Bicaudal D
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).
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).
Egalitarian protein colocalizes with BicD protein at all stages of oogenesis.
Immunoprecipitation experiments show that both proteins are part of a protein
complex. Egl and BicD proteins localize to the oocyte in three stages that correlate
with the stepwise polarization of the oocyte. It is proposed that the Egl-BicD protein
complex links microtubule polarity and RNA transport. During early oogenesis, the
complex is required to transport factors promoting oocyte differentiation; during later
stages of oogenesis the complex directs the sorting of RNA molecules required for
anterior-posterior and dorsoventral patterning of the embryo (Mach, 1997).
How the asymmetry between the two pro-oocytes arises is unknown, but it has been proposed that it could be generated during the first division of the cystoblast to give rise to a two-cell
cyst. During this division, a vesicular structure called the spectrosome (see Drosophila Spectrin) associates with one pole of the mitotic spindle and is asymmetrically partitioned between the two daughter cells. Since each of these cells gives rise to one pro-oocyte and seven nurse cells, this asymmetry might determine which pro-oocyte is fated to become the oocyte. Whatever mechanism generates the initial asymmetry, it seems that the key step in the selection of the oocyte is the accumulation of Bicaudal-D and Egalitarian proteins in a single cell. Null mutants in either gene block the localization of the protein encoded by the other to the presumptive oocyte and prevent all other known steps in oocyte differentiation, such as the formation of an active MTOC in this single cell and the subsequent microtubule-dependent localization of oocyte-specific transcripts,
such as Oskar. Although it is unclear at what point in the pathway of oocyte selection
the spindle genes (see Drosophila homeless) act, they must function upstream of the process that results in the localization
of Bic-D (and presumably Egl) to a single cell. It is most likely that the spindle proteins are directly involved in this process: (1) the spn double mutant combinations delay but do not block the choice between the two pro-oocytes, suggesting that they do not remove the initial asymmetry, but slow down its expression. (2) A reduction in Bic-D or Egl activity later in oogenesis leads to the same ventralized phenotype that is produced by the single spn mutations. This raises the possibility that the spn gene products interact with Bic-D and Egl at two different stages of oogenesis, first to select the oocyte and then to regulate Gurken expression once the oocyte has formed (Gonzalez-Reyes, 1997).
A slow migrating isoform of Bic-D is phosphorylated; an Ala-40 to Val mutation in a Ser/Ala rich region of the protein interferes with this phosphorylation process. This mutation also interferes with the accumulation of BicD in the pro oocyte and with oocyte differentiation (Suter, 1991).
In a yeast two-hybrid screen an interaction was identified between Drosophila lamin Dm0, a structural nuclear protein, and BICD, a protein involved in oocyte development. The interaction can be reconstituted in vitro and takes place between segments of both proteins predicted to form coiled coils. The affinity for lamin Dm0 of the minimal binding site on BICD is modulated in a complex fashion by other BICD segments. A point mutation, F684I, that causes the dominant, bicaudal, Bic-D phenotype inhibits lamin binding in the context of the minimal lamin-binding site, but not in a larger BICD fragment. The minimal lamin-binding site of BICD binds to a few other coiled-coil proteins, but binding to these proteins is not influenced by the F684I point mutation, suggesting that the interaction with lamin may play a role in Bic-D function. Structural studies demonstrated that BICD is 60%-70% alpha-helical, is a dimer, and consists of two parts: a thin rod-shaped part of about 32 nm, and a thicker rod-shaped part of about 26 nm. Likely, the thinner rod-shaped part of full-length BICD consists of the N-terminal half of the protein, and the lamin-binding site is located within the thicker rod-shaped part (Stuurman, 1999).
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).
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).
Microtubule arrays direct intracellular organization and define cellular polarity. This study shows a novel function of glycogen synthase kinase-3β (GSK-3β) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. The data revealed that GSK-3β is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3β or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3β knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3β and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization (Fumoto, 2006).
The Drosophila body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. This study investigated the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. Exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. In vivo, Rab6 is found in a complex with Bicaudal-D, and Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila (Coutelis, 2007).
During polarized exocytosis, secretory vesicles emerging from the TGN are
targeted via molecular motors and cytoskeletal tracks to the plasma membrane,
where they are tethered. Subsequently, their fusion with the plasma membrane
permits the secretion of the vesicle contents, as well as the incorporation of
vesicular lipids and proteins into the plasma membrane, allowing membrane
growth and the establishment of specific domains. The exocyst complex plays a
crucial role in the incorporation of particular membranes and membrane
proteins at specific sites or in active domains of the plasma membrane.
Consistent with this, Drosophila sec5 mutant egg chambers display mislocalization of other exocyst components, cytoplasmic clusters of actin and a loss of plasma membranes. Thus, Sec5 protein is at the core of the exocyst complex in Drosophila, as is the case in yeast and in mammals (Coutelis, 2007).
Both sec5 null (sec5E10) and strongly affected
rab6D23D egg chambers display actin and general
organization defects, and arrest development during early oogenesis. Similarly, sec5 hypomorphic (sec5E13) and rab6D23D egg chambers that develop past stage 7 display phenotypes ranging from wild type to a loss
of nurse cell cortical actin and the concomitant presence of ring canal
clusters in the nurse cell cytoplasm. The striking parallel between the rab6 and
sec5 phenotypes, together with the finding that a loss of Rab6
affects Sec5 localization, suggests that the varying degrees of membrane loss
observed in rab6D23D egg chambers reflects the relative
reduction of exocyst-complex function in the egg chamber. Thus, during
Drosophila oogenesis, Rab6 promotes Sec5 localization and therefore
appears to be important for exocyst-complex organization and function.
However, consequent to loss of rab6 function, a striking difference was observed between nurse cells and oocyte in the severity of plasma membrane collapse and Sec5 mislocalization. It is hypothesized that the oocyte acts as a major source of membrane in rab6D23D egg chambers and/or that multiple exocytic pathways cooperate within the germline cyst to promote cyst development (Coutelis, 2007).
Differences in membrane content between the oocyte and the nurse cells, as
well as between the individual nurse cells, are observed as early as the
germarium stage in wild-type egg chambers. The fusome, a membranous
Spectrin-rich structure derived from the spectrosome, which itself is a
precursor organelle present in the germline stem cells, grows asymmetrically
through the ring canals during the divisions of the germline cyst, linking
each cystocyte. It is thought that the oocyte is the four-ring-canal cell
that retains the greater part of fusome during the first division. Furthermore, a Drosophila Balbiani body has recently been discovered, which, together with the fusome, organizes the specific enrichment of organelles in the oocyte throughout oogenesis. It is therefore possible that, in rab6 clones, in which the fusome
appears normal, such a mechanism of enrichment of organelles
in the oocyte concomitantly ensures that the concentration in the oocyte of
any perduring Rab6 protein, thus privileging the growth of the plasma membrane
of the oocyte over that of the nurse cells. Supporting this notion is the
observation that GFP-tagged Rab6 expressed in the germline is enriched in the
oocyte from the early stages of oogenesis (germarium region 2) onwards. Together, the combined actions of a residual Rab6-dependent and of additional Rab6-independent pathways might also permit most rab6D23D oocytes to maintain sufficient
vesicular trafficking to develop past stage 7 (Coutelis, 2007).
The stereotypic organization of affected rab6D23D egg
chambers at mid-oogenesis is striking. The oocyte is connected to open syncytia via its four ring canals, suggesting that the membranes linking nurse cells and oocyte are the most resistant. Furthermore, the
growth of the remaining membranes indicates that additional vesicular material
is delivered and incorporated into these plasma membranes. This suggests that,
in these rab6D23D egg chambers, sustained vesicle trafficking in the oocyte causes new membrane addition to the oocyte plasma membrane. It is hypothesized that, due to the continuity of the plasma membrane defining the cyst, the oocyte acts as a source of membrane that spreads by lateral diffusion throughout the plasma membrane of the cyst, allowing its growth (Coutelis, 2007).
It appears that Rab6-independent exocytic pathways also contribute to the
delivery of vesicular material to the plasma membrane in the
Drosophila egg chamber. Indeed, Syx1A is detected on the remaining
plasma membrane of both rab6-null and sec5 egg
chambers, supporting the existence of a Rab6- and Sec5-independent
exocytic pathway mediating protein export. This selective loss of Sec5 from
nurse cell membranes in rab6 open syncytia, together with the known
functions of the exocyst, suggest a simple explanation for the defects caused by a lack of Rab6 function in oogenesis. It is hypothesized that Rab6-dependent and -independent pathways might differ qualitatively in the proteins whose traffic they mediate, or
quantitatively in their relative contributions to the delivery of the same
cargo between nurse cells and oocyte. These differences may account for the
observed differential requirement for Rab6 in the localization of Sec5 in
nurse cell, versus oocyte, plasma membranes (Coutelis, 2007).
Our analysis has revealed two separate functions of Rab6: one is a general
role in the organization and growth of the egg chamber, and the other is its
specialized role in MT cytoskeleton polarization and oskar mRNA
localization. This second function appears specific to Rab6 because, in sec5 mutant egg chambers, Staufen localization is normal and the MT cytoskeleton is correctly organized. Only oskar mRNA, and not Oskar protein, is ectopically detected in
rab6D23D egg chambers. This suggests an
impairment of oskar mRNA localization, rather than a defect in its
anchoring, in which case Oskar protein would be detected with the detached RNA.
Defects in oskar mRNA localization, which relies on MT polarity,
could be due to a failure in the focusing of the MT cytoskeleton that is
observed in rab6 egg chambers (Coutelis, 2007).
In Drosophila and mammalian cells, BicD is known to regulate MT
organization. At mid-oogenesis, Rab6 and BicD cooperation could direct MT
organization and/or promote the vesicular transport necessary for oocyte
polarization and oskar mRNA localization. Given the implication of
membrane trafficking in the asymmetric localization of mRNAs, it also possible
that polarized membrane transport along the oocyte MT network directs
oskar mRNA to the posterior of the oocyte, by hitch-hiking along
trafficking vesicles (Coutelis, 2007).
In MDCK cells, definition of apical and basolateral plasma membrane domains
is required during polarization for the arrangement of MT along an
apical-basal axis. Vesicular trafficking is crucial to establish, specify and
maintain these membrane domains. By analogy, at stage 7, the polarizing signal from the posterior follicular cells to the Drosophila oocyte that causes repolarization of the MT cytoskeleton might do so by inducing the definition of anterior-lateral and
posterior membrane domains. It is therefore possible that, in
rab6D23D oocytes, as in epithelia, defects in vesicular
trafficking and TGN sorting underlie the observed defects in MT-network
organization. Consistent with this idea, a mispolarized MT cytoskeleton is
also observed in oocytes lacking Rab11. Thus,
vesicular trafficking and the specification of membrane domains may be
required for repolarization of the MT network and for the localization of
molecular determinants in the Drosophila oocyte at mid-oogenesis (Coutelis, 2007).
The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. This study describes a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. Evidence is provided that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFα homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. These results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport (Januschke, 2007).
