upon its overexpression in all of the experiments. Thus, the distinction between net symmetric and asymmetric transcript distribution could reflect subtle differences in the affinities of mRNAs for rather nonselective recognition factors (Bullock, 2006).

Egl, BicD, and Dlc levels could be important for minus-end motility because mRNAs dissociate from them during transit and efficient transport requires reassociation, or because of a function for different numbers of molecules in the transport complex from the outset. To discriminate between these two possibilities, an investigation was carried out to see whether mRNAs assembled in an environment where there is sufficient BicD are sensitive to a subsequent drop in the levels of BicD in the cytoplasm. This is not the case; h transcripts injected into an embryo overexpressing BicD and then withdrawn ~1 min later continue to be transported efficiently through the cytoplasm of BicDHA40/r5 acceptor embryos in which BicD levels are otherwise limiting (Bullock, 2006).

This finding is associated with neither diffusion of the BicD from the donor embryo following transplantation, confirmed using an epitope tag specific to the overexpressed BicD, nor the transplantation procedure, because mRNA transferred either between BicDHA40/r5 or between wild-type embryos behaves similarly to when transcripts are simply injected into these genotypes. Likewise, the movement of h particles exposed to cytoplasm overexpressing Egl or Dlc is not sensitive to the drop in the concentration of these proteins upon transfer to a wild-type embryo (Bullock, 2006).

These experiments indicate that the only point at which levels of these three proteins is critical is at the initial assembly of transport complexes and suggest that different RNA signals modulate motor activity in a perduring fashion by recruiting different numbers of Egl, BicD, and dynein molecules to each mRNP. Indeed, both Egl and BicD are found complexed with other molecules of themselves in vivo. Higher-order assemblies of dynein also exist in the embryo; measurements of stall forces of minus-end runs of lipid droplets reveal quantized steps of 1.1 pN -- equivalent to that of a single motor -- up to ~6 pN. Furthermore, the increases in both minus-end run length and velocity observed for localizing mRNAs and upon augmenting levels of transport proteins are consistent with observations of increasing numbers of active dyneins working together in vitro (Bullock, 2006).

To directly test whether the extent of minus-end motility of cargoes is associated with the amount of transport proteins nucleated, transcripts of wild-type h or the more efficiently localizing construct hSL1x3 was injected into embryos and the amount of Egl and BicD assembled on individual mRNA particles in transit was assayed. Consistent with such a model, concentration of Egl and BicD above cytoplasmic levels can be observed on many particles of hSL1x3 mRNA, but never on particles of h. Because it is not possible to observe motor components above background levels in these injection experiments, their recruitment to different RNA signals was investigated following incubation with cytoplasmic extracts in vitro. These experiments reveal that localization efficiency of mRNAs does indeed correlate with the amount of dynein components, as well as Egl and BicD, that they assemble (Bullock, 2006).

Together, these data provide evidence of a novel mechanism in which apical localization signals bias bidirectional motor movement by controlling the number of Egl, BicD, and dynein molecules incorporated into each mRNP. Because short-range bidirectional transport can occur in the absence of RNA localization signals, it is envisaged that these signals regulate minus-end motility, at least in part, by recruiting dynein motors in addition to those involved in distributing uniform mRNAs. Egl and BicD could function as adaptors that mediate the association of these additional dyneins with localization signals. Consistent with such a role, tethering mammalian BicD sequences to cargoes is sufficient to stimulate dynein recruitment and transport, and recruitment of Dhc to localizing mRNAs in vitro is reduced, but not abolished, upon immunodepletion of BicD from extracts (Bullock, 2006).

Nonetheless, the finding that increasing levels of Egl, BicD, or Dlc can modulate transport argues against a strict linear pathway of assembly. One intriguing explanation, which could also account for the substantial differences in the relative amounts of Egl and BicD assembled on individual mRNA particles, is that not all of the binding sites within the RNA:motor assembly must be saturated before transport is initiated. Modulating the number of mRNA elements or the concentration of each of the transport proteins could therefore alter the average number of fully functional Egl/BicD/dynein complexes assembled on each mRNP. Such a probabilistic mechanism could also account for the large variation in motile behaviors exhibited by particles of the same mRNA (Bullock, 2006).

Although differential motor recruitment appears to be one important mechanism to generate different classes of mRNA motion, the data hint at the existence of additional regulatory processes. The anti-BicD antibody largely uncouples minus-end run lengths from velocity, and overexpression of BicD alters transitions between travel states, but not run lengths or velocity. Thus, BicD is likely to have additional roles in regulating dynein activity. This could be through its binding to the dynactin complex, which is likely to play a key role in coordinating minus- and plus-end-directed motor activity. Indeed, Egl and BicD levels could regulate the hypothesized switch mechanism that is proposed to coordinate opposite polarity motor activities and determine when runs end (Bullock, 2006).

In many cell types, mRNAs exhibit net transport toward the plus ends of microtubules. These distributions could result from modulation of a related bidirectional transport complex using mRNA elements that preferentially nucleate or stimulate plus-end-directed motor activity. The findings also suggest that different organelles, vesicles, and macromolecules could assume a wide range of polarized distributions within the same cell by balancing opposite polarity motor activities through numerical differences in the same repertoire of transport proteins (Bullock, 2006).

Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate

In many cell types, polarized transport directs the movement of mRNAs and proteins from their site of synthesis to their site of action, thus conferring cell polarity. The cytoplasmic dynein microtubule motor complex is involved in this process. In Drosophila, the Egalitarian (Egl) and Bicaudal-D (BicD) proteins are also essential for the transport of macromolecules to the oocyte and to the apical surface of the blastoderm embryo. Hence, Egl and BicD, which have been shown to associate, may be part of a conserved core localization machinery in Drosophila, although a direct association between these molecules and the dynein motor complex has not been shown. This study reports that Egl interacts directly with Drosophila dynein light chain (Dlc), a microtubule motor component, through an Egl domain distinct from that which binds BicD. It is proposed that the Egl-BicD complex is loaded through Dlc onto the dynein motor complex thereby facilitating transport of cargo. Consistent with this model, point mutations that specifically disrupt Egl-Dlc association also disrupt microtubule-dependant trafficking both to and within the oocyte, resulting in a loss of oocyte fate maintenance and polarity. These data provide a direct link between a molecule necessary for oocyte specification and the microtubule motor complex, and supports the hypothesis that microtubule-mediated transport is important for preserving oocyte fate (Navarro, 2004).

