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First published online 18 April 2007
doi: 10.1242/dev.02832
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The Wellcome Trust Centre for Cell Biology, Michael Swann building, The University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
Author for correspondence (e-mail:
ilan.davis{at}ed.ac.uk)
Accepted 12 February 2007
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, Oogenesis, Oocyte, Nurse cells, Intracellular mRNA localization, Microtubules, Actin, Cytoplasmic Dynein, Bicaudal D, Egalitarian, Ring canals
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The oocyte
nucleus is transcriptionally inactive during most of oogenesis
(King and Burnett, 1959).
Consequently, the majority of the contents of the growing oocyte originates
from the polyploid nurse cells, with the exception of yolk granules and some
specific signals that are secreted from the overlying follicle cells
(Spradling, 1993). Such
long-distance transport of cellular components, including mRNA, between
interconnected cells or within very large cells is a common and important
process in many biological systems
(Pollard and Earnshaw, 2002).
Nurse cell-to-oocyte transport occurs through actin-rich ring canals and can
be divided into four distinct phases
(Mahajan-Miklos and Cooley,
1994). First, in early oogenesis, oocyte specification in the
germarium depends on the selective transport of Bicaudal D (BicD) and
Egalitarian (Egl) (Mach and Lehmann,
1997; Suter and Steward,
1991), which, together with microtubules (MTs), are required for
oocyte specification (Koch and Spitzer,
1983; Theurkauf et al.,
1993). Second, during a period of 2 days in mid-oogenesis, from
stages 2 to 7, both the nurse cells and the oocyte grow at the same rate
(Spradling, 1993), and a
variety of important transcripts that encode axis specification factors are
transported into the oocyte. During the third phase, at stages 8-10A of
oogenesis, this process continues, except the relative size of the oocyte
increases in relation to the nurse cells because of the transport of yolk from
the follicle cells and cytoplasm from the nurse cells. The fourth and last
transport phase involves the `dumping' of the entire nurse cell contents into
the oocyte at stage 10B, leading to the doubling of the oocyte volume within
30 minutes (Mahajan-Miklos and Cooley,
1994).
It has been proposed that MTs provide the tracks along which
minus-end-directed motors, such as Dynein, could transport proteins,
organelles and mRNAs into the oocyte
(Theurkauf et al., 1993). This
hypothesis is supported by the fact that strong mutations in components of the
Dynein complex, including Dhc, Lis-1, BicD and Egl, result in egg chambers
devoid of oocytes (Gepner et al.,
1996; Liu et al.,
1999; McGrail and Hays,
1997; Swan et al.,
1999). However, no direct evidence for these proposals has been
obtained and the exact mechanism by which these proteins affect this process
is unclear. Moreover, the specificity of a putative Dynein-dependent nurse
cell-to-oocyte transport system for different cargoes, such as organelles and
mRNAs, remains elusive, as does the exact path of transport.
Key transcripts that must be transported from the nurse cells to the oocyte
during mid- to late-oogenesis include: gurken (grk) mRNA,
which encodes a TGF
homologue responsible for setting up the primary
axes (Gonzalez-Reyes et al.,
1995; Neuman-Silberberg and
Schüpbach, 1993); bicoid (bcd) mRNA,
encoding the anterior morphogen (Driever
and Nüsslein-Volhard, 1988); and oskar
(osk) mRNA, encoding a key factor responsible for posterior
patterning and germ cell determination
(Ephrussi et al., 1991). Once
in the oocyte, the minus-end-directed MT-dependent molecular motor,
cytoplasmic Dynein (Dynein), is required for the localization of grk
(MacDougall et al., 2003) and
bcd (Januschke et al.,
2002b) mRNA. By contrast, the plus-end-directed molecular motor
Kinesin I is required for osk mRNA localization to the posterior of
the oocyte. The transport of each of these mRNAs into the oocyte has been
suggested to involve the same mechanism as that of apical transport of
pair-rule transcripts in the blastoderm embryo
(Bullock and Ish-Horowicz,
2001), which was previously shown to involve MTs
(Lall et al., 1999) and Dynein
(Wilkie and Davis, 2001). The
former study showed the signal-dependent nurse cell-to-oocyte transport of
ectopically expressed pair-rule transcripts, which are ordinarily not
expressed during oogenesis. Taking these observations together, they suggest
the existence of a similar Dynein-dependent process that could mediate nurse
cell-to-oocyte mRNA transport during oogenesis. However, the role of Dynein in
the movement of transcripts, such as bcd, osk and grk mRNA,
that are expressed in oogenesis, remains unclear.