In vertebrate cells, Rab6 is associated with the Golgi and the trans-Golgi network (TGN) membranes. To investigate the subcellular localization of Drab6 in the Drosophila germ line, the expression pattern was monitored of
transgenic lines expressing Drab6 fused to GFP and RFP. It was observed that
during oogenesis, the global distribution of Drab6 evolved. Drab6 first
accumulated transiently in a central position during stages 7/8, then is
uniformly distributed at the beginning of stage 9 to end up juxtaposed to the
entire oocyte cortex. It is noteworthy that promoters of different strengths give similar expression patterns. In addition, the genomic null allele rab6D23D is fully rescued by the different lines expressing Drab6 (Januschke, 2007).
Drab6 does not colocalize extensively with ER membranes (labeled with
PDI-GFP). Instead, it seems to be differentially associated with two types of Golgi units as revealed by its association with Lava Lamp (Lva and GalT). Lva, a cis-Golgi marker, colocalizes with Drab6, mainly at the cortex of the oocyte and in nurse cells. A GFP trap protein corresponding to a UDP-galactose:beta-N-acetylglucosamine beta-1,3-galactosyltransferase (GalT), enriches predominantly in Golgi membranes, exhibits a distribution similar to that of GFP-Drab6: it accumulates in the center of the oocyte at stage 8, where it colocalizes with Drab6, and is later confined to the cortex. Importantly, the distribution of Lva and GalT is similar in both matαtubGFP-Drab6, ubiRFP-Drab6 and control oocytes. Given that Lva and GalT markers are not present in the Golgi cisternae that are evenly distributed throughout the oocyte, as documented by electron microscopy (EM) analysis, they might be the hallmark of distinct functional Golgi units, with Drab6 being able to interact with both types of Golgi. Unlike Lva, the distribution of which is only mildly affected, GalT and acetylglucosamine-modified proteins [detected by the wheat germ agglutinin lectin (WGA)] expressed by Golgi structures are abnormally distributed in Drab6 mutants. Moreover, ultrastructural analysis by EM revealed that the ER is abnormally swollen in Drab6 mutant oocytes, and that the Golgi mini-stacks are markedly curved, with partially inflated cisternae (Januschke, 2007).
These morphological effects led to an investigation of the role of Drab6 in the secretory pathway. The polarized secretion of the TGFα-like growth factor Grk was monitored. Grk secretion is restricted to the anterodorsal corner through a rapid transit from the ER towards the Golgi apparatus. In GFP-Drab6-rescued egg chambers, Grk and Drab6 colocalize. In Drab6 mutant oocytes, grk mRNA localization is the same as in wild type. Grk protein, however, is slightly more abundant than in controls and an important fraction extends ventrally. Polarized secretion of Grk lead to the formation of two dorsal appendages on the egg shell. In the absence of Drab6, mislocalized Grk induces ventralization, instead of a dorsalization (multiple dorsal appendages on the egg shell) as observed when Grk is ectopically secreted. Hence, this argues for a specific failure of Grk delivery to the plasma membrane. This phenotype is specific to Drab6 because it could be fully rescued by the GFP-Drab6 transgene (Januschke, 2007).
Next, the intracellular localization of Grk was examined in the absence of
Drab6. Grk accumulated frequently in large ring-like particles in the Drab6 mutant, but not in control oocytes. These Grk 'rings', similar to those of yolk granules, did not contain Lva, suggesting that Grk is not blocked in the Golgi. Grk actually accumulates in Drab6 mutants on vesicles stained by LysoTracker, which labels either lysosomes or late endosomes containing yolk granules. Hence, two independent approaches suggest that Grk is not blocked in the Golgi, but is mislocalized to post-Golgi compartments, probably endosomes (Januschke, 2007).
Interestingly, the secretory impairment was also confirmed by
Lycopersicon esculentum tomato lectin (LE) detecting modified
proteins in the Golgi. In the absence of Drab6, LE revealed abnormal vesicular
structures in the oocyte and nurse cells that fail to reach the cortex. EM analysis also demonstrated rupture of the plasma membrane between neighboring nurse cells. Finally, it was observed that GFP-Drab6-rescued egg chambers exhibit an accumulative enrichment of Drab6 at the plasma membrane during oogenesis, which is particularly evident in nurse cells. This is consistent with the involvement of Drab6 in secretion towards the plasmalemma (Januschke, 2007).
The existence of three important and novel aspects of
Drab6 function during oogenesis has been established as follows: (1) Consistent with its localization in vertebrate cells, Drab6 is
predominantly localized to the Golgi complex in Drosophila, but
overlaps with Golgi markers that have distinct localizations, suggesting that
Drab6 might associate with distinct functional Golgi units. Drab6 might also
play a role in membrane exchange between Golgi and ER and in Golgi
organization, according to EM analysis, which is again consistent with
known functions of mammalian Rab6. (2) By controlling the migration of Golgi units towards the cell
cortex, Drab6 controls the delivery of membrane to the plasmalemma, as shown
in Drab6 mutants in which glycosylated proteins labeled by WGA and LE
lectins accumulate in large vesicular structures. This pattern is similar to
the mislocalization profile of Grk in the absence of Drab6.
(3) In the oocyte, Drab6 is required for the anterodorsal secretion of
Grk, which leads to the differentiation of the follicle cells required for the
morphogenesis of the dorsal appendages of the egg shell. In the absence of
Drab6, it was observed that Grk is mislocalized to late endosomal or lysosomal
compartments, demonstrating that Drab6 also affects post-Golgi traffic. In
vertebrates, one of the Rab6 isoforms (Rab6A') is also involved in
endosome-to-Golgi transport. Additionally, a role for Ypt6p (the only copy of Rab6 in
the yeast S. cerevisiae) has also been documented as being involved
in fusion of endosome-derived vesicles with the late Golgi (Siniossoglou, 2001). It remains to be established whether Drab6 functions directly in the secretory pathway or if the effects observed in Drab6 mutants on post-Golgi trafficking are a consequence of defects in endosome-to-Golgi trafficking (Januschke, 2007).
In order to identify potential Drab6-binding proteins, a yeast
two-hybrid screen was performed using as bait Drab6Q71L, a GTPase-deficient mutant. Sixty-two distinct truncated clones of BicD, lacking parts of the amino-terminus, interacted with Drab6Q71L. The intersection
of all identified fragments defined a minimal interacting domain, mapping to
amino acids 699-772 in the coiled-coil motif H4 of BicD, shown for murine BicD to interact with the mammalian Rab6. In order to validate this interaction, glutathione S-transferase (GST) pull-down assays were performed, using lysates from wild-type ovaries. GST-Drab6 specifically retained BicD; GST alone and GST-Rab1 did not bind BicD. Furthermore, preloading GST-Drab6 with the non-hydrolyzable GTP analog, GTP-γ-S, yielded an improved interaction with BicD. It is concluded, therefore, that in vitro, BicD interacts through its carboxy-terminus preferentially with the active form of Drab6 (GTP-bound), as has been shown for mammalian Rab6 (Januschke, 2007 and references therein).
Time-lapse recording showed that in the oocyte and nurse cells, RFP-Drab6 and BicD-GFP colocalize to multiple large aggregates with low dynamics. Further GFP-Drab6 accumulation in the center depends on the presence of BicD during stage 8, as observed in a BicDmom background. Interestingly, in such BicDmom oocytes, Grk is found in ring-like structures remote from the nucleus, as observed in Drab6 mutant oocytes (Januschke, 2007).
Since BicD and Rab6 have been shown to be involved in MT-based transport, experiments were conducted to discover
whether Drab6-positive structures require MTs for movement. Time-lapse microscopy revealed that large aggregates are less dynamic than the highly motile small particles. Colchicine MT depolymerization severely reduced the movement of Drab6 particles, which form large clusters, indicating that Drab6 is actively transported along MTs. The MT motors Kinesin I [Kinesin heavy chain (Khc)] and Dynein have been shown to be involved in polarizing the Drosophila oocyte. Inactivating the Dynein complex by the overexpression of Dynamitin prevents accumulation of Drab6 at the oocyte cortex, but does not significantly reduce Drab6 movements. By contrast, in Khc7.288 germ line clones, Drab6 does not localize in the center of the oocyte during stage 7/8 but forms abnormal aggregates around the mispositioned nucleus. For reasons not currently fully understood, the speed of Drab6 particles is significantly reduced compared with controls or Dynamitin-overexpressing oocytes (Januschke, 2007).
Drab6 and BicD interact in a yeast two-hybrid screen and
in GST pull-down assays and colocalize in vivo. Moreover, there are
indications that Drab6 requires BicD for correct subcellular localization,
which suggests that Drab6 interacts with BicD in Drosophila as it does in mammals. Strikingly, it was found that lack of each protein compromises Grk secretion in a very similar way. Overexpression of Dynamitin, to impair Dynein function, induces ectopic accumulation of Grk and ventralization of the egg shell (Januschke, 2002). Therefore, in Drosophila, BicD/Dynein and Drab6 are likely to be involved together in Grk secretion to the anterodorsal corner of the oocyte (Januschke, 2007).
It is important to mention that colocalization of the two proteins is
limited. Moreover, lack of BicD or Drab6 yields different phenotypes. BicD mutation affects oocyte determination and the position of the oocyte nucleus, but has no impact on MT organization in mid-oogenesis, which is not the case in the Drab6 mutant. A genetic interaction between BicD's co-factor Egalitarian and Kinesin I has already been demonstrated, suggesting that Drab6 might interact with Dynein and Kinesin I via BicD (Januschke, 2007).
Interestingly, it was noticed that in the absence of Drab6, osk mRNA
is not correctly localized in the oocyte. gurken and bicoid mRNAs are, however, unaffected, and osk mRNA localization to the oocyte center is frequent when the MT network is not correctly polarized. In Drab6 mutant oocytes, the defective posterior localization of the MT plusend marker Khc-ß-Gal indicates a defect in MT organization (Januschke, 2007).
Given that Drab6 is required for late Grk signaling at the anterodorsal
corner of the oocyte, it might also be involved in early germ line to soma signaling mediated by Grk, which controls MT organization. This is thought unlikely. In the absence of this signaling, posterior follicle cells differentiate into anterior follicle cells and, as a consequence, the posterior structure of the egg shell, the aeropyle, is substituted with an anterior structure, the micropyle. An aeropyle is always observed at the posterior of eggs derived from Drab6 mutant oocytes. Additionally, removing Drab6 from the posterior follicle cells does not affect oocyte polarity. Hence, Drab6 is possibly involved in MT organization at the posterior pole. Interestingly, Rab6 family interactors such as Rab6IP2/ELKS are capable of interacting with CLASPs at the cortex of HeLa cells, suggesting a link between Rab6 protein and MT organization at the cortex (Januschke, 2007).
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. 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. Egl and BicD also associate with dynein light chain (Dlc) and the dynein/dynactin complex, respectively, and are recruited to injected localizing mRNAs in embryos to bias the net movements of a bidirectional mRNA transport complex apically. 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. 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 (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. This is based on the findings that the CTD can be functionally substituted by heterologous motifs for organelle recognition 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. 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) prevents recruitment of Egl to bcd injected into embryos. 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. 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. 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. 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, 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. The N terminus of BicD can efficiently capture dynein/dynactin components from cell extracts, 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. 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).