To determine how the Egl protein affects molecular transport, attempts were made to identify domains in Egl critical for its function. The Egl protein sequence contains a putative 3'-5' exonuclease domain (RNase D). To determine if this domain is functional, the five conserved catalytic residues, which are critical for exonuclease activity in other proteins containing this domain, were mutated. Either singly or in combination, these mutations had no effect on Egl function during oogenesis. Hence, 3'->5' exonuclease activity may not be essential for Egl function. Nevertheless, the RNase D domain probably has a role in Egl function, because deletion of a 164-amino-acid region encompassing the domain abolished Egl function, without affecting the stability of the protein or its ability to bind to BicD (Navarro, 2004).

An important clue to Egl transport function emerged when a yeast two-hybrid screen demonstrated that Egl, through its carboxyl terminus, associates with Dlc, a component of the dynein microtubule motor complex. This was confirmed by co-immunoprecipitation from human embryonic kidney (293T) cells that expressed a portion of the Egl protein (amino acids 880->993) along with Dlc. To demonstrate that this association occurs in vivo, Dlc was co-immunoprecipitated from ovary extracts using an Egl antibody. The complex could be precipitated from wild-type extracts but not egl-null extracts, confirming the specificity of the Egl antibody (Navarro, 2004).

The specific sequences in both Egl and Dlc required for their interaction were identified. For Egl, sequencing of a previously uncharacterized egl allele (egl3e) demonstrated a mis-sense substitution (S954L) near its C terminus, within a 'dynein light chain interaction motif' found in several proteins that associate with dynein light chain. This mutation markedly reduces the interaction of Egl with Dlc in both a directed yeast two-hybrid assay and in co-immunoprecipitation from co-transfected 293T cells. Furthermore, with ovary extracts from flies expressing only the egl3e mutation, the anti-Egl antibody failed to co-immunoprecipitate any detectable Dlc in three out of four experiments and only a trace in the fourth. The motif in Dlc necessary for its interaction with Egl was also identified by mutational analysis. Dlc mutant 74 (F73S) bound Egl more weakly than wild-type Dlc (approximately 50%), and mutant 52 (F62S) did not bind detectably to Egl. This motif of Dlc (located between amino acids 57 and 86) also interacts with Bim and the intermediate chain of the dynein motor complex. These results confirm the specificity of the interaction between Dlc and Egl, and demonstrate that the interaction occurs through a Dlc interaction domain near the C terminus of Egl and a well established binding motif in Dlc (Navarro, 2004).

Egl also associates with BicD, but through an amino-terminal Egl domain. To determine whether the interaction between Egl and Dlc required BicD, the Egl->Dlc complex was co-immunoprecipitated from egl4e mutant extracts, where the Egl->BicD interaction is disrupted. The Egl4e mutant protein interacted with Dlc. Thus, the Egl->Dlc complex is not dependent on the Egl->BicD association. Furthermore, the egl4e and egl3e alleles show partial complementation in trans. This finding, as well as the observation that more than one Egl molecule may be present in the Egl->BicD complex, suggests that Egl3e and Egl4e multimers may be able to bring BicD and the dynein motors into the same complex, or that BicD and Dlc can bring multiple Egl molecules into the motor complex, thereby providing partial Egl function (Navarro, 2004).

Oocyte specification commences at the anterior tip of the ovary in the germarium, where stem cell divisions produce a new stem cell and a differentiating cystoblast. Four synchronous divisions yield a 16-cell cyst, each cell of which is connected by intercellular bridges. Whereas the future oocyte accumulates oocyte-specific markers and enters meiosis, its 15 sister cells become polyploid nurse cells. Several key events coincide with oocyte determination, for example, the synaptonemal complex (a marker for meiosis) forms between homologous chromosomes in four cyst cells and then restricts to the future oocyte, and a microtubule network is established that extends from the nurse cells through the intercellular bridges into the oocyte. Transport along this network results in the accumulation of oocyte-specific factors in the future oocyte. To analyse the role of the Egl->Dlc interaction during oogenesis, the phenotype of egl3e flies was compared with that of egl-null mutants, which fail to develop an oocyte. The accumulation of oocyte-specific markers such as Orb and the state of the oocyte nucleus was examined. The oocyte nucleus was judged by synaptonemal complex formation and karyosome condensation (which visualizes the DNA compaction typical of the oocyte nucleus). In egl-null mutant ovaries all 16 cells contained Orb and initially entered meiosis and formed a synaptonemal complex, but in later egg chambers the synaptonemal complex dissolved and all 16 cells became polyploid nurse cells. In contrast, egl3e mutant egg chambers initially restricted Orb to one cell, but Orb accumulation was delayed, less robust and eventually not maintained when compared with wild type. In approximately 70% of germaria examined, the synaptonemal complex formed and was restricted to one cell by stage one (as in wild-type germaria), but the localization of the synaptonemal complex to the oocyte was not maintained (as with Orb). In the remaining 30%, restriction of the synaptonemal complex to a single cell was delayed (Navarro, 2004).

Egl function may extend beyond oocyte specification. Although in the strong allelic combination (egl3e/eglPR29) most egl3e egg chambers lost Orb from the oocyte by stage 5 of oogenesis, a weaker allelic combination (egl3e/eglwu50) produced more mature egg chambers that retained Orb localization. In these chambers, the karyosome did not form properly, and often the chromatin appeared fragmented or looked decondensed. Interestingly, the amount of Orb that accumulated in the oocyte correlated with the degree of chromosome condensation. Thus, the chromatin in egg chambers with less Orb was less condensed and appeared more 'nurse-cell like'. Such chambers may have been reverting to nurse-cell fate. Taken together, analysis of the egl3e mutation suggests that egl is not only required for the initial oocyte specification, but also for the subsequent maintenance of oocyte fate (Navarro, 2004).

Does all Egl function require Dlc association? The egl3e phenotype clearly is weaker than egl null mutations, but this point mutation does not completely disrupt the Egl->Dlc association. Hence, an Egl protein was generated with two mutations (S954L and S958R) in the Dlc-binding site (egldlc2pt) that fails to bind to Dlc in vitro. Transgenic flies expressing the double-point-mutant protein at levels above 50% of wild type in an otherwise egl-null background showed a phenotype distinct from that of egl-null mutants and more similar to egl3e-mutant ovaries. In addition, localization of Orb to the oocyte was delayed. In the majority of cases, the synaptonemal complex formed in all 16 cells in germarial region 2A, but was restricted to one cell by stage 1. Similarly to egl3e mutants, all cells reverted to nurse cell morphology by stage 5/6. In the remaining cases (29%), synaptonemal complex localized to two cells in region 2B and then dissolved. Thus, not all Egl function relies on Dlc association (Navarro, 2004).