The transport of bcd and osk mRNA into the oocyte has been known
for some time to be MT dependent (Pokrywka
and Stephenson, 1991). Furthermore, bcd mRNA forms
particles that move into the oocyte at speeds suggestive of an
active-transport process (Cha et al.,
2001). Exu, a factor required genetically for bcd mRNA
localization (St Johnston et al.,
1989), was shown to be required within the nurse cells for
injected bcd RNA to localize correctly once in the oocyte
(Cha et al., 2001) and to move
as particles from the nurse cells to the oocyte
(Theurkauf and Hazelrigg,
1998). However, the role of motor proteins in nurse cell-to-oocyte
transport was not addressed directly, although a Dynein-binding protein,
Swallow (Schnorrer et al.,
2000), and the Dynein co-factor Dynactin
(Januschke et al., 2002a) were
shown to be required for bcd mRNA localization in the oocyte. Thus,
although the requirement for motor proteins in mRNA localization in the oocyte
has been demonstrated conclusively, the mechanism and path of the nurse
cell-to-oocyte transport of specific mRNAs remains poorly studied. It is also
not known whether mRNAs are simply swept along by the flow of cytoplasm from
nurse cell to oocyte. Although some selectivity of cytoplasmic transport has
been observed (Bohrmann and Biber,
1994), it is also possible that cellular components are
transported into the oocyte via the pressure created by the synthesis of
cytoplasmic components in the nurse cells.
Here, we define the path of movement and mechanism of grk mRNA transport from nurse cells to oocyte. By injecting fluorescently labelled grk RNA into nurse cells and assaying its movement, we show that grk RNA is first transported rapidly and directly towards the ring canals, accumulating in a discrete zone in front of the ring canals, followed by its rate-limiting transport through the ring canals. We show that this transport is MT-, BicD- and Dynein-dependent, and that grk RNA recruits the Dynein-associated proteins Egl and BicD in the nurse cells. We show further that bcd and osk RNA are likely to be transported by a similar mechanism. We propose that Dynein-dependent transport of grk, bcd and osk transcripts towards the ring canal allows the more-efficient transport of key axis specification transcripts than would the slower transport of cytoplasmic components, which occurs throughout much of oogenesis.
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MATERIALS AND METHODS |
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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).
For controls, siblings of the cross (w, Df(2L)TW119/CyO or w,
BicDr8/CyO; P [w+hsBic-D]-94/+) and OrR flies were
used. To confirm the absence of BicD protein in Bic-Dmom
ovaries, each fly had one ovary dissected and fixed for BicD immunostaining,
whereas the other ovary was used for the in vivo study of grk RNA
nurse cell-to-oocyte transport.
RNA in situ hybridization
In situ hybridization was performed as previously described
(MacDougall et al., 2003;
MacDougall et al., 2001).
Digoxigenin anti-sense RNA probes were transcribed using full-length
grk (G. Schupbach, Princeton University, Princeton, NJ), osk
(A. Ephrussi, EMBL, Heidelberg, Germany) and bcd (C.
Nusslein-Volhard, Max Planck Institute, Tübingen, Germany) cDNAs. In each
experiment, at least 40 ovarioles (stage 2-9) and ten egg chambers (stage 10A
and older) were examined from OrR or
dhc6-6/dhc6-12 females.
Synthesis of fluorescent and biotinylated RNA
RNAs were in vitro transcribed using T3, T7 and SP6 polymerases and
UTP-AlexaFluor546, UTP-AlexaFluor488, UTP-Cy3 or UTP-Biotin as previously
described (Wilkie and Davis,
2001). Plasmids templates were: full-length grk (G.
Schupbach); bcd (C. Nusslein-Volhard); K10 3' UTR (S.
Bullock, MRC LMB, Cambridge, UK); I factor (also known as
I-element; V. Van de Bor, University of Nice, Nice, France); and a 3
kb fragment of lacZ amplified from pG5-L-G3
(Thio et al., 2000) and cloned
into pGEM-T.
Injection of RNAs and inhibitors
Ovaries were dissected in Series 95 halocarbon oil (KMZ Chemicals) and egg
chambers were adhered to coverslips using tungsten needles. Fluorescently
labelled RNA (250-500 ng/µl) was injected into nurse cell cytoplasm using
Femtotip needles (Eppendorf). At least two batches of RNA were injected for
each experiment, on separate occasions. Colcemid was co-injected with
fluorescently labelled RNA at 100 µg/ml into Tau-GFP egg chambers.