The expression of the RNA-binding factor Fragile X mental retardation protein (FMRP) is disrupted in the most common inherited form of cognitive deficiency in humans. FMRP controls neuronal morphogenesis by mediating the translational regulation and localization of a large number of mRNA targets, and these functions are closely associated with transport of FMRP complexes within neurites by microtubule-based motors. However, the mechanisms that link FMRP to motors and regulate its transport are poorly understood. This study shows that FMRP is complexed with Bicaudal-D (BicD) through a domain in the latter protein that mediates linkage of cargoes with the minus-end-directed motor dynein. In Drosophila the motility and, surprisingly, levels of FMRP protein are dramatically reduced in BicD mutant neurons, leading to a paucity of FMRP within processes. Functional evidence is provided that BicD and FMRP cooperate to control dendritic morphogenesis in the larval nervous system. These findings open new perspectives for understanding localized mRNA functions in neurons (Bianco, 2010).
BicD proteins (BicD in Drosophila and BicD1 and BicD2 in
mammals) play roles in the transport of a subset of cargoes
by the minus-end-directed microtubule motor dynein. The N-terminal two-thirds of BicD interact with dynein and its accessory complex dynactin, and the C-terminal third (the C-terminal domain [CTD]) mediates mutually exclusive association
with different cargoes. The best-characterized roles of BicD proteins are in the bidirectional transport of Golgi vesicles and a subset of asymmetrically localized Drosophila mRNAs, which are mediated by binding of the CTD to the
membrane-associated G protein Rab6 and the RNA-binding
protein Egalitarian (Egl), respectively. The interactions
of the BicD CTD with both proteins are inhibited by the
K730M substitution in the Drosophila BicD protein, which
is a null mutation in vivo. K730M does not, however, inhibit
binding of the BicD CTD to other copies of BicD, indicating
that it specifically effects association of BicD with motor cargoes (Bianco, 2010).
In an attempt to elucidate the basis of linkage of other cargoes
to dynein, a GST pull-down from fly embryonic
extracts was performed with the Drosophila BicD CTD (amino acids 536-782) and an equivalent K730M mutant protein as a specificity
control. Mass spectrometry revealed that a protein of 80-85
kDa reproducibly recruited only by the wild-type CTD was Drosophila FMRP (27 unique peptides), and this was confirmed by western blotting. Endogenous
BicD and FMRP were specifically coimmunoprecipitated
from Drosophila embryonic extracts. Unlike known Egl-interacting proteins, FMRP was not coimmunoprecipitated with a GFP-tagged Egl protein. This finding,
together with the observation that binding of both Egl and
FMRP to BicD is impaired by the K730M mutation, suggests that BicD:FMRP complexes are largely, or completely, distinct from BicD:Egl complexes (Bianco, 2010).
The ability to detect FMRP in CTD pull-downs and BicD
immunoprecipitations from extracts was abolished by treatment
with RNase. In contrast, the complex of Egl with BicD was not sensitive to RNase treatment. Thus, the stable association of BicD and FMRP
in extracts is dependent on RNA. Nonetheless, a weak interaction of the BicD CTD with a subfragment of FMRP (aa 220-618) was found in yeast two-hybrid assays. This interaction was specific, as shown by the fact that it was disrupted by the K730M mutation within the BicD CTD. These findings raise the possibility of a direct contact of BicD and FMRP in vivo that is stabilized by the association of FMRP
with RNA targets and possibly other RNA-associated proteins (Bianco, 2010).
These results suggest that BicD could be a functional
interactor of FMRP in vivo. Subsequent studies therefore focused on neurons, where FMRP plays a prominent role. As previously observed, endogenous FMRP is
enriched in puncta within the cell body and neurites of
Drosophila primary neurons cultured from larval brains. Endogenous BicD was also found in puncta in these cells, but these were much more frequent than those
containing FMRP. Although there was overlap of a subset of FMRP puncta with BicD puncta, the widespread distribution of BicD precluded
a meaningful interpretation about the extent of complex
formation of BicD and FMRP in fixed primary neurons (Bianco, 2010).
Therefore neuronal cultures were established from brains of
transgenic larvae expressing FMRP::GFP and BicD::mCherry
and time-lapse microscopy was used to assay for cotransport of
puncta containing both proteins. These fluorescent fusion proteins retain function
and account for ~20% and 50% of the levels of total FMRP and BicD proteins, respectively, in transgenic larval brain extracts (Bianco, 2010).
Both BicD::mCherry and FMRP::GFP were widely distributed
in the cytoplasm of the primary neurons, but bidirectionally
transported FMRP::GFP puncta were found in all cells and
92.4% ± 3.2% of them were cotransported with a puncta of
BicD:mCherry. Thus, FMRP and BicD can be contained within the same motile transport complexes in neurons. The motility of FMRP::GFP in these experiments
will be described in more detail below. Only 77.2% ± 4.6%
of motile BicD::mCherry puncta were cotransported with
a puncta of FMRP::GFP (155 particles in 20 cells), indicating
that BicD may transport additional cargoes in these cells
and/or that a subset of BicD::mCherry complexes may contain
only nonfluorescent, endogenous FMRP (Bianco, 2010).
Next whether BicD has a functional role in FMRP:motor complexes in neurons was explored by assessing the subcellular localization of FMRP in third instar BicD mutant larvae. Because the high expression of FMRP expression in neighboring
nonneuronal cells obfuscates the distribution of the endogenous protein in thin neuronal processes, UAS-FMRP::GFP was expressed at low levels by using a panneuronal GAL4 driver. In neurons of zygotic BicD null mutant
larvae, which also lack detectable maternal BicD protein, the amount of FMRP::GFP within the neurites was greatly reduced compared to wild-type. Surprisingly, there was also a much weaker FMRP::GFP signal in the cell body of BicD mutant neurons relative to wild-type. Western blotting of third instar larval brain extracts confirmed a striking reduction in levels of both FMRP::GFP and endogenous
FMRP in the absence of BicD (Bianco, 2010).
Strong mutations in genes encoding the dynein and kinesin-1
motor proteins, which should inhibit microtubule-based FMRP
transport in Drosophila, did not alter the amount of FMRP. These findings, together with observations from interfering with dynactin function, suggest that
the reduction in FMRP protein levels in BicD mutants is caused
by a specific role of BicD rather than an indirect consequence
of inefficient FMRP transport (Bianco, 2010).
Levels of the Fmr1 mRNA, which encodes FMRP, were indistinguishable
in wild-type and BicD mutant brain extracts, as revealed by quantitative RT-PCR. Thus, the requirement for BicD in maintaining FMRP protein levels is
not associated with RNA decay or transcription. Further
evidence against a defect in Fmr1 transcription in BicD
mutants is provided by the strong reduction in the levels of
the GFP-tagged FMRP protein, which is transcribed under the control of yeast-derived UAS promoter elements. The FMRP::GFP transgene also lacks the untranslated
sequences from the Fmr1 gene, revealing that BicDs regulation of FMRP protein
amount is mediated through the Fmr1 coding sequence. BicD may therefore influence FMRP protein stability through an unknown mechanism. However, it currently cannot be rule out that BicD regulates the translation of FMRP; at least in
mammals, the coding sequence of Fmr1 mRNA contains
a translational control element, which negatively regulates
protein production by binding FMRP. Distinguishing
between these and other possibilities will require long-term
studies. Interestingly, the underlying mechanism appears to
be restricted to certain cell types as shown by the fact that
FMRP levels in cultured Drosophila D-Mel cells (a derivative
of S2 cells) were not reduced by RNAi-mediated depletion of BicD (Bianco, 2010).
To investigate whether BicD also has a role in controlling
FMRP motility, the distribution of residual FMRP::GFP was examined in BicD mutant primary cultured larval neurons. There was a strong decrease in the proportion of FMRP::GFP particles that localized to neurites in BicD mutants compared
to wild-type, with FMRP particles also less likely
to reach the most distal regions of the mutant processes. The changes in FMRP distribution are unlikely to result from differences in cellular morphology or general effects on trafficking processes because the length and
complexity of neurites, as well as the distribution of mitochondria,
was comparable in BicD mutant and wild-type neurons (Bianco, 2010).
To test directly whether BicD is required for FMRP motility,
time-lapse imaging of FMRP::GFP particles was performed in
cultured larval neurons. FMRP particles in wild-type neurons were
usually stationary during several minutes of filming, but
some occasionally underwent periods of rapid, directed movement. Motile particles in the processes exhibited persistent motion both toward and away from the cell body, with some particles rapidly switching directions. There was
no overall bias in the length of directed, continuous movements
(run lengths) toward and away from the cell body, consistent with a completely mixed microtubule polarity in both neurites and the soma (Bianco, 2010).
Consistent with BicDs well-characterized role in dynein/
dynactin-mediated transport, inhibition of dynactin function
by neuron-specific expression of a dominant-negative version
of the p150Glued subunit (DGlued) strongly reduced the
motility of FMRP puncta and their localization into neuronal processes. Despite the strong difference in efficiency of FMRP transport, the amounts of FMRP were indistinguishable between DGlued and wild-type extracts. This observation provides further evidence that the role of BicD in regulating FMRP protein levels is not due to a general effect of inhibiting transport (Bianco, 2010).
BicD is complexed with dynein and the plus end motor
kinesin-1 on at least some bidirectional cargoes, and
a kinesin-1 family member associates with FMRP complexes
and contributes to their transport in mammalian neurons.
In Drosophila primary neurons with a strong kinesin-1 heavy
chain mutant genotype (Khc17/27), there was a striking alteration
of FMRP appearance compared to wild-type cells, with
discrete particles not detectable above the diffuse cytoplasmic
signal. This observation, which is reminiscent of the reduced size of a kinesin-1 mRNP cargo in Drosophila oocytes, raises the possibility that both dynein
and kinesin-1 cooperate in FMRP transport in Drosophila neurons (Bianco, 2010).
BicD may have a direct role as a constituent of FMRP:motor
complexes. Alternatively, reduced levels of FMRP in BicD
mutants might have an indirect effect by reducing the probability
of FMRP encountering other transport factors. To
attempt to discriminate between these possibilities, advantage was taken of the observation that overexpression of BicD, even to a very large extent, does not alter the total amount of FMRP. This presumably reflects wild-type levels of BicD being nonlimiting for the function in controlling FMRP levels (Bianco, 2010).
2-fold overexpression of BicD (tagged with mCherry)
dramatically increased the run lengths and net displacements
of motile FMRP::GFP particles in cultured neurons, compared
to the wild-type. Run lengths in processes
were similar for movements both toward and away from the
cell body upon BicD overexpression. Nonetheless,
there was increased targeting of FMRP into distal
processes compared to wild-type. Once again, this presumably reflects the ability of long-distance, unbiased bidirectional transport to aid cargo spreading.
These results demonstrate that BicD is able to control motility
and subcellular localization of FMRP independently from the
role in regulating overall levels of the protein (Bianco, 2010).
The results of quantification of particle motility, together
with observations that (1) FMRP is recruited by means of
the domain of BicD involved in linking cargoes to dynein and (2) FMRP colocalizes in motile particles with BicD in vivo, provides strong
evidence that BicD plays a direct role in FMRP:motor
complexes. In the case of other cargoes studied, BicD is not
obligatory for their linkage to motor complexes but increases
their travel distances significantly. The residual directed transport of FMRP particles in BicD mutant neurons suggests that BicD may play a similar stimulatory role in this context. Other components of FMRP-containing transport
particles presumably also contribute to linkage with motor proteins (Bianco, 2010).