To determine whether localization of the dynein motor complex itself requires Egl->Dlc interaction, the localization of dynein heavy chain (Dhc) was followed. In wild-type ovaries, Dhc localizes to the oocyte in germarial region 2B, where it is initially concentrated anteriorly but moves to a posterior position at stage 1 and subsequently surrounds the oocyte nucleus. In egl-null mutants, Dhc localized to a single cell in region 2 and remained as a distinct anterior focus until stage 4/5, when it disappeared from the oocyte. This data is in contrast to Bolivar (2001), who did not observe enrichment of Dhc within the oocyte in the allelic combination eglwu50/Df, but supports the observation of Therkauf (1993), who reported that a microtubule-organizing centre initially forms in egl mutants but is not maintained. In the egldlc2pt lines, Dhc localized to the 'oocyte' as in wild type, whereas Orb was only faintly and transiently enriched in the oocyte. All egg chambers lost their enrichment of Dhc by stage 5/6, when all cyst cells resembled nurse cells. These results suggest that the Egl->Dlc association is required for the transport of cargo such as Orb to the oocyte, but is not essential for the initial localization of motor complex components (Navarro, 2004).

If the Egl->Dlc interaction is critical for oocyte development, dlc mutants would be expected to have an ovary phenotype similar to egl mutants. As dlc-null mutants are lethal, germ line clones were generated using the FLP/FRT system to study loss of Dlc function during early oogenesis. dlc mutant clones produced egg chambers with a 16 nurse cell phenotype. The oocyte-specific markers Orb and Egl were not localized to the oocyte, and all 16 cells became polyploid nurse cells. Interestingly, the synaptonemal complex initially was restricted to a single cell but was not maintained beyond stage 1. The synaptonemal complex defects observed in dlc-null mutants more closely resemble the phenotypes of egl mutants that cannot interact with Dlc than that of egl-null mutants. These results suggest that Egl->Dlc association is critical for the directed transport of factors required to maintain oocyte fate, but may not be essential to generate the initial asymmetry necessary for oocyte specification (Navarro, 2004).

Interestingly, dlc-null mutants, similarly to egl-null mutants, fail to accumulate any oocyte-specific markers, whereas egldlc2pt mutants show transient and weak localization of these markers to a single cell. Although this subtle difference in phenotype could reflect residual (albeit undetectable) binding between Egl and Dlc, an alternative model is favored which is based on the observation that Egl mutant protein defective in Dlc interaction could still interact with BicD. In this scenario, Egl can enter the motor complex independently of its association with Dlc, perhaps through its interaction with BicD, which similarly to its mammalian homologues may be able to interact directly with motor complex components (Navarro, 2004).

A more direct role for Egl and BicD in RNA cargo localization within the oocyte was initially suggested by the interplay between egl and dominant alleles of BicD. BicDD alleles cause a dominant bicaudal phenotype due to the mislocalization of the posterior determinant oskar (osk) RNA to the anterior pole. egl-null mutations suppress this phenotype, whereas it is enhanced by extra copies of wild-type egl. It was found that the BicDD phenotype is also suppressed by lowering the gene dosage of dlc, Dhc or the dynein-associated protein Lis-1. Among the progeny of BicDD females mutant for one copy of a dynein complex component, many more larvae hatch and more embryos develop normal segmentation than in the control. Hence, the dynein minus-end-directed microtubule motor complex is strongly implicated in osk RNA transport (Navarro, 2004).

The function of BicD and Egl may be coupled to transport in both directions along microtubules. In contrast to the minus-end-directed microtubule motor-associated proteins, reduction in the dose of proteins such as Barentz (Btz) and Staufen (Stau), which have been proposed to link osk RNA to plus-end-directed motors and the plus-ended motor protein kinesin heavy chain (Khc), enhanced the BicDD phenotype. These findings suggest that the BicDD phenotype may be due to an imbalance between the Egl->BicD->Dlc complex favouring microtubule minus-end directed transport and the Khc->Btz->Stau plus-end directed transport machinery, which is required to localize oskar to the posterior pole of the oocyte. Indeed, Egl and BicD may regulate this balance between plus- and minus-end-directed forces during normal localization of RNA and proteins in the oocyte, because osk RNA localization to the posterior pole is defective in egl3e and BicDmom mutant oocytes (Navarro, 2004).

This study presents evidence that Egl interacts directly with Dlc, identifies the motifs required for their association, and shows that this interaction is required for the maintenance of oocyte fate and the establishment of oocyte patterning. However, neither the Egl->Dlc interaction nor microtubule-based transport in general are required for the establishment of the initial asymmetry between the oocyte and its fifteen nurse sisters. As null mutations in egl or BicD but not mutations in Dlc or in the Dlc interaction domain of Egl impair restriction of the synaptonemal complex to a single cell, Egl function in restricting the synaptonemal complex seems to be independent of microtubule-directed transport and probably precedes the localization of Egl or BicD to the future oocyte. Mutational analysis further suggests that restriction of the synaptonemal complex requires an Egl->BicD interaction but not a physical interaction between Egl and Dlc. Thus, Egl and BicD mediate initial pro-oocyte selection independently of the microtubule network, and then in association with the dynein motor complex maintain oocyte fate by presumably controlling directed transport of cargo towards the future oocyte (Navarro, 2004).

A mammalian homologue of BicD, BICD2, has been shown in tissue culture to mediate minus end-directed transport along the microtubule network by interacting both with components of the dynactin->dynein motor complex and with cargo, such as organelles. The current results suggest that, in Drosophila, Egl serves as the primary adaptor coupling the Egl->BicD complex to the dynein motor complex. Future experiments should uncover the relative role of Egl and BicD in linking cargo to this transport machinery (Navarro, 2004).

A hairy RNA localization signal modulates the kinetics of cytoplasmic mRNA transport

In several Drosophila cell types, mRNA transport depends on microtubules, the molecular motor dynein and trans-acting factors including Egalitarian and Bicaudal-D. However, the molecular basis of transcript recognition by the localization machinery is poorly understood. The features of hairy pair-rule RNA transcripts that mediate their apical localization have been characterized using in vivo injection of fluorescently labelled mRNAs into syncytial blastoderm embryos. A 121-nucleotide element within the 3'-untranslated region (HLE) is necessary and sufficient to mediate apical transport. The signal comprises two essential stem-loop structures, in which double-stranded stems are crucial for localization. Base-pair identities within the stems are not essential, but can contribute to the efficiency of localization, suggesting that specificity is mediated by higher-order structure. Using time-lapse microscopy, the kinetics of localization has been measured; impaired localization of mutant signals is due to delayed formation of active motor complexes and, unexpectedly, to slower movement. These findings, and those from co-injecting wild-type and mutant RNAs, suggest that the efficiency of molecular motors is modulated by the character of their cargoes (Bullock, 2003).