To study the requirement of MTs in grk RNA ring canal accumulation,
colcemid (100 µg/ml) was pre-injected over each Tau-GFP egg
chamber followed by grk RNA injection into the nurse cells 10 minutes
later. AlexaFluor568-coupled Phalloidin was injected at a concentration of 4
units/ml (0.13 µM) into wild-type nurse cells to visualize ring canals and
to facilitate imaging of cytoplasmic movements at the same plane of focus as a
ring canal.
Immunostaining of injected egg chambers
Biotinylated RNAs were injected into wild-type nurse cells and fixed after
15-20 minutes. Egg chambers were fixed for 20 minutes by placing 3.7%
formaldehyde in PBT (0.1% Tween) over the halocarbon oil. All subsequent
staining steps were performed in a watchglass under a dissecting microscope to
avoid losing the injected egg chambers during the washes. Biotin-labelled RNA
was detected with Avidin-conjugated AlexaFluor488 or AlexaFluor546 (Molecular
Probes). BicD and Egl were visualized with anti-BicD (B. Suter) and anti-Egl
(R. Lehmann, New York University School of Medicine, NY), which were used at
dilutions of 1/20 and 1/4000, respectively. Secondary antibodies were
AlexaFluor488- and AlexaFluor568-coupled (Molecular Probes). In all
recruitment experiments, at least three egg chambers were analyzed for each
condition. Alexa568-coupled Phalloidin (1/50) was used to visualize actin.
Four-dimensional imaging and deconvolution
Fixed egg chambers were mounted in Vectashield (Vector Laboratories).
Imaging was performed on a wide-field DeltaVision microscope (Applied
Precision, Olympus IX70 microscope, Coolsnap HQ camera from Roper). Images
were acquired with a 20x/0.75NA or 100x/1.35NA objective lens and
subsequently deconvolved (Davis,
2000) using Sedat/Agard algorithms with Applied Precision
software. Several egg chambers were imaged in parallel depending on the time
resolution needed. For high-power imaging of MTs, Tau-GFP and
Tau-GFP/+; dhc6-6/dhc6-12 egg chambers were
sectioned through the ring canal at 1 µm intervals with a 100x/1.35NA
objective. Live imaging of RNA particles at 100x/1.35NA was performed at
1 second intervals for approximately 3 minutes, after which time
photobleaching prevented the identification of particles for tracking.
Particle tracking
RNA particles were manually tracked with SoftWoRx (Applied Precision) and
Metamorph (Universal Imaging Corporation) software as previously described
(MacDougall et al., 2003).
Nurse cell cytoplasmic movements near the ring canals were analyzed as for RNA
particles, at 100x/1.35NA and 1 second time intervals, by differential
interference contrast (DIC) (n=4 egg chambers). Trails of RNA or
cytoplasmic particles were generated using SoftWoRx. Speeds of particle
movements are shown as average speeds±standard error (s.e.). It was
difficult to estimate the proportion of RNA particles that move in directed
tracks, because stationary particles are difficult to distinguish from
autofluorescent cytoplasmic bodies. By contrast, rapidly moving RNA particles
of a similar intensity of fluorescence are easily identified.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
grk RNA in the oocyte (MacDougall
et al., 2003). Fulllength grk RNA was injected into the
nurse cells at different stages of oogenesis. At all stages tested, ranging
from 6 to 10A, injected grk RNA was able to move into the oocyte from
the nurse cells and then localize correctly within the oocyte
(Fig. 1A,C,E, and see Movies 1,
2, and Table S1 in the supplementary material) in a manner similar to the
endogenous transcript (Fig.
1B,D,F). At early stages, before the reorganization of MTs and the
migration of the oocyte nucleus, injected grk RNA localized correctly
in a posterior crescent near to the oocyte nucleus
(Fig. 1A,B), where MT minus
ends are thought to be concentrated
(Micklem et al., 1997). By
contrast, when grk RNA was injected into the nurse cells of stage
8-10A egg chambers (Fig. 1C-F),
it became localized in a dorso-anterior crescent near the oocyte nucleus in a
very similar pattern to its localization when injected into the oocyte, and to
the endogenous transcript at the same stage of oogenesis
(MacDougall et al., 2003). We
found that the efficiency of transport of injected grk RNA into the
oocyte was higher in the earlier stages and that some injected RNA remained in
the nurse cells, within the time scale of the experiment, which was limited,
by egg chamber survival in halocarbon oil, to 1-2 hours.