The functional significance of the BicD:FMRP
interaction in dendrites was explored by focusing on the role of FMRP in dendritic
morphogenesis. The well-characterized model
system for dendritic development in the Drosophila third instar
larva, the dorsal class IV dendritic arborization (da) neuron
ddaC, was explored (Bianco, 2010).
Dorsal ddaC neurons within zygotic BicD mutant larvae had
a much less extensively branched dendritic arbor than wildtype
cells. A similar inhibition of the dendritic branching program was observed in three different zygotic Fmr1 null mutant genotypes. Intermediate terminal branching
defects were also found in ddaC neurons heterozygous for
Fmr1D50M. This phenotype, which could be suppressed by the FMRP::GFP transgene, underscores the importance of correct
FMRP protein levels for neuronal morphogenesis (Bianco, 2010).
These results demonstrate that both BicD and FMRP are
required for efficient branching of the dendritic arbor in dorsal
ddaC neurons. Interestingly, FMRP negatively regulates
dendritic elaboration in mushroom body neurons in adult
brains. It has also previously been reported that mutating
Fmr1 increases branching of ventral da neurons, although
effects on specific classes of neurons within the cluster have
not been reported. The differential requirements for Fmr1 in controlling
the morphology of different neuronal cells is consistent
with previous findings. It has been showed that Fmr1 mutations cause overextended axons in LNv cells but a failure of axon extension in DC neurons. Cell type-specific effects of FMRP on neuronal morphogenesis may reflect differences in
the repertoire of its mRNA targets (Bianco, 2010).
BicD overexpression specifically in class IV da neurons
significantly increased the number of dendritic branches in
the distal regions of arbors in dorsal ddaC neurons compared
to wild-type. This result, together with the diminished branching in BicD mutant neurons, reveals a correlation between the amount of available BicD and the
degree of arborization of ddaC and that BicD can function
autonomously within neurons to control this process (Bianco, 2010).
Strikingly, the ability of overexpressed BicD to augment dendritic branching of ddaC appears to be due predominantly to its interaction with FMRP, as evidenced by a strong suppression of the BicD overexpression phenotype in Fmr1 null mutants, with neuronal morphology not significantly different to the Fmr1 mutant alone. Because BicD overexpression does not alter FMRP protein levels, increased branching is likely to be influenced by BicDs ability to control FMRP motility. Live cell imaging revealed that BicD promotes long-distance bidirectional transport of FMRP complexes on microtubules, thereby facilitating the exploration of neuronal processes. Such a mechanism may increase the probability of encounters of these complexes with factors that activate translation of associated mRNAs, which in some contexts could be responsive to local signaling. Nonetheless, the reduction of overall FMRP protein levels is highly likely to contribute to BicD loss-of-function phenotypes in da neurons, as potentially is the altered transport of FMRP-independent cargoes (Bianco, 2010).
A DEAD-box protein, Me31B, forms a cytoplasmic RNP complex with oocyte-localizing RNAs. During early oogenesis, loss of Me31B causes premature
translation of oocyte-localizing RNAs within nurse cells, without affecting their transport to the oocyte. In early egg chambers that lack Me31B, at least two mRNAs in particles, OSK and Bicaudal-D mRNAs, are prematurely translated in nurse cells, though the transport of these RNAs to the oocyte is Me31B independent. These results suggest that Me31B mediates
translational silencing of RNAs during their transport to the oocyte. These data provide evidence that RNA transport and translational control are
linked through the assembly of RNP complex (Nakamura, 2001).
A visual screen was conducted with an ovarian GFP-cDNA library, in which fusion genes are expressed in germline cells during oogenesis. Transgenic flies were generated with this library and proteins were identified that distribute in a granular pattern during oogenesis. Screening ~3000 independent lines, one was isolated in which GFP signals were detected as cytoplasmic particles during oogenesis. The particles were dispersed in the cytoplasm of both nurse cells and oocytes but never detected within nuclei. The particles were frequently observed passing through ring canals, suggesting that the particles are assembled in nurse cell cytoplasm and transported to the oocyte (Nakamura, 2001).
The cDNA from this line was identified as me31B. In the cDNA fusion, almost the entire coding region of me31B, which lacks only the first four codons, was fused in frame with that of gfp. Me31B, a DEAD-box protein and therefore a putative ATP-dependent RNA helicase, was isolated as a gene expressed extensively during oogenesis. Me31B is a part of an evolutionally conserved DEAD-box protein group, which includes human RCK/p54 (71% identical), Xenopus Xp54 (73%), Caenorhabditis elegans C07H6.5 (76%), Schizosaccharomyces pombe Ste13 (68%) and Saccharomyces cerevisiae Dhh1 (68%). Furthermore, Me31B is phylogenetically close to two evolutionally conserved proteins, eIF4A and Dbp5/Rat8p but far from Vasa, which functions in germline development (Nakamura, 2001).
To examine distribution of the endogenous Me31B, antibodies were generated that specifically recognized Me31B. The distribution pattern of endogenous Me31B is identical to that of GFP-Me31B. No detectable signal in somatic follicle cells is observed at any stage of oogenesis. Me31B is first detected at a low level in germarium region 2B, where the signal is concentrated in the pro-oocytes. The signal remains concentrated in the oocyte until mid-oogenesis. In early egg chambers, a low level Me31B signal is detected in nurse cell cytoplasm. In both nurse cells and oocytes, the signal appears to be granular. Me31B signals in nurse cell cytoplasm become more evident from stage 5-6, when Me31B expression is drastically increased. In addition, Me31B is frequently enriched around nurse cell nuclei. Later, Me31B accumulates at the posterior pole of stage 10 oocytes. However, this posterior accumulation is transient, as revealed by uniform distribution of the signal in cleavage embryos. By cellular blastoderm stage, Me31B becomes undetectable in the entire embryonic region. No zygotic expression of Me31B was detected during embryogenesis (Nakamura, 2001).
Because Me31B is probably an RNA-binding protein that is transported to the oocyte, it was asked whether Me31B forms a complex with oocyte-localizing RNAs. Colocalization of OSK mRNA with Me31B was examined. OSK mRNA starts to accumulate in oocytes from germarium region 2B, with the concentration of OSK increasing over time. Posterior accumulation of OSK mRNA in the oocyte begins from stage 8 onwards. By fluorescent in situ hybridization, OSK mRNA exhibits particulate signals in the cytoplasm of both nurse cells and oocytes, and is frequently concentrated around nurse cell nuclei. This distribution pattern of OSK mRNA is essentially identical to that of Me31B, with colocalization present until OSK mRNA localizes to the posterior pole of stage 10 oocytes (Nakamura, 2001).
Colocalization of Me31B with other RNAs was also examined. Ovaries were doublestained for Me31B and BicD mRNA. In early egg chambers, BicD mRNA also produces particulate signals, and appears to localize in Me31B-containing particles. This colocalization becomes apparent from stage 5-6, when BicD mRNA expression is elevated. The oocyte-localizing RNAs examined [BCD, NOS, Oo18 RNA-binding (ORB), Polar granule component (PGC) and Germ cell-less (GCL)] all produce particulate signals in the cytoplasm of both nurse cells and oocytes, and colocalize with Me31B. In contrast, Vasa mRNA, which is not specifically transported to the oocyte, does not appear to be colocalized with Me31B. These results indicate that Me31B forms cytoplasmic particles that contain oocyte-localizing RNAs (Nakamura, 2001).
The complicated and redundant phenotypes observed in me31B- egg chambers in mid-oogenesis are unlikely to be the primary effect of loss of me31B function. Earlier phenotypes of me31B- egg chambers were examined using a FLP/FRT system to generate homozygous germline clones that are marked by the loss of Vas-GFP fusion protein. Based on Hoechst and phalloidin staining, me31B- egg chambers are morphologically normal until stage 4-5. From stage 6 onwards, oocytes in me31B- egg chambers fail to grow normally. At this stage, these egg chambers begin to degenerate. In early me31B- egg chambers, Exu signal is concentrated to the oocytes. Distributions of OSK and BicD mRNAs in me31B- egg chambers were examined. Both OSK and BicD mRNAs also accumulate in the oocytes of me31B- egg chambers until the chambers degenerate. Particulate signals for these RNAs are detectable in nurse cell cytoplasm in these egg chambers. These results indicate that Exu, OSK and BicD mRNAs can be transported to the oocyte even in the absence of Me31B. It is concluded that in early egg chambers, Me31B is dispensable for the transport of the molecules that form a complex with Me31B (Nakamura, 2001).
Whether loss of Me31B affects translation of OSK and BicD mRNAs was examined. Ovaries were immunostained with an anti-Osk antiserum. Although OSK mRNA is expressed during almost all stages of oogenesis, its translation is repressed to keep Osk protein level very low during early oogenesis. In me31B- egg chambers, Osk signal is significantly increased compared with that in the neighboring me31B+ egg chambers (Nakamura, 2001).
A similar increase of BicD signal in me31B- egg chambers is more evident. In wild-type egg chambers, BicD protein, like BicD mRNA, is highly concentrated in the oocytes starting from germarium region 2B. In the egg chambers lacking me31B, increased BicD signal is detected in nurse cell cytoplasm. These results suggest that loss of Me31B in germline cells causes derepression of OSK and BicD mRNA translation during their transport to the oocyte (Nakamura, 2001).
Both BIC-D RNA species are present in very early embryos suggesting a maternal origin. The level of both Bic-D species drops in the 2-4 hour interval, and by 4-8 hours there is virtually no BIC-D mRNA in the embryo. After 8 hours, the larger, 4.4 kb transcript reappears and is detected at all stages of the life cycle, including in adult males. A third transcript of 5.7 kb is present in late embryos, pupae and adult males. The function(s) of zygotically expressed Bic-D is currently unknown (Suter, 1989).
In adult female cystocytes the transcript is found up to stage 7 only in the oocyte, where it is concentrated around the nucleus. At about stage 8, an increase of signal is observed in the nurse cell complex, and the accumulation of Bic-D mRNA increases up to stage 11 when the nurse cells empty their cytoplasmic contents into the oocyte. At stage 8, the oocyte begins to increase in size due to the deposition of yolk and nurse cell contents, and it is during this period that the BIC-D transcript is localized to the anterior end of the oocyte, the mRNA forming a cap covering the anterior end of the oocyte. By stage 11-12, when deposition is terminated, the mRNA is no longer localized in the oocyte, but is found nearly uniformily distributed. This distribution is maintained throughout the last stages of oogenesis (Suter, 1989).
Drosophila oocytes develop within cysts containing 16 cells that are interconnected by cytoplasmic
bridges. Although the cysts are syncytial, the 16 cells differentiate to form a single oocyte and 15 nurse
cells; several mRNAs that are synthesized in the nurse cells accumulate specifically in the oocyte.
To gain insight into the mechanisms that generate the cytoplasmic asymmetry within these cysts, cytoskeletal organization was examined during oocyte differentiation. Shortly after formation of the 16
cell cysts, a prominent microtubule organizing center (MTOC) is established within the syncytial
cytoplasm; at the time the oocyte is determined, a single microtubule cytoskeleton connects the
oocyte with the remaining 15 cells of each cyst. Recessive mutations at the Bicaudal-D and
egalitarian loci, which block oocyte differentiation, disrupt formation and maintenance of this
polarized microtubule cytoskeleton. Microtubule assembly-inhibitors phenocopy these mutations, and
prevent oocyte-specific accumulation of Oskar, Cyclin B and 65F mRNAs. It is proposed that formation
of the polarized microtubule cytoskeleton is required for oocyte differentiation, and that this structure
mediates the asymmetric accumulation of mRNAs within the syncytial cysts (Theurkauf, 1993).