Efficient recognition of the h transcript depends on two stem-loops, SL1 and SL2a; each is necessary for robust localization, but neither is sufficient alone. The mutagenesis data demonstrate that evolutionarily conserved double-stranded stems of SL1 and SL2a are indispensable for proper transcript transport. However, secondary structure of the stems is not the sole determinant of signal activity because transversions that alter base-pair identities lead to inefficient localization. In addition, although many predicted single-stranded regions are inessential for signal activity, a mutant in which all of these bases are removed or altered localizes only weakly. Thus, higher-order RNA structure is also likely to be important for specific recognition by the localization machinery (Bullock, 2003).

Specificity could reside in the tertiary conformation of the RNA, as has been demonstrated for various well-characterized RNA-protein interactions. Such three-dimensional structures would be difficult to infer solely from mutagenesis data, especially because of the large assortment of potential non-canonical interactions between bases (Bullock, 2003).

Intermolecular RNA interactions could also be involved in h mRNA recognition. Transcript oligomerization appears to be important for localization of bcd transcripts to the anterior of the late oocyte/early embryo, although the signals and trans-acting factors driving this transport process seem to be distinct from those acting in Egl/BicD-mediated early export into the oocyte. Studies of mutant h RNA transcripts have not yet revealed evidence that oligomerization is necessary for transport. Mixtures of up to three different transcripts were injected; rescue of a non-localizing mutant RNA signal by a co-injected localizing transcript has never been detected. Nonetheless, time-lapse studies show transport of injected h RNA in particles that contain numerous transcript molecules, although the imaging is not sensitive enough to detect cargoes of individual molecules. Indeed, particle formation is not sufficient to direct formation of an active transport complex: non-localizing mRNAs are also found in particles (Bullock, 2003).

Although K10 and bcd localization signals share no obvious primary sequence similarities with the HLE, they share structural features, suggesting that they are recognized similarly. The K10 localization signal is only 44 nt long and, unlike the HLE, comprises only a single stem-loop region; nonetheless, it recruits Egl and BicD. bcd transcripts also harbor a stem-loop (the 57-nt stem-loop V) that is required for early transport from nurse cells into the oocyte and for apical localization of injected bcd transcripts in the embryo and their association with Egl and BicD. Like h, the activities of both the bcd and K10 stem-loops rely heavily on double-stranded stems in which exact base-pair identities contribute to, but do not determine, efficient localization; base-pair transversions in all the stems can compromise the efficiency of localization. In common with the h SL1, the bcd stem-loop V is not sufficient for localization, but is fully active when dimerized (Bullock, 2003 and references therein).

The apparent complexity and redundancy of the HLE supports a model for signal recognition in which multiple protein-RNA contacts are needed for the formation of a specific, stable complex. In the HLE, weak binding sites for the machinery may be distributed in SL1 and SL2a. Thus, transcripts with two h SL1 domains are at least as active as those with a wild-type HLE. SL2a may provide quantitatively weaker binding signals; it is unable to support any localization either alone or when multimerized. One possibility is that SL1 alone establishes low affinity interactions with the localization machinery, and binds with high affinity together with SL2. The same mode of recognition could also apply for K10, if, unlike h and bcd, the requisite sites are located within a single stem-loop (Bullock, 2003).

Despite the overall similarities of the structural requirements for localization of bcd, K10 and h, no significant shared base-pair identities were identified within essential regions of the signals. The possibility that different transcripts are recognized by distinct RNA-binding factor(s) and recruited to shared components of the machinery cannot be excluded. However, the same localization signals are active in a variety of cell types. Also, stem-loops from different transcripts, each of which is relatively inactive in isolation, can complement to mediate completely efficient localization when combined in the same transcript. Thus, the view is favored that different transcripts share similar higher-order features, such as tertiary RNA conformations of the stems or RNA oligomers, which are recognized by the same factor(s). Multiple RNA motifs per signal and/or RNA or protein oligomerization would lead to the formation of the multiple protein-RNA contacts that confer specificity (Bullock, 2003).

RNA/BicD/Egl association appears to be a prerequisite for transport. BicD is unlikely to bind RNA directly because it lacks a known RNA-interaction domain, but BicD could hetero- or homo-oligomerize via its heptad repeat domains and thereby increase the numbers of protein-RNA contacts. Egl includes a domain with homology to certain 3'-5' exonucleases and a variety of other nucleic acid-interacting proteins, and thus might recognize RNA directly. However, its ability to recognize specific RNA sequences or structures has yet to be demonstrated (Bullock, 2003).

The fusome and microtubules enrich, Par-1 in the oocyte, where it effects polarization in conjunction with Par-3, BicD, Egl, and Dynein

After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Par-1 is shown to become restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. baz is required for Par-1 relocalization within the oocyte and the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization (Vaccari, 2002).

During oocyte specification, localization of the determinants BicD, Egl, and Orb to the early oocyte relies on the asymmetric distribution of microtubules in the cyst, evident as a dense focus of MT in the oocyte. Depolymerization of the MT by colchicine abolishes the localization of BicD, Egl, and Orb and results in egg chambers with 16 nurse cells and no oocyte. It was therefore asked if the restriction of Par-1 to the oocyte during the transition from region 2a to region 2b is also MT dependent. Ovaries of flies fed with colchicine for 12 hr fail to localize Par-1 and Orb to the oocyte, indicating that Par-1 restriction to the oocyte is indeed MT dependent. This is in contrast to the localization of Par-1 to the fusome, which occurs independently of MT (Vaccari, 2002).

The distribution of Par-1 within the oocyte was further examined by focusing on the transition between regions 2b and 3, when par-1-dependent polarization of the oocyte occurs. In germarial region 2b, Par-1 is enriched anterior to the oocyte nucleus. In region 3, the protein is mainly detected at the posterior of the oocyte, where it remains. During this relocalization, Par-1 colocalizes completely with BicD in the germline. Because par-1 is required for BicD relocalization within the oocyte, the distribution of Par-1 was examined in BicD hypomorphs that allow differentiation of an oocyte. Par-1 is detected but mislocalized in an anterior dot within the BicD mutant oocytes. Hence, Par-1 and BicD are interdependent for their relocalization to the posterior of the oocyte region 3b (Vaccari, 2002).

To assess whether the MT cytoskeleton mediates relocalization of Par-1 and BicD within the oocyte, wild-type ovaries were dissected a short time after treatment with colchicine. A screen was carried out for region 3 egg chambers in which the focus of oocyte MT was destroyed. In these, BicD and Par-1 remain anterior to the oocyte nucleus, indicating that MTs are required for oocyte polarization (Vaccari, 2002).

The MT motor Dynein has been reported to influence development of the germline cyst. Loss-of-function mutants in dhc64C, encoding the heavy chain of the minus end-directed molecular motor Dynein, fail to develop an egg chamber because of mitotic failure in the germarium. However, hypomorphic dhc64C mutants develop an oocyte and 15 nurse cells. In a high percentage of such egg chambers, both Par-1 and BicD remain at the anterior of the oocyte in region 3. Hence, after its initial requirement in cyst formation, the minus end-directed motor Dynein is involved in the relocalization of Par-1 and BicD to the posterior of the oocyte (Vaccari, 2002).

Thus, impairment of the MT cytoskeleton and mutations in BicD and dhc64C affect Par-1 relocalization within the oocyte. Conversely, in par-1 mutants, the MT cytoskeleton is not focused in the oocyte, BicD fails to relocalize, and Dynein is not enriched in the oocyte. The mutual interdependence of these genes and the MT suggests that all these components cooperate to form a polarization complex in the oocyte. Interestingly, the N1 antibody begins to detect Par-1 only when its function is genetically required, suggesting that, in region 2, the kinase may undergo a change in conformation or in its association with other factors (Vaccari, 2002).

The presence and localization of Par-1 in the oocyte at the time of its determination and polarization complements the previously reported localization of Par-1 on the fusome prior to oocyte determination and establishes Par-1 as a unique oocyte marker, for at least two reasons. (1) Absence of any one of the oocyte determinants, BicD, Egl, or Orb, prevents the concentration of the two other determinants in this cell. In contrast, in the absence of Par-1, it is the relocalization of the determinants within the oocyte that is specifically affected. (2) BicD, Egl, and Orb are not present on the fusome, and the observed enrichment of these determining factors in the oocyte is the result of the enrichment of their RNAs in this cell during its specification. In contrast, no par-1 RNA is detected in the germline at such early stages. The idea that Par-1 is initially loaded on the fusome, where it perdures during the cyst divisions, and that it is later preferentially inherited by the oocyte, is favored. Taken together, the facts that par-1 mutants show no fusomal defects and that accumulation of Par-1 itself in the oocyte requires MT suggest that Par-1 does not affect the oocyte MT cytoskeleton from its fusomal location. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where it acts in conjunction with BicD, Egl, and Dynein to effect polarization (Vaccari, 2002).

Role of Bicaudal D and Egalitarian in RNA transport in oogenesis and embryogenesis

Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the same machinery and RNA signals drive specific accumulation of maternal RNAs in the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos; interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor complex that drives transcript localization in a variety of tissues (Bullock, 2001).

During Drosophila oogenesis, specification of the oocyte is associated with selective accumulation of RNA determinants supplied by the neighboring, interconnecting ovarian nurse cells. Subsequently, deposition of mRNA transcripts at selected sites within the oocyte leads to localized translation of the proteins that establish the prospective embryonic body axes. gurken (grk) transcripts reside first posteriorly and then anterodorsally, and sequentially establish the anteroposterior and dorsoventral axes. bicoid (bcd) and oskar (osk) transcripts localize to the anterior and posterior of the oocyte, respectively, to pattern the anteroposterior body axis (Bullock, 2001).

Asymmetric RNA localization is also evident during zygotic development, especially in the unicellular syncytial blastoderm embryo. At this stage, several transcripts including those of the pair-rule and wingless (wg) segmentation genes lie exclusively apically of the layer of several thousand peripheral nuclei. Localization of these transcripts seems to be mediated by signals within their 3' untranslated regions (UTRs), and to be driven on microtubules by the minus-end-directed molecular motor, dynein. The linkers and other factors that provide the cargo specificity are unknown. Nor is it clear if transcript localization in blastoderm embryos relates to that in other types of cells (Bullock, 2001).

There is a rapid apical localization of fluorescently labelled fushi tarazu ( ftz) pair-rule transcripts injected into the basal cytoplasm of the cycle 14 blastoderm embryo. Although these experiments indicated a requirement for nuclear proteins fluorescein, labelling compromizes the structure of the transcripts, and pair-rule [even-skipped, hairy (h), ftz, paired and runt] and wg transcripts labelled with several other fluorochromes localize apically within 5-8 min without the need for exogenous protein. Indeed, injected unlabelled transcripts also localize apically. The protein-free assay retains specificity for apical transport, since transcripts that are normally unlocalized [Krüppel (Kr), huckebein] or enriched in the basal cytoplasm (string) are not transported apically and instead diffuse away from the site of injection (Bullock, 2001).

The injection assay was used to investigate whether any maternal transcripts that localize in the oocyte are recognized by the localization machinery of blastoderm embryos. Five such transcripts [bcd, grk, nanos (nos), osk and female sterile (1) K10 (K10)] were tested, and all accumulate in the apical cytoplasm after injection. With the exception of osk transcripts -- only a small proportion of which localize apically -- the efficiency of localization of these transcripts appears indistinguishable from that of pair-rule transcripts. Maternal transcripts also localize apically when zygotically expressed from endogenous transgenes. Preinjection with colcemid severely inhibits apical localization of the injected maternal transcripts, indicating that their localization in blastoderm embryos, like that of the pair-rule transcripts, is dependent on intact microtubules (Bullock, 2001).

Further experiments show that the same signals mediate transcript transport during oogenesis and apical localization in blastoderm embryos. Focus was placed on transcripts of the K10 gene, which localize through a 44-nucleotide region of the 3' UTR (transport/ localization sequence; K10TLS) -- the shortest signal thus far shown to be active during oogenesis. This signal, which is predicted to form a stem-loop structure, mediates all aspects of K10 transcript localization, that is, transport of transcripts from the nurse cells into the oocyte from stage 2 and localization at its anterior pole between stages 8-10 (Bullock, 2001).

The K10TLS is sufficient to drive apical localization in blastoderm embryos. Reporter stg and Kr transcripts, into which the K10TLS (stg-K10TLS and Kr-K10TLS) was inserted, localize apically in a way that is indistinguishable from pair-rule transcripts. Moreover, the same regulatory signals are used for transcript localization during oogenesis and in the embryo. A 5-nucleotide transversion in the K10 transcript that disrupts base pairing of the K10TLS stem-loop abolishes all aspects of localization during oogenesis, and prevented K10 transcripts from localizing in the blastoderm injection assay. Kstem5'3', in which compensatory mutations restore base pairing in the stem, directs weak but significant localization in embryos. The same signal also partially restores localization during oogenesis. These results suggest that the same machinery is used in both cases (Bullock, 2001).

The common aspect of maternal RNA localization measured in these experiments is unlikely to be transport within the oocyte, because the maternal transcripts tested are distinctly distributed in late stage oocytes by means of different motors and accessory factors. However, all the transcripts -- with the possible exception of grk -- are synthesized in adjacent nurse cells and reach the oocyte by transport along microtubules. To test whether this process is analogous to apical localization in blastoderm embryos, a bcd transcript was used containing a single nucleotide change (4496G->U). This change prevents early oocyte-specific transport (stages 4-6) without disrupting later (stage 6 onwards) import of transcripts into the oocyte or their subsequent accumulation at the anterior cortex. This mutation inhibits apical bcd localization in blastoderm embryos, suggesting that transcripts localize in this injection assay through the same machinery that transports transcripts into the early oocyte (Bullock, 2001).