Some of the spatial cues that direct early patterning events in Drosophila embryogenesis are
maternal mRNAs localized in the oocyte during oogenesis. For example, Bicaudal-D, fs (1) K10, and
Orb RNAs are transiently localized at the anterior oocyte margin in mid oogenesis, and oskar RNA is
localized at the posterior oocyte margin beginning in mid oogenesis. Using inhibitors of cytoskeletal
function, it is found that microtubules, but not microfilaments, are required for localization of these
mRNAs during oogenesis, results similar to those described for Bicoid RNA. However, the
RNAs show a differential sensitivity to microtubule inhibitors. Anterior localization of Bicaudal-D, fs
(1) K10, and Orb RNAs is completely disrupted following even mild drug treatments. Bicoid RNA
localization is intermediate in its response to microtubule drugs, while Oskar RNA localization is much
more resistant. In addition, the localized RNAs respond differently to taxol, a microtubule stabilizing
agent. The differences among these RNAs suggest that factors other than microtubules are required to
maintain the positions of localized RNAs in the oocyte. Microtubules are also required for the
preferential accumulation of these transcripts in the previtellogenic oocyte, consistent with the idea that
these mRNAs are transported by a microtubule-dependent mechanism to the oocyte (Pokrywka, 1995).
Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions, transgenic flies were made expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different
phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation (Pare, 2000).
The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote,
subsequently have been shown to regulate cellular polarity in diverse
cell types by means of an evolutionarily conserved protein complex
including PAR-3, PAR-6, and atypical protein kinase C (aPKC).
The Drosophila homologs of par-1, par-3 (bazooka [baz]), par-6 (DmPar-6), and pkc-3
(Drosophila aPKC; DaPKC) each are known to play
conserved roles in the generation of cell polarity in the germ line as
well as in epithelial and neural precursor cells within the embryo. In
light of this functional conservation, the potential role
of baz and DaPKC in the regulation of
oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of
initial anterior-posterior polarity within germ-line cysts and
maintenance of oocyte cell fate. Germ-line clonal analyses indicate
both proteins are essential for two key aspects of oocyte
determination: the posterior translocation of oocyte specification
factors and the posterior establishment of the microtubule organizing
center within the presumptive oocyte. Baz and DaPKC
colocalize to belt-like structures between germarial cyst cells.
However, in contrast to their regulatory relationship in the
Drosophila and C. elegans embryos, these
proteins are not mutually dependent for their germ-line localization,
nor is either protein specifically required for PAR-1 localization to
the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).
Oocyte differentiation requires the polarized accumulation of oocyte
specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in
the localization of these factors, mutant germ-line clones
for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst
cells in region 2a, and then both molecules are targeted first to the
two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In
contrast, ORB fails to translocate from the anterior to a posterior
crescent in both baz and DaPKC null germ-line cysts in germarial
region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in
baz and DaPKC null germ-line clones in germarial
region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).
The posterior assembly of a functional MTOC has been directly
implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the
failure to translocate these factors to a posterior crescent in region
3 baz or DaPKC mutant cysts may result from a defect
in microtubule reorganization within these mutant cysts. In contrast to
wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of
oocyte specification factors in these mutants are likely caused, at
least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).
The Bic-D protein
contains four well-defined heptad repeat domains characteristic of intermediate
filament proteins, and several of the mutations in Bic-D map to these conserved domains. A structure-function analysis of Bic-D has been undertaken by testing the function of mutant Bic-D transgenes (Bic-DH) deleted for each of the heptad repeat domains in a Bic-D null background. These transgenic studies indicate that only the C-terminal heptad repeat deletion results in a protein that has lost zygotic and ovarian functions. The three other deletions result in proteins with full zygotic function, but with affected ovarian function. The functional importance of each domain is well correlated with its conservation in
evolution. The analysis of females heterozygous for Bic-DH and the existing alleles Bic-DPA66 or Bic-DR26 reveals that Bic-DR26 as well as some of Bic-DH transgenes have antimorphic effects. The yeast two-hybrid interaction assay shows that Bic-D forms homodimers. Furthermore, Bic-D exists as a multimeric protein complex consisting of Egl and at least two Bic-D monomers (Oh, 2000).
Besides causing a bicaudal phenotype (see biological overview), null alleles of BicD show a zygotic requirement. Null flies can be raised on an enriched medium, but on normal medium no adult flies hatch. Null adults raised on enriched medium are generally lethargic and uncoordinated, and they usually die within two or three days after eclosion. Bic-D is required to establish oocyte identity in one cystocyte and is essential, not only for the oocyte-specific accumulation of oocyte markers, but also for the posterior migration of the oocyte (Ran, 1994).
To analyze the role of BicD later in oogenesis, Drosophila lines have been constructed in which BicD expression is under the control of the hsp70 promoter. In these flies, BicD can be induced early in oogenesis, allowing an oocyte to be made. Then by shifting flies to non-inducing conditions, BicD levels are depleted for the remainder of oogenesis. Using this system, it can be seen that BicD is indeed required in the later stages of oogenesis. In ovaries from mutant females, oocyte growth is reduced, apparently due to defects in nurse-cell-to-oocyte transport. Smaller oocyte size results in the misalignment of follicle cells and the underlying germ line, leading to ventralization of dorsal follicle cells (as seen by a defect in dorsal/ventral patterning) and to defects in centripetal cell migration, giving rise to chorion defects. In addition, BicD is required for the localization of specific mRNAs at both the anterior and posterior of the oocyte (Swan, 1996).
The chromosomal region 36C on 2L contains two maternal-effect loci, dorsal (dl) and Bicaudal-D
(Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral
and anterior-posterior axes, respectively. To analyze the region genetically, X-ray-induced
dorsal alleles were isolated, which were recognized by virtue of the haplo-insufficient temperature-sensitive
dorsal-dominant phenotype in progeny of single females heterozygous for a mutagenized chromosome.
From the 20,000 chromosomes tested, three deficiencies were isolated: two inversions with breakpoint in
dl and one apparent dl point mutant. One of the deficiencies, Df(2L)H20 (36A6,7; 36F1,2) was used to
screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D.
44 lethal mutations were isolated defining 11 complementation groups. Maternal-effect mutations were recovered of four dl alleles, as well as six alleles of quail and one allele of kelch, two
previously identified maternal-effect genes. Through complementation tests with various viable mutants
and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically
visible bands. The region was subdivided into seven subregions by deficiency breakpoints. One lethal
complementation group as well as the two maternal loci, Bic-D and quail, are located in the same
deficiency interval as is dl (Steward, 1986).
To understand the role of Bicaudal-D in the establisment of oocyte polarity, a screen was carried out for genetic enhancers of Bic-D mutation. This screen identified the mutation E415 as a dominant genetic enhancers of Bic-D.
The mutation E415 maps to a genetic locus that codes for the Drosophila homolog of Lissencephaly-1 (DLis-1). Mutations in the human Lissencephaly-1 (Lis-1) gene cause Miller-Dieker syndrome. This syndrome is characterized by a
smoothened brain surface and disorganized cortical layers, resulting from
failed neuronal migration during brain development. Homologs of Lis-1
have also been identified in the filamentous fungus Aspergillus nidulans and in Saccharomyces cerevisiae, and both function with the microtubule minus-end-directed motor dynein/dynactin in nuclear migration (Swan, 1999).
Patterning of the Drosophila embryo depends on the correct localization
of patterning determinants within the oocyte, beginning in mid-oogenesis.
Starting in stage 8, specific mRNAs begin to accumulate either at the posterior
end of the oocyte or along the anterior cortex; this pattern is dependent
on Bic-D and microtubules. The distribution of anteriorly localized factors (orb, egl, nos and bcd mRNAs and Orb protein) and posteriorly localized factors (osk mRNA and Osk protein) were studied in E415 ovaries. All of these factors show disruptions in their normal subcellular localization within the oocyte. These effects on localization could be due to effects
on transport, anchoring or stability of transcripts in the mutant (Swan, 1999).
In stage 7 of oogenesis, the oocyte nucleus migrates from the posterior to the future
dorsal-anterior corner of the oocyte. In about half of stage-9 and stage-10 E415/E415 mutant egg chambers, the oocyte nucleus is positioned incorrectly. This could be due to a failure in nuclear migration or anchoring. Since the oocyte nucleus takes up almost the entire oocyte in stage-8 and earlier mutant egg chambers, it is not possible to determine whether nuclear positioning is affected before stage 9. Bic-D is also required for positioning of the oocyte nucleus2, and therefore E415 could function in nuclear positioning through the Bic-D-Egl complex. Consistent with this view, Bic-D and Egl proteins accumulate at high levels in a region between the oocyte cortex and nucleus: this localization is abolished in E415 mutants. Microtubules are also required for oocyte nuclear positioning; whereas microtubule organization in E415 homozygous mutants appears to be unaffected in early oogenesis, some alterations in microtubules are observed after stage 7. In wild-type oocytes, microtubules appear to be concentrated at the anterior cortex, apparently reflecting their nucleation from anterior sites. Microtubules still appear to be organized in this way in E415 mutants, but are often less focused (Swan, 1999).
DLis-1 shares with the dynein heavy chain gene, Dhc, a requirement
in oocyte determination, indicating that the dynein heavy chain may function like its fungal homologs in a pathway with dynein/dynactin. A specific allele of Dhc (Dhc6-6) dominantly suppresses the rough-eye phenotype produced by a mutation in the dynactin component Glued (Gl1 mutation) whereas a deficiency removing Dhc has no effect. This allele-specific interaction
is evidence that Gl and Dhc may act in the same pathway. Similarly,
Dhc6-6 behaves as a strong dominant suppressor of the DLis-1E415 homozygous phenotype, resulting in fertility, proper nuclear positioning and near normal oocyte growth. A deficiency of Dhc and the point mutation Dhc 6-12 have no effect on the DLis-1E415 phenotype. Therefore Dhc shows the same allele-specific interaction with DLis-1 as it does with Gl, indicating that DLis-1 may function in a genetic pathway with Dhc, and implicating dynein in nurse-cell-to-oocyte transport and nuclear positioning in the oocyte. Tests were also made for genetic interactions between Gl and DLis-1 and between Gl and Bic-D. The antimorphic Gl1 mutation confers lethality in a DLis-1E415 homozygote and in a Bic-DPA66/Df(2L)TW119 background, indicating
that both DLis-1 and Bic-D may function in the same essential
process as dynactin (Swan, 1999).
To determine how DLis-1 could interact with dynein/dynactin, the localizations of DLis-1 and Dhc proteins were studied in wild-type oocytes and
in oocytes with mutations in either gene. DLis-1 signal is concentrated along
the cortex of wild-type oocytes from as early as stage 5 of oogenesis. This localization is not detectable in DLis-1 E415/DLis-1E415 ovaries, indicating that the antiserum specifically recognizes DLis-1. To determine whether DLis-1 localization is dependent on dynein function,
DLis-1 distribution was studied in Dhc6-6/
Dhc6-12 mutants. This hypomorphic allelic combination
has no effect on the cortical accumulation of DLis-1 protein.