This proposal is supported by the finding that pair-rule transcripts accumulate in the early oocyte if synthesized ectopically during oogenesis. r5f3 females express a hybrid transcript containing a portion of the ftz coding sequence and the entire 3' UTR under the control of the constitutively active RpA1 promoter. r5f3 transcripts accumulate specifically in the oocyte from stage 3, and concentrate at the anterior cortex of the oocyte between stages 8 and 10B, after which they become distributed throughout the oocyte. This pattern of localization is indistinguishable from that of K10 transcripts and closely follows the distribution of the minus ends of microtubules. Localization of r5f3 is dependent on an intact microtubule cytoskeleton, since it is inhibited by prior treatment with colchicine. A hybrid ftz transcript (r5f3-1) lacking the 3' UTR, and therefore the signal for apical localization in blastoderm embryos, is retained in the nurse cells and not transported to the oocyte (Bullock, 2001).

These results indicate that blastoderm localization signals can drive transcript transport during oogenesis. This view is supported by more detailed analysis of maternally expressed pair-rule transcripts. The injection assay reveals a minimum region between positions 1,374 and 1,579 in ftz that is necessary and sufficient for localization in blastoderm embryos. A similar region of ftz seems to be required for localization of transcripts into the oocyte. Furthermore, h and runt transcripts, driven maternally by the Hsp70 promoter, also accumulate specifically in the oocyte and later reside at its anterior cortex, whereas Kr or truncated h transcripts lacking most of the 3' UTR fail to localize either in blastoderm embryos or during oogenesis (Bullock, 2001).

These data suggest that components of the blastoderm localization machinery are also likely to function in RNA transport into the early oocyte. Genetic screens for maternal mutations that affect formation of the embryonic axis have identified egl and BicD as genes required for oocyte differentiation and for specific RNA accumulation in the oocyte. However, their exact activities are uncertain. BicD protein includes multiple heptad repeats, which may mediate oligomerization and interactions with other proteins; Egl includes a domain shared with 3'-5' exonucleases. During oogenesis, these two proteins form complexes together and colocalize at the minus ends of microtubules. The integrity of the microtubule cytoskeleton is defective in egl and BicD mutants, which has been proposed to explain subsequent defects in RNA localization. Alternatively, Egl and BicD might act directly in RNA transport. However, evidence that distinguishes between these two possibilities is lacking (Bullock, 2001).

Whether Egl and BicD are present in early embryos was examined. Both proteins are supplied maternally to the embryo. They are noticeably enriched apical to the nuclei at blastoderm stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the proteins is present in the basal cytoplasm (Bullock, 2001).

Whether endogenous Egl and BicD can associate with injected localizing transcripts, as might be expected if they are components of the RNA localization machinery, was tested. Injection of h transcripts leads to marked enrichment of Egl and BicD protein levels at the sites of RNA localization. Similar results are found on injection of the other tested maternal and zygotic localizing transcripts ( ftz, bcd, grk, K10, nos, osk and wg). Both proteins accumulate basally at the site of injection within 1-2 min. Protein recruitment is not inhibited in embryos preincubated with colcemid, showing that it is not dependent on intact microtubules. Thus, the proteins are recruited locally before transport and are transported together apically with transcripts (Bullock, 2001).

Interaction of injected transcripts with Egl/BicD is mediated by intact localization signals: protein recruitment to Kr-K10TLS and stg-K10TLS was detected, but not to Kr, stg, Kstem5' and bcd4496G->U, or to a h transcript containing a 21-base-pair (bp) deletion within the localization signal that abolishes localization. When localization is weak, recruitment of Egl and BicD was only detected by transcripts that have localized apically (for example, osk). The above results suggest that only transcripts that bind Egl/BicD can be transported apically (Bullock, 2001).

Whether BicD and Egl are required for apical localization in blastoderm embryos was examined. Strong BicD alleles block oogenesis early, and weaker mutant mothers that lay fertilized eggs (BicDHA40/BicDR26 and BicDH3/BicDR26) retain sufficient BicD activity for a normal apical distribution of endogenous pair-rule transcripts. However, the reduced BicD activity in these embryos no longer supports efficient transport of injected transcripts: 62% of BicDHA40 /BicDR26 and 73% of BicDH3/BicDR26 embryos show no or weak localization 5-8 min after injection, compared with 10% of wild-type embryos. Moreover, an antibody against BicD blocks RNA transport. Preinjection into the basal cytoplasm of anti-BicD antibody 4C2 strongly inhibits the localization of injected h, ftz, grk and stg-K10TLS transcripts in 70%-75% of embryos. The microtubule cytoskeleton is not obviously affected by the brief (~20 min) antibody treatment, indicating that the effects on RNA transport are probably direct. Injection of anti-BicD antibody prevents apical localization of endogenous pair-rule transcripts, also leading to anteroposterior smearing of their distribution. Thus, apical transcript localization seems to be important in restricting the range of activity of pair-rule genes, and allowing their combinatorial control of Drosophila segmentation (Bullock, 2001).

Injecting blastoderm embryos with anti-Egl also inhibits apical localization of both exogenous and endogenous pair-rule transcripts, without overtly disrupting the microtubule network. Moreover, its effect is more potent in embryos from mothers containing only a single copy of the egl gene, indicating that the antibody disrupts RNA localization by inhibiting the activity of Egl. Egl and BicD are probably also involved in transporting other cargoes. The arrangement of peripheral nuclei is disrupted after injection of antibodies to either of the two proteins, consistent with data showing a requirement for BicD in nuclear migration in eye imaginal disc cells. Embryos injected with either antibody undergo abnormal morphogenesis, which is also indicative of Egl and BicD transporting additional cargoes (Bullock, 2001).

These results indicate that Egl and BicD are principal elements of a complex that transports RNA in blastoderm embryos. Egl and BicD appear to be present as pools of excess cytoplasmic protein that associate selectively with localizing transcripts and are transported together apically. Protein recruitment occurs before transport and does not require microtubule integrity; rather, transport depends on Egl and BicD activity. Egl and BicD probably act directly to mediate RNA transport associated with establishment and maintenance of the oocyte. Thus, mutant transcripts that are defective in export from nurse cells into the oocyte fail to recruit Egl or BicD in blastoderm embryos. grk transcripts are also recognized by the Egl-BicD-microtubule transport pathway, which is consistent with the hypothesis that nurse cells are a source of these transcripts for the early oocyte and that they do not derive exclusively from the oocyte nucleus (Bullock, 2001).

Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of protein/RNA co-transport. The complex may have an additional role in anchoring transcripts at their destination. Alternatively, maintenance of localized transcripts might not depend on an independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).

The structural basis of how the transport machinery and RNA signals recognize each other is unclear. The shortest signal defined to date, the K10 transport/localization sequence, probably relies on both primary and secondary structure. Thus, mutating bases in the stem (Kstem5') inactivate the signal, and compensatory mutations that restore base pairing in the stem (Kstem5'3') reactivate the signal. However, the Kstem5'3' signal is only partially active, indicating that primary sequence and possibly tertiary structure are also important. Nor could shared structural features be identified in several maternal and zygotic localization signals. This could be due to promiscuous or multiple adapter proteins, or because the motor protein complex allows alternative RNA contacts. Egl or BicD may contribute directly to determining RNA selectivity. Neither includes a well characterized RNA-binding motif, but Egl includes a domain found in a variety of nucleic-acid-recognizing proteins. Other components of the complex may also contribute to selective RNA recognition in blastoderm embryos. However, none of the proteins currently implicated in localizing maternal transcripts are likely candidates for such adapters, being absent in blastoderm embryos [Orb, Swallow (Swa), Exuperantia (Exu)], not required for early transport of transcripts into the oocyte (Stau, Exu, Swa), or not recruited to localizing pair-rule transcripts (Bullock, 2001).

Dhc, Egl and BicD have markedly similar distributions during oogenesis and in blastoderm embryos, and seem to function together in specifying oocyte identity. It is proposed that an Egl/BicD complex links specific RNAs to dynein and the microtubules. The same machinery may operate elsewhere in Drosophila. For example, inscuteable transcripts, which localize asymmetrically in neuroblasts, also localize apically when injected into blastoderm embryos. Indeed, germline transcripts localize apically when expressed in follicle cells. Egl and BicD homologs have been identified in Caenorhabditis elegans and mammals, and might comprise part of an evolutionarily conserved cytoskeletal system for transporting transcripts and other cargoes (Bullock, 2001).

An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila

To determine whether Egalitarian and Bicaudal D directly affect the extent to which OSK mRNA mislocalizes, the distribution of OSK mRNA was examined in BicD-Dominant mutants. Reducing the amount of egl wild-type product decreases ectopic localization of osk to the anterior and increasing the amount of egl wild-type product enhances the mislocalization of OSK to the anterior. Because the effect of BicD-Dominant mutants depends on egl wild type function, it is concluded that egl and BicD act in the same pathway and that the two function in concert to control OSK mRNA localization. It is also thought that Egl and BicD have a role in dorsoventral polarity, as mutation of the two genes reduce the level of Gurken mRNA. Localization of GUR is known to require an intact microtubule cytoskeleton (Mach, 1997).

Egl is localized to a single cell and is required for the development of that cell as the oocyte; however, three observations indicate that egl is not the oocyte determinant. First, increasing the copy number of egl+ increases the levels of Egl protein within the germarium but does not cause multiple cells within the cluster to obtain oocyte fate. Second, in egl null mutations all 16 cells initially follow the prooocyte fate and form synaptonemal complex before reverting to the nurse cell fate. Third, in BicDr26 mutants, Egl protein is highly enriched in a single cell but this cell does not follow the oocyte fate. Thus Egl protein alone is not sufficient to control oocyte fate. Therefore the idea is favored that Egl and BicD determine oocyte fate as a protein complex by controlling the distribution of molecules that regulate oocyte and nurse cell differentiation (Mach, 1997).

An early sign of oocyte determination is the formation of the synaptonemal complex. However, germ cells with three or four ring canals located adjacent to the oocyte form synaptonemal complexes transiently but eventually follow the nurse cell fate. This transient expression of one aspect of oocyte fate may reflect the distribution of an oocyte determinant. At this stage of oogenesis, in germarial regions 2A and 2B, Egl and BicD proteins become enriched in the future oocyte. If Egl and BicD are involved in localizing oocyte determining factors, lower amounts of these factors in cells adjacent to the oocyte may account for this transient entry into meiosis. Consistent with this hypothesis, in egl mutants all 16 cells form synaptonemal complexes transiently (Carpenter, 1994). According to this model, egl may be required not for the activation or synthesis of oocyte-determining factors, but rather for producing a critical concentration of these factors in the future oocyte. Differentiation of the nurse cell-oocyte cluster requires the establishment of two cell fates: the premeiotic oocyte and polyploid nurse cells. In egl and BicD mutant ovaries the cell that would normally become an oocyte develops as a sixteenth nurse cell; thus, the same factors that promote oocyte determination may also repress nurse cell fate. Common to both oocyte and nurse cell fate decisions is a change from normal cell cycle regulation: The oocyte arrests in meiotic prophase and the nurse cells become polyploid by DNA replication without cytokinesis. A cyclinE mutation perturbs this decision, causing one of the cells that would normally become a nurse cell to develop as a second oocyte; thus, determination of oocyte and nurse cell fate may be linked to the differential distribution or activation of cell cycle regulators. It is therefore intriguing to speculate that the Egl-BicD complex is involved in the distribution of these regulators (Mach, 1997).

Specification of the oocyte as different from its 15 sister cells and positioning of the oocyte posterior to the nurse cells are essential for the subsequent polarization of the oocyte. Oocyte determination requires egl and BicD; oocyte positioning requires the function of a number of genes such as dicephalic, homeless, and spindleC. Oocyte determination both requires and stabilizes a polarized microtubule network that leads to microtubule-mediated transport of RNA from the nurse cells into the future oocyte. Among these RNAs are grk, osk, and bicoid, which encode essential regulators of the two embryonic axes. The anterior-posterior axis is thought to be set when Grk protein, a TGF-alpha-like molecule, is secreted from the oocyte and signals to the underlying follicle cells to promote posterior follicle cell fate. A yet unknown signal returned from the follicle cells leads to the repolarization of the microtubule network in the oocyte such that the MTOC at the posterior cortex is lost and microtubules now extend with the minus end from the anterior toward the posterior. This repolarization has two consequences: (1) microtubule polarity leads to the sorting of RNA molecules along the anterior-posterior axis, that is, bicoid RNA becomes localized to the anterior pole and osk RNA becomes localized to the posterior pole of the oocyte; (2) grk RNA moves in close association with the oocyte nucleus to the dorsal anterior margin of the oocyte. In another intercellular signaling step, Grk protein, secreted from the oocyte, now promotes dorsal follicle cell fate (Mach, 1997).

Each step -- first determination of oocyte fate, then specification of the anterior-posterior axis, and finally specification of the dorsoventral axis -- requires both RNA transport along a polarized microtubule network and the function of the Egl-BicD complex. The distribution of Egl and BicD proteins resembles that of the minus-ends of microtubules, and mutations in either egl or BicD disrupt microtubule stability or the initiation of the microtubule organizing center in the oocyte. Consequently, many RNAs that are transported into the oocyte during early oogenesis do not accumulate in a single cell in egl and BicD mutants (Mach, 1997).