DLis-1 localization was mapped in egg chambers treated with the microtubule-destabilizing
drug colchicine. Under conditions that disrupt microtubules and dynein localization, cortical DLis-1 signal is still present, indicating that its cortical localization or maintenance does not depend on localized dynein or microtubules. In contrast, Dynein localization is dependent on DLis-1. In wild-type egg chambers, Dhc localizes to the presumptive oocyte in region 2 of the germarium and remains enriched in the oocyte throughout oogenesis. In DLis-1E415 mutants, this
specific accumulation is completely abolished. This strong effect of DLis-1E415 on Dhc localization contrasts with its subtle effect on the accumulation of other oocyte-specific factors, indicating that
DLis-1 may specifically regulate Dhc localization and that the localization
of most oocyte-specific factors to the oocyte is independent of DLis-1
and Dhc function. Thus there appear to be two distinct microtubule-dependent nurse-cell-to-oocyte transport mechanisms at work during oogenesis. One mechanism involving Bic-D, DLis-1 and Dhc is involved in bringing oocyte determinants into the presumptive oocyte, and, subsequently, is responsible for oocyte growth in stages 1-7 of oogenesis. A second microtubule-based transport
mechanism would function during these stages in the transport of oocyte-specific
mRNAs and proteins into the oocyte, possibly using other dyneins. In wild-type egg chambers at stages 7-9, Dhc accumulates along the
oocyte cortex and around the oocyte nucleus. Later in stage 9, Dhc accumulates mainly at the posterior and
anterior oocyte margins. These aspects of Dhc localization are disrupted
in DLis-1 mutants and, therefore, DLis-1 is necessary for most or
all aspects of dynein localization within the female germ line (Swan, 1999).
Lis-1 and Bic-D function in nuclear migration in neurons. Given the role of Lis-1 homologs in fungi, it has been suggested that the requirement for human Lis-1 in neuronal migration could also reflect a role in nuclear migration. In the developing cerebral cortex and cerebellum, migrating neurons project out a cytoplasmic extension towards their target, and then their nucleus translocates along this extension. An analogous process occurs during neural development in Drosophila. In the
third-instar eye imaginal disc, undifferentiated cells lie at the basal surface
and extend processes apically. As photoreceptor cells differentiate
posterior to the morphogenetic furrow, their nuclei translocate to the apical
surface of the eye disc. In serial confocal sections nuclei start
to appear ~4 µm below the apical surface and are no longer visible beyond
8 µm. In flies homozygous for a
pupal-lethal allele of DLis-1, nuclei also start to appear 4
µm below the apical surface, but many nuclei are also found more basally. This phenotype is identical to that of mutants for Glued,
suggesting that DLis-1 functions with dynein/dynactin in nuclear migration
in these neural cells. Given that DLis-1 is 70% identical to the human
Lis-1, these findings strongly support the possibility that the failure in
neuronal migration in Miller-Dieker syndrome also results from a failure in
dynein/dynactin-dependent nuclear migration (Swan, 1999).
As in the ovary, DLis-1 appears to function with Bic-D in nuclear
migration during eye development. Bic-D mutant eye discs also exhibit a severe
defect in nuclear positioning, with photoreceptor nuclei being found at all
levels basal to 4 µm and, frequently, in the axons that project basally from
these cells. The requirement for DLis-1 and Bic-D in nuclear positioning in the
developing eye imaginal disc could reflect a role for these genes in
microtubule organization. Microtubules in the eye disc extend longitudinally
along the apical-basal axis, but the polarity of these microtubules is not
known. To establish the orientation of these microtubules, third-instar eye imaginal discs were immunostained with antibodies to gamma-tubulin. In wild-type imaginal discs, gamma-tubulin
is found at high levels along the apical cortex and within this region in a
single strong focus about 2 µm below the apical cortex and 2 µm apical to
the photoreceptor nuclei. In
DLis-1K13209 mutants, gamma-tubulin still accumulates at the
apical surface, indicating that microtubules are orientated normally within
the mutant photoreceptors. However, the subapical focus of
gamma-tubulin is more diffuse and is undetectable in many
photoreceptors, indicating a requirement for DLis-1 in focusing
microtubule minus ends. A similar effect has been noted in Glued mutants, suggesting that DLis-1 and dynein/dynactin also function in the
same pathway in focusing microtubule minus ends in the eye. Interestingly,
whereas Bic-D mutants consistently exhibit a more marked defect in
nuclear positioning, localization of gamma-tubulin is normal in these
mutants (Swan, 1999).
Several models for nuclear localization have been advanced. This
analysis of DLis-1 allows for the proposal of a model for nuclear migration in
the Drosophila oocyte in which a cortical protein (DLis-1)
anchors microtubules via the minus-end-directed microtubule motor
dynein: DLis-1 appears to function at the cortex, and its localization to the
cortex is independent of microtubules and dynein. The dynein
heavy chain, Dhc, also associates with the oocyte cortex and this
localization requires DLis-1. Association of the nucleus with
these microtubules would then allow it to be anchored to the cell cortex.
The cortical localization of Dhc is also microtubule dependent,
and this could indicate that dynein uses microtubules to reach the cortex.
Bic-D-Egl could mediate the interaction between DLis-1 and dynein or the
interaction between microtubules and the oocyte nucleus. As well as
localizing to the oocyte cortex, Dhc also associates with the oocyte nucleus
throughout oogenesis, indicating that it may function as well in linking the
nucleus to microtubules (Swan, 1999).
The oocyte is the only cell in Drosophila that goes through
meiosis with meiotic recombination, but several germ cells
in a 16-cell cyst enter meiosis and form synaptonemal
complexes (SC) before one cell is selected to become the
oocyte. Using an antibody that recognises a component of
the SC or the synapsed chromosomes, an analysis was carried out of how meiosis becomes restricted to one cell, in relation to
the other events in oocyte determination. Although Bicaudal-D
and egalitarian mutants both cause the development of cysts with
no oocyte, they have opposite effects on the behavior of
the SC: none of the cells in the cyst form SC in BicD null
mutants, whereas all of the cells do in egl and orb mutants.
Furthermore, unlike all cytoplasmic markers for the
oocyte, the SC still becomes restricted to one cell when the
microtubules are depolymerised, even though the BicD/Egl
complex is not localised. These results have lead to the proposal of a
model in which BicD, Egl and Orb control entry into
meiosis by regulating translation (Huynh, 2000).
In the course of a study on the role of inscuteable (insc) during
oogenesis, it has been found that an anti Insc antibody recognizes a nuclear
structure that is present in some of the germ cells in regions
2a to 3 of the germarium. However, this staining does
not disappear in germline clones of protein null allele insc 22,
indicating that it is due to a cross-reaction of the antibody. Nevertheless, the staining pattern is very reminiscent of that expected for a component of the
synaptonemal complex (SC), and therefore the staining was analyzed
further, since this would be the first marker identified for the SC
structure in Drosophila. Several lines of evidence indicate that the antiserum does
indeed label the SC or a component associated with its formation. (1) The nuclear staining colocalizes with DNA, and has a morphology that corresponds exactly with the observed behaviour of the SC in electron micrographs. The staining is dotty in very early region 2a when the SC starts to form, becomes more thin and thread-like when the chromosomes are fully synapsed, and then becomes more compact in region 3, when the meiotic chromosomes condense to form the karyosome. (2) This structure
first appears at the stage when the cysts enter into meiosis. The mitotic cysts in region 1 of the germarium express Bam protein, but this disappears after the final division when the cysts move from region 1 to region 2a of the
germarium. The nuclear staining is only detectable in cysts that no longer show any Bam expression, indicating that it labels a postmitotic
structure. (3) The spatial distribution of the signal within the cyst precisely
follows that described for the SC at the EM level. The signal first appears in two cells in early region 2a and spreads to four cells per cyst in the middle of 2a, before it is restricted to two cells, and finally to one cell in region 2b. Ovaries from females that are mutant for C(3)G were examined, since these are the only characterized
mutants that completely abolish the formation of the
SC at the electron microscope level. C(3)G encodes the fly
homolog of yeast Zip1 and mammalian SCP1,
components of the transverse filament of the SC, and the effects
of the C(3)G mutation on the SC are therefore likely to be direct
(Szauter and Hawley, personal communication to Huynh, 2000). The nuclear
structure stained by this antibody is absent in C(3)G mutant cysts, even though the localization of Orb protein to the oocyte occurs normally. Thus, the
antibody acts as a marker for the formation of the SC, although
the molecular nature of the epitope recognized is not known (Huynh, 2000).
A detailed analysis of the behavior of the SC in comparison
to that of cytoplasmic markers for oocyte determination, such as
Orb and Bic-D proteins, reveals a number of distinct steps in the
restriction of oocyte fate to one cell. The SC first
appears in early region 2a cysts in the nuclei of two cells, which
are presumably the pro-oocytes. The punctate appearance of the
SC suggests that they are at the zygotene stage of meiotic
prophase 1. The next one or two cysts per germarium have four
cells in synapsis. Two of these cells have four ring canals (the
pro-oocytes) and contain an almost continuous SC, typical of the
pachytene stage, while the two cells on either side, presumably
the cells with three ring canals, contain a zygotene-like SC. In the
middle of region 2a, the SC disappears from the two cells with
three ring canals, but the two pro-oocytes still have complete
SCs, and accumulate Orb and Bic-D proteins. Soon afterwards,
Orb and Bic-D become concentrated in only one of these cells,
providing the first sign that this pro-oocyte has been selected to
become the oocyte. However, the SC still appears identical in
both pro-oocytes at this stage. The SC disappears from
one pro-oocyte as the cyst enters region 2b, and the cell that
remains in meiosis is always the one that has already
accumulated Orb or Bic-D. Finally, SC becomes more compact
in region 3 and a hole forms in its middle, before it disappears
soon after the cyst leaves the germarium.
This comparison of the behavior of nuclear and
cytoplasmic markers for the oocyte reveals two important
features about how oocyte fate becomes restricted to one cell.
(1) The two pro-oocytes are already different from the other
14 cells in the cyst in early region 2a, as they both start to form
SC at this stage. BicD and Orb only accumulate in these cells
in mid 2a, about two cysts further down the germarium.
(2) Orb and Bic-D become restricted to the oocyte
before any sign of oocyte identity can be deduced from the
behavior of the SC (Huynh, 2000).
A cyst can progress through the normal pattern of SC
localization to one cell in the presence of high concentrations
of colcemid, suggesting restriction of SC to one cell is not mediated by microtubules. Unlike the microtubules, BicD, orb and egl mutations disrupt
all steps in the restriction of the SC to one cell, and this leads
to two important conclusions: (1) BicD and Egl must have
a function that is independent of microtubules, even though
they are required for the establishment or maintenance of the
MTOC in the oocyte; (2) this
function of BicD, Egl and Orb does not depend on their own
localisation to the oocyte, since all three proteins are
completely delocalized after colcemid treatments, yet the SC
still becomes restricted to one cell.
Although both BicD and egl mutations give rise to cysts in
which all 16 cells appear identical, they have different effects
on the behavior of the SC itself. In BicD null germline clones,
none of the cells form a detectable SC, whereas all cells reach
the full pachytene stage in egl mutants (Huynh, 2000).