The role of the Egl-BicD complex in anterior-posterior axis formation is suggested by its effect on localization of osk RNA. egl and BicD mutants abolish transport of osk into the oocyte. In BicD mutants, the BicD-Egl complex directs ectopic localization of osk RNA to the anterior of the oocyte (Ephrussi, 1991). The osk localization signal in the osk 3'-UTR contains separable regions for oocyte and posterior localization of osk. Because the Egl-BicD complex affects the initial localization of osk to the oocyte the model is favored that the complex mediates ectopic localization via the oocyte localization signal. The model would predict that during normal oogenesis, osk RNA is released from the BicD-Egl complex once the complex relocates to the anterior pole. As a consequence, osk RNA localization at the anterior is only transient and osk RNA becomes anchored stably at the posterior pole through sequences in the osk 3' UTR necessary for posterior localization. In BicD mutants, however, the BicD-Egl protein complex is somehow altered such that it is unable to release osk RNA after the complex has moved to the anterior and thus osk RNA remains at the anterior. One prediction of this hypothesis is that anterior localization of osk RNA in BicD mutants depends on the oocyte localization domain and not the posterior localization domain within the osk 3' UTR (Mach, 1997).

This hypothesis would explain why in BicD mutants osk is localized to the anterior pole independent of gene functions such as staufen, cappuccino, and spire, which are required for the normal posterior localization of osk (Mach, 1997).

The Egl-BicD complex is also involved in establishment of the dorso-ventral axis. egl affects eggshell morphology and that this phenotype can be attributed to a defect in grk RNA localization. Similarly, 90% of eggshells produced by BicD mutant females have fused dorsal appendages, indicating ventralization as a result of reduced function of the grk pathway. Thus the Egl-BicD complex may not only affect initial transport of grk RNA into the oocyte where Grk sets the anterior-posterior axis but may also affect grk RNA localization to the anterior during mid-oogenesis when Grk sets the dorsoventral axis (Mach, 1997).

Although it is possible that the Egl-BicD complex affects RNA localization solely by stabilizing microtubule structure, the hypothesis is favored that association of the Egl-BicD complex with microtubules stabilizes microtubules and that the complex then acts as a link between microtubules and the RNA localization machinery. If egl and BicD act directly to localize RNAs, these proteins may either bind RNA or associate with an RNA-binding protein, such as Orb, which has a distribution strikingly similar to that of Egl and BicD and a localization that depends on Egl and BicD function (Mach, 1997).


Search PubMed for articles about Drosophila Egalitarian

BolĂ­var, J., et al. (2001). Centrosome migration into the Drosophila oocyte is independent of BicD and egl, and of the organisation of the microtubule cytoskeleton. Development 128(10): 1889-97. PubMed ID: 11311168

Bullock, S. L. and Ish-Horowicz, D. (2001). Conserved signals and machinery for RNA transport in Drosophila oogenesis and embryogenesis. Nature 414(6864): 611-6. 11740552

Bullock, S. L., et al. (2004). Differential cytoplasmic mRNA localisation adjusts pair-rule transcription factor activity to cytoarchitecture in dipteran evolution. Development 131: 4251-4261. PubMed ID: 15280214

Carpenter, A.T.C. (1994). egalitarian and the choice of cell fates in Drosophila melanogaster oogenesis. Ciba Found. Symp. 182: 223-254. PubMed ID: 7835153

Clark, A., Meignin, C. and Davis, I. (2007). A Dynein-dependent shortcut rapidly delivers axis determination transcripts into the Drosophila oocyte. Development 134: 1955-1965. PubMed ID: 17442699

Claussen, M. and Suter, B. (2005). BicD-dependent localization processes: From Drosophilia development to human cell biology. Ann. Anat. 187: 539-553. PubMed ID: 16320833

Cohen, R. S., Zhang, S. and Dollar, G. L. (2005). The positional, structural, and sequence requirements of the Drosophila TLS RNA localization element. RNA 11: 1017-1029. PubMed ID: 15987813

Dienstbier, M., Boehl, F., Li, X. and Bullock, S. L. (2009). Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor. Genes Dev. 23(13): 1546-58. PubMed ID: 19515976

Ephrussi, A., Dickinson, L. K. and Lehmann, R. (1991). Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66: 37-50. PubMed ID: 2070417

Hoogenraad, C. C., et al. (2001). Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes. EMBO J. 20: 4041-4054. 11483508

Hoogenraad, C. C., et al. (2003). Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport. EMBO J. 22: 6004-6015. PubMed ID: 14609947

Hughes, J. R., Bullock, S. L. and Ish-Horowicz, D. (2004). Inscuteable mRNA localization is dynein-dependent and regulates apicobasal polarity and spindle length in Drosophila neuroblasts. Curr. Biol. 14: 1950-1956. PubMed ID: 15530398

Macdonald, P. M. and Kerr, K. (1997). Redundant RNA recognition events in bicoid mRNA localization. RNA 3: 1413-1420. PubMed ID: 9404892

Mach, J. M. and Lehmann, R. (1997). An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila. Genes Dev. 11: 423-435. PubMed ID: 9042857

Maybeck, V. and Röper, K. (2009). A targeted gain-of-function screen identifies genes affecting salivary gland morphogenesis/tubulogenesis in Drosophila. Genetics 181(2): 543-65. PubMed ID: 19064711

Navarro, C., et al. (2004). Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate. Nat. Cell Biol. 6(5): 427-35. 15077115

Oh, J., Baksa, K. and Steward, R. (2000). Functional domains of the Drosophila Bicaudal-D protein. Genetics 154(2): 713-24. 10655224

Pearson, J. and Gonzalez-Reyes, A. (2004). Egalitarian and the case of the missing link. Nat. Cell Biol. 6: 381-383. PubMed ID: 15122261

Tekotte, H. and Davis, I. (2002). Intracellular mRNA localization: Motors move messages. Trends Genet 18: 636-642. PubMed ID: 12446149

Theurkauf, W. E., Alberts, B. M., Jan, Y. N. and Jongens, T. A. (1993). A central role for microtubules in the differentiation of Drosophila oocytes. Development 118: 1169-1180. PubMed ID: 8269846

Vaccari, T. and Ephrussi, A. (2002). The fusome and microtubules enrich, Par-1 in the oocyte, where it effects polarization in conjunction with Par-3, BicD, Egl, and Dynein. Curr. Biol. 12: 1524-1528. 12225669

Van De Bor, V,, et al. (2005). gurken and the I factor retrotransposon RNAs share common localization signals and machinery. Dev. Cell 9: 51-62. PubMed ID: 15992540

Biological Overview

date revised: 30 October 2009

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

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