BicD and Egl are part of the same protein
complex, and it is therefore surprising that they have opposite
phenotypes. It is suggested that BicD and Egl may have different functions. BicD
is required to enhance SC formation in the cells that normally
enter meiosis, whereas Egl functions to repress SC formation
in the other cells of the cyst. The strongest mutations in orb
have a very similar effect on SC formation as do egl mutants,
suggesting that Orb protein is also involved in this repression.
Given the colocalization of Orb with Egl and BicD, it will be
interesting to determine whether it is part of the same protein
complex (Huynh, 2000).
The discovery that the restriction of SC to one cell requires
neither microtubules nor the localization of BicD, Egl and Orb
raises the question of how this asymmetry arises. It has
previously been suggested that BicD and Egl function in the
transport of meiosis promoting factors and oocyte determinants
from the future nurse cells into the oocyte. Although this could
still be the case if this transport occurs either very early in
region 2a or along some non-microtubule cytoskeletal network,
such as actin, this model cannot easily explain why BicD and
egl mutations have opposite effects on SC formation. An alternative model is preferred in which BicD, Egl and
Orb are required to interpret a pre-existing asymmetry that is
set up in region 1 (Huynh, 2000 and references therein).
The divisions that give rise to the cyst are asymmetric with
respect to the fusome, and recent data strongly suggest that this
structure, or some factor associated with it, somehow marks
the future oocyte. If this is
correct, this unidentified mark could regulate the BicD/Egl
complex, so that it performs different functions in the different
cells of the cyst. For example, the Egl-dependent activity of the
complex could repress SC in the cells that do not inherit the
factor, and the BicD-dependent activity could enhance its
formation in the cells that do, thereby explaining the different
phenotypes of the null mutations in the two genes. It is
interesting to note that BicD protein is phosphorylated, and that
mutations that disrupt this phosphorylation give rise to egg
chambers with 16 nurse cells. Thus,
this post-translational modification could be responsible for the
spatial regulation of the activity of the BicD/Egl complex (Huynh, 2000).
Although these results suggest that BicD and Egl have
functions that are independent of the microtubules, the nature
of this activity is unclear. However, a number of lines of
evidence suggest that these proteins may be involved in
translational control. (1) BicD was originally identified
because two single amino acid changes in the gene produce a
dominant bicaudal phenotype in which Oskar mRNA is mis-expressed
at the anterior of the oocyte. Since Oskar translation is
normally repressed unless the RNA is localized to the posterior
pole, these mutant BicD proteins must not only trap Oskar RNA
at the anterior, but also relieve translational repression.
Mutations in egl suppress the BicD gain-of-function
phenotype, while extra copies of egl enhance it, indicating that
the ectopic translation of Oskar mRNA requires the formation
of the BicD/Egl complex. The second argument for a role of BicD and Egl in
translational control comes from the discovery that orb null
mutations give a very similar phenotype to egl mutants. Orb
protein, which contains two RNA-binding motifs, has recently
been shown to associate with the 3'UTR of Oskar mRNA, and
is required for its efficient translation. Similarly, the Xenopus Orb homolog,
CPEB, binds to elements in the 3'UTRs of a number of
mRNAs, and induces the polyadenylation and translational
activation of these mRNAs during oocyte maturation. Furthermore, the Spisula solidissima (clam)
homolog plays a role not only in translational activation, but
also in repression, since it binds to masking elements in the
3'UTRs of cyclin mRNAs to prevent their translation before
fertilization. Thus,
Orb functions as a regulator of translation, and can act as both
a repressor and an activator in other species. This raises the
possibility that the BicD/Egl complex exerts different effects
in the cells of the cyst by controlling the inhibitory and
activating functions of Orb. For example, Orb could repress the
translation of factors required for SC formation in the future
nurse cells, and activate their translation in the pro-oocytes and
oocyte. If this model is correct, the selection of the oocyte
would occur by a similar mechanism to the other asymmetries
that are generated later in oogenesis, which are also all based
on the translational regulation of asymmetrically localized
mRNAs, such as Bicoid, Gurken and Oskar (Huynh, 2000 and references therein).
The behavior of the SC indicates that the determination of the
oocyte occurs in two steps. The two pro-oocytes must have
been selected by early region 2a, because they already behave
differently from the other 14 cells of the cyst at this stage, but
the development of the cyst remains symmetric until the end
of 2a, when BicD and Orb disappear from the losing pro-oocyte.
It has been proposed that the choice between
the two pro-oocytes could depend on competition between
these cells as they progress through meiosis, with the cell that
is more advanced becoming the oocyte and then inhibiting its
neighbor. However, the results presented here argue against this model:
(1) cytoplasmic factors, such as BicD and Orb, are
concentrated in one cell before there is any visible difference
between the SCs in the two pro-oocytes; (2) the
cytoplasmic aspects of oocyte determination occur normally in
C(3)G mutants, which completely lack the SC, and
in meiW68 mutants, which fail to initiate
meiotic recombination. Thus, any
competition between these two cells must be independent of
SC formation and recombination (Huynh, 2000 and references therein).
Although meiosis is not required for oocyte determination,
it can clearly influence this process, as demonstrated by the
results on the spn genes. Several lines of evidence indicate that
mutations in spnB, C and D disrupt the repair of dsDNA breaks
during meiotic recombination, activating a checkpoint pathway
that inhibits Gurken mRNA translation and the formation of the
karyosome. The results presented here strongly suggest that this checkpoint also
inhibits the determination of the oocyte, since the SC becomes
restricted to one cell much later than in wild type in these
mutants. This phenotype also allows the
time when recombination occurs to be narrowed down. This process cannot begin
until the SC forms in early region 2a, but the double-strand
DNA breaks have to be repaired before the two cells with three
ring canals exit meiosis, since this stage is delayed in spnC
mutants, indicating that the checkpoint pathway has already
been activated (Huynh, 2000).
Activation of the meiotic checkpoint causes a change in the
mobility of Vasa protein, leading to the suggestion that the
patterning defects seen in spn mutants result from the
inhibition of Vasa by this pathway. The results presented here show that the SC becomes restricted to one
cell at the normal time in most vasa mutant cysts. Thus, the
delay in oocyte determination in spn mutants cannot be a
consequence of the inhibition of Vasa, suggesting that the
checkpoint pathway has additional targets that control oocyte
selection (Huynh, 2000).
One problem in the study of cyst development in region 2 has
been the difficulty in ordering the various developmental
events that occur in this region. Using this marker for the SC,
the behavior of this structure
relative to the localization of cytoplasmic factors like Orb and
BicD could be followed, and these could be correlated with the data from EM studies on
the behavior of the SC, and the centrioles. On the
basis of this comparison, a number of
distinct stages in the restriction of oocyte fate to one cell can be distinguished: (1) The
first cyst in region 2a shows no sign of SC, but
Bam protein has already disappeared. (2) The two pro-oocytes
reach the zygotene stage of meiosis in early region 2a, and
start to form SC. (3) Soon afterwards, the
two cells with three ring canals also form SC. The SC in the
pro-oocytes has reached its maximum length, indicating that
they have reached the pachytene stage. The dsDNA breaks
generated during recombination must have already been
repaired, since the meiotic checkpoint can arrest the pattern of
SC staining at this stage. EM data also suggest that
intracellular transport begins at this point, since the first signs
of the migration of the centrioles towards the pro-oocytes
can be seen when the two cells with three ring canals
are in meiosis, and this may correlate with the first appearance
of a focus of microtubules in the cyst in the middle of region
2a. (4) The SC disappears from the two
cells with three ring canals in the middle of region 2a, but the two
pro-oocytes still have complete SCs. Orb and Bic-D
start to accumulate in the pro-oocytes at this stage.
The centrioles have migrated to either side of the largest ring
canal, which connects the two pro-oocytes, and the first signs
of 'nutrient streaming' appear, since elongated mitochondria
can be seen inside the ring canals in electron micrographs. (5)
All of the steps in cyst development so far are symmetric,
relative the largest ring canal, and the first asymmetry
becomes evident in cysts numbers 5 and 6, when Orb and Bic-D
become concentrated in one cell. The centrioles also start
to move into the oocyte, and the largest ring canal is
presumably open, because mitochondria can now be seen
inside it. However, both pro-oocytes still contain an identical
intact SC at this stage. (6) As the cyst enters region 2b, one
pro-oocyte loses its SC and reverts to the nurse cell pathway
of development. The pro-oocyte that remains in
meiosis and becomes the oocyte is always the cell that has
already accumulated Orb and Bic-D. The cytoplasm of the
oocyte now contains all of the centrioles, BicD and Orb
proteins, and an obvious MTOC, which nucleates
microtubules that extend into the other 15 cells of the
cyst. Thus, both the nucleus and cytoplasm of the oocyte are
clearly different from the other cells of the cyst by this stage.
Immediately afterwards, the oocyte starts to behave differently
from the other cells in the cyst, as it moves to the posterior
during the transition between region 2b and region 3. At
the same time, the karyosome forms, and the SC becomes
more compact, before disappearing soon after the cyst leaves
the germarium (Huynh, 2000).
During early Drosophila oogenesis, one cell from a cyst of
16 germ cells is selected to become the oocyte, and
accumulates oocyte-specific proteins and the centrosomes
from the other 15 cells. The microtubule
cytoskeleton and the centrosomes follow the same
stepwise restriction to one cell as other oocyte markers.
Surprisingly, the centrosomes still localize to one cell after
colcemid treatment, and in BicD and egl mutants, which
abolish the localization of all other oocyte markers and the
polarization of the microtubule cytoskeleton. In contrast,
the centrosomes fail to migrate in cysts mutant for Dynein heavy chain 64C, which disrupts the fusome. Thus, centrosome migration is independent of the organization of the microtubule cytoskeleton, and seems to depend instead
on the polarity of the fusome (Bolivar, 2001).
Since BicD and egl abolish the polarization of the MT
cytoskeleton in the cyst, the only candidate for a polarized
structure that could direct centrosome migration in these
mutants is the fusome. In fact, the cells that accumulate the centrosomes of egl and BicD mutant cysts possess the largest portion of the degenerating fusome. This observation demonstrates
that, as in wild type, the asymmetry established during
fusome morphogenesis persists until the stages when the
centrosomes migrate in BicD and egl cysts.
In order to test a direct role for the fusome in centrosome
movement, the behavior of the centrosomes was analyzed in a
mutant that affects the integrity of the fusome. Germline clones
of null alleles of Dynein heavy chain 64C divide correctly and
produce cysts that show a very similar phenotype to BicD and
egl. These mutant cysts contain 16 nurse cells in which oocyte
cytoplasmic markers do not accumulate in a single cell. In addition to this phenotype, a null allele of Dhc64C affects the integrity of the
fusome. Dhc64C mutant cysts possess a normal-looking
fusome in region 1 and early in region 2a. However, the fusome
of older region-2a cysts shows a fragmented appearance.
Interestingly, the centrosomes of these cysts fail to migrate to
a single cell, strongly suggesting that centrosome migration
requires an intact fusome (Bolivar, 2001).
Dhc64C is necessary for the localization of centrosomes and cytoplasmic markers to the oocyte. It was then
investigated if Dhc64C is also required for the restriction of
meiosis to the oocyte and the distribution of the
synaptonemal complex was analysed in Dhc64C germline clones. Like BicD mutants, Dhc64C is required for the formation of
the SC. Thus, lack of function of dynein blocks the three
asymmetries present in region-3 oocytes, suggesting that the
restriction of meiosis to the oocyte, the organization of a polarized
MT centered in this cell, and the migration of centrosomes to the
oocyte, depend upon the polarization of the fusome (Bolivar, 2001).
Since the MT cytoskeleton seems to be polarized toward the
oocyte prior to the migration of the centrosomes, this suggests
that most of the centrosomes of region-2 cysts might have
lost their MT nucleating properties. These post-mitotic
centrosomes thus would act differently from their region-1
counterparts, which retain the ability to grow microtubules, at
least during the mitotic divisions of the cyst. A test was performed to see whether the molecular composition of post-mitotic
centrosomes is different to mitotic ones. The distribution of Centrosomin (Cnn), a marker for the active centrosomes of mitotic cells, was examined. Cnn, like
gamma-tubulin, is present in region-1 centrosomes. In
contrast, Cnn is absent or barely detectable in region-2 and -3
cysts. This change in composition of centrosomes
depends upon the activity of egl, since in egl mutant cysts Cnn
reappears in post-mitotic region-2b centrosomes and by region 3 they possess a noticeable staining with the alpha-cnn
antibody. Although no explanation for this difference is available, it suggests that the correct determination of the oocyte among the cells
of the cyst affects the composition of the germline
centrosomes (Bolivar, 2001).
Bicaudal-D (Bic-D) is required for the transport of determinant mRNAs and proteins to the presumptive oocyte, an essential step in the differentiation of the oocyte. Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins. Examined were the ovarian phenotypes of females expressing mutant Bic-D proteins (Bic-DH) deleted for each of the heptad repeat domains. The altered migration of follicle cells observed in mutant ovaries suggests that Bic-D functions in the germline and directs the inward migration of somatic follicle cells. In the germarium Bic-D is required for the organization of the egg chamber and the structural integrity of the oocyte and nurse cells. Examination of the polarized microtubule network in Bic-DH ovaries shows that Bic-D function is required for both the establishment of the polarized microtubule network and its maintenance throughout oogenesis. To explain the multiple functions suggested by the pleiotropic Bic-D phenotype, it is proposed that Bic-D protein forms a filamentous structure and represents an integral, essential part of the cytoskeleton (Oh, 2001).
Bic-D function in the germline is required for follicle cell migration around the germline cyst. Follicle cell migration begins in germarial
stage 2a-2b and happens about the same time the oocyte
differentiates from the other cystocytes. So it appears that
Bic-D may be essential in region 2a-2b in all cystocytes.
Alternatively, the differentiation of the oocyte and possibly
its migration to the posterior end of the cyst indirectly
control the packaging of the germline cyst by the follicle
cells. Even though the abnormal packaging of cysts is
observed in only 20% of egg chambers, these observations
suggest that signaling between the germline and soma
occurs as soon as the 16-cell cyst is formed.
A number of genes, Notch, Delta, daughterless, hedgehog, brainiac, egghead, and tou-can,
control the migration of follicle cells around the
16-cell germline cyst. This process requires cell-cell interactions and
signaling between the germline cysts and the somatic
follicle cells and results in the differentiation of the follicle
cells into two specialized cell types, polar and stalk. Mutations
in the genes listed above show defects in follicle cell
intercalation between the cysts in the germarium, resulting
in compound egg chambers where multiple cysts are packaged
into a single follicle. This mispackaging is possibly a result of the failure to differentiate polar and stalk cells (Oh, 2001 and references therein).
Bic-D function is not required for Notch signaling, since
in Bic-DH the stalk and polar cells develop normally. The
results also indicate that the abnormal migration of follicle
cells around Bic-D mutant germline cysts is unlikely to be
a result of a defect in E-cadherin. A possible explanation
for the abnormal migration of the follicle cells in Bic-DH
is that the cytoskeletal organization is disturbed, causing
mislocalization of a germline to soma signal that regulates
the migration of the follicle cells around the cyst. The
theory that in Bic-DH mutants the cytoskeleton is misorganized
is also consistent with the frequently observed mis-positioned oocyte and with the protrusion of nurse cell nuclei into the cytoplasm of the oocyte (Oh, 2001).
The follicle cell migration phenotype is due to loss of
function of Bic-D and not to an antimorphic effect of the
deleted proteins because the abnormal migration of follicle
cells is also observed in Bic-D null germline clones,
and abnormal numbers of germ cells are also observed in
Bic-DPA66 and Bic-DR26 mutants although at very low frequencies. Most importantly, increased dosage of the transgenes does not enhance the phenotype; in fact, it relieves the severity of the phenotypes (Oh, 2001).
A range of polyploidy has been observed in oocyte chromosomes
in Bic-DH mutants. The partial polyploidy of the
oocyte nucleus is first seen in egg chambers older than stage
3 and often is the only apparent defect; the accumulation of
yolk proteins and the morphology of these egg chambers
seem normal. Antibody staining shows that the degree of polyploidy of the oocyte correlates with the level of localized Bic-D in the oocyte. The polyploidy phenotype is probably due to a defect in the localization of factors
required for the maintenance of the meiotic status of the
oocyte during germarial or vitellarial stages, reflecting the
continuous requirement of Bic-D function. A similar phenotype
is observed in encore (enc) and some alleles of
ovarian tumor (otu). Since otu is required for the
organization of the actin cytoskeleton in both early and late
stages of oogenesis, the partial polyploidy phenotype in otu
mutants may also be derived from defects in the transport of factors (Oh, 2001).
Bic-D and Egl protein colocalize at all stages of oogenesis.
Co-immunoprecipitation experiments show that Bic-D and
Egl protein form a protein complex. The colocalization of Bic-D protein with Egl is
maintained in Bic-DH1, Bic-DH2, and Bic-DH3, suggesting that deletion of these domains does not affect the formation of Bic-D/Egl complex. In Bic-DH4
germline clones, Bic-D and Egl fail to localize. The last heptad domain could
therefore mediate both the formation of the Bic-D/Egl
complex and the regulation of the localization of Bic-D to the oocyte (Oh, 2001).
All the phenotypes observed in Bic-DH
ovaries, the abnormal migration of follicle cells, the different levels of ploidy
of the oocyte, mislocalization of Gurken RNA and protein, the mislocalization of Bic-D and Egl proteins, and the failure in maintenance of the microtubule
network, indicate that Bic-D is required for the organization
of the egg chamber and maintenance of the structural
integrity of the oocyte-nurse cell complex. It is proposed that
Bic-D function may be required for cytoskeletal polarity in
the cysts and egg chambers (Oh, 2001).
Studies of the function of the Lis-1 gene support the
hypothesis that Bic-D regulates the cytoskeletal organization
of germline cells. Lis-1 germline and CNS clones show
almost identical phenotypes as do clones lacking dynein
heavy-chain function, and Lis-1 and dynein heavy chain appear to interact. Some of the ovarian Lis-1 phenotypes are similar to Bic-D ovarian and
eye phenotypes, suggesting that dynein, Lis-1, and Bic-D are essential for the formation and function of the microtubule network (Oh, 2001).
However, it is not clear how Bic-D functions in the formation and maintenance of the microtubule network. Bic-D may be a microtubule-associated protein that initiates
microtubule reorganization in germarial cysts and maintains the microtubule network.
However, the following observations do not support this
theory. First, in co-immunoprecipitation experiments,
the association of Bic-D with tubulin is not observed, and
Bic-D protein does not cosediment with tubulin on sucrose
density gradients. Neither is Bic-D identified as a microtubule associated protein in microtubule preparations from embryonic extracts. Finally, mutations in Bic-D cause more pleiotropic phenotypes than does the disruption of the
microtubule network by colchicine treatment (Oh, 2001).
It is proposed that Bic-D protein may be an integral and essential part of the cytoskeleton, rather than having a specific function in RNA transport: (1) Bic-D protein is similar in sequence to various intermediate filament proteins
that form cytoskeletal structures. (2) The selective accumulation of Bic-D and Egl protein in the presumptive oocyte in region 2a and 2b is the first sign of cyst polarity, following the break down of the fusome. This accumulation
precedes and is essential for the formation of the MTOC in
the pro-coocyte, suggesting that the polarity of the cyst,
reflected by the selective accumulation of Bic-D and Egl, is
somehow translated into the cytoskeletal polarity reflected
in the polarized microtubule network. (3) The distribution
of Bic-D protein is similar to that of the actin and microtubule
networks in the oocyte, suggesting that it could be part
of a cytoskeleton. (4) Several phenotypes observed in Bic-DH
ovaries, such as the abnormal migration of the follicle cells
and the protrusion of nurse cell nuclei into the oocyte, are
consistent with Bic-D having a structural role in the cytoskeletal
organization of the oocyte and egg chamber. (5)
The abnormal localization patterns of Bic-DH1 and Bic-DR26 proteins suggest that Bic-D may form a filament-like structure. Both mutant Bic-D proteins retain the ability to localize to the presumptive oocyte, but the level of localized proteins is significantly higher than that of wild-type
protein. This higher accumulation of the proteins could
represent hyperaggregated forms of the filament, in turn
possibly resulting in an abnormal cytoskeleton. (6) The
phenotype of Bic-DH2, ranging from egg chambers that are
similar to those produced by Bic-D null mutants to normal
eggs, suggests that Bic-D protein may aggregate to form a
higher order cytoskeletal structure. Two molecules of Bic-D
protein bind colinearly to form a rod-shaped dimer in vitro. The pleiotropy of
phenotypes detected in Bic-DH2 may arise from mutant
Bic-D monomers sometimes associating out of register
relative to their heptad repeats, leaving ends available for
further interaction. Interaction between such molecules
could lead to polymerized filaments with null or only
partial function. In other cases the mutant protein could
form colinear dimers that may have full function. (7) The
putative structural organization of Bic-D protein, two effector
domains separated by a linker, is consistent with the theory that Bic-D could form filamentous structures serving as a scaffold for generating the
asymmetric distribution of the factor(s) that establish and
maintain the polarized microtubule network (Oh, 2001).
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).
Nuclear translocation, driven by the motility apparatus consisting of the cytoplasmic dynein motor and microtubules, is essential for cell migration during embryonic development. Bicaudal-D (Bic-D), an evolutionarily conserved dynein-interacting protein, is required for developmental control of nuclear migration in Drosophila. Nothing is known about the signaling events that coordinate the function of Bic-D and dynein during development. This study shows that Misshapen (Msn), the fly homolog of the vertebrate Nck-interacting kinase is a component of a novel signaling pathway that regulates photoreceptor (R-cell) nuclear migration in the developing Drosophila compound eye. Msn, like Bic-D, is required for the apical migration of differentiating R-cell precursor nuclei. msn displays strong genetic interaction with Bic-D. Biochemical studies demonstrate that Msn increases the phosphorylation of Bic-D, which appears to be necessary for the apical accumulation of both Bic-D and dynein in developing R-cell precursor cells. It is proposed that Msn functions together with Bic-D to regulate the apical localization of dynein in generating directed nuclear migration within differentiating R-cell precursor cells (Houalla, 2005).
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BicaudalD:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 1 June 2024
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