To determine whether the movement of injected grk transcript from
the nurse cells to the oocyte is specific, we injected a number of other
transcripts, including control RNAs. We first injected bcd RNA, which
had previously been shown to move efficiently into the oocyte
(Cha et al., 2001).
bcd RNA injected into the nurse cells moved into the oocyte at an
equivalent rate to grk RNA (Fig.
1G,H and see Table S1 in the supplementary material) and localized
to the anterior of the oocyte, in the same pattern as the endogenous
transcript. We then injected fluorescently labelled lacZ RNA and
found that these control transcripts failed to accumulate substantially in the
oocyte, and that the small amount of RNA that entered the oocyte was evenly
distributed in the cytoplasm (Fig.
1I and see Table S1 in the supplementary material). We conclude
that injected grk and bcd RNA are specifically transported
efficiently from the nurse cells into the oocyte.
To elucidate the path and details of the movement of grk and bcd RNA from the nurse cells, we studied the movement at higher magnification and time resolution. We found that, in nurse cells, injected grk RNA forms particles that move in directed paths to a specific location near the oocyte, which appeared to be ring canals (Fig. 2A and see Movie 3 in the supplementary material). By contrast, injected lacZ RNA did not accumulate in similar locations. To test directly whether grk RNA localized at ring canals, we fixed the injected egg chambers 20 minutes after the injection of Biotin-labelled grk RNA and stained them with AlexaFluor488-Phalloidin to co-visualize the ring canals with RNA. We found that, in 88% of egg chambers (n=8), grk RNA accumulated at a region in front of the ring canals (Fig. 3A, and see Movie 4 and Table S2 in the supplementary material). By contrast, injected lacZ RNA failed to accumulate in this manner (n=12).
To test whether the accumulation of grk RNA at the ring canals is
a consequence of directed movement towards this region of the nurse cells, we
co-visualized injected grk RNA with Actin-GFP in living egg chambers.
We found that grk RNA particles moved in directed paths, rather than
randomly, towards the ring canals, which they accumulated in front of before
passing through (Fig. 3C-E).
These paths are most easily appreciated when viewing the time lapse movies
(Movies 3, 4 in the supplementary material). The movement towards and through
ring canals occurred at different speeds
(Fig. 3), suggesting that they
involve distinct mechanisms of RNA transport. These speed differences are
consistent with previous observations of Exu-GFP particles
(Theurkauf and Hazelrigg,
1998). We found that K10
(MacDougall et al., 2003) and
I factor (also known as I-element - FlyBase)
(Van De Bor et al., 2005) mRNA
behave similarly when injected into a nurse cell (see Fig. S1A-C and Tables
S1, S2 in the supplementary material).
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grk mRNA transport within nurse cells is Dynein and MT dependent
To determine whether grk mRNA transport within nurse cells and its
accumulation at the ring canals requires MTs, we injected grk RNA
into egg chambers expressing transgenic Tau-GFP
(Micklem et al., 1997), which
decorates the MTs (Fig. 4B,C).
We found that MTs are organized with a focus of high concentration at the ring
canal (Fig. 4A) at the region
where injected grk RNA accumulates
(Fig. 4B,C). We then
co-injected the MT depolymerizing drug Colcemid with fluorescent grk
RNA and found that the accumulation near the ring canals and the movement into
the oocyte was inhibited rapidly (Fig.
4D,E). Tau-GFP imaging showed that, 10 minutes after Colcemid
injection, MTs were mostly depolymerized
(Fig. 4D). We found that MT
depolymerization also abolished the directionality of grk-particle
movement towards the ring canals (Fig.
4F,G). We conclude that the specific movement of grk RNA
particles in the nurse cells along straight paths to the ring canals is MT
dependent.
To test whether cytoplasmic Dynein, the major minus-end MT motor in the
cell, is required for grk RNA movement towards the ring canals, we
injected grk RNA into hypomorphic allelic combinations of Dynein
heavy chain (Dhc64C), the large force-generating ATPase subunit of the Dynein
motor. These viable mutant combinations do not show dorso-ventral polarity
defects, but the females are sterile
(McGrail and Hays, 1997). We
found that, in dhc6-6/dhc6-12 mutant egg
chambers, injected fluorescent grk RNA moved towards the ring canals
in a directed fashion, but at slower rates than in wild type (n=14).
Injected grk RNA also moved into the oocyte and localized, but,
again, at a slower rate (Fig.
5A-D and see Movies 5, 6 in the supplementary material). To
determine whether endogenous grk mRNA transport and/or localization
is impaired in dhc hypomorphic mutants, we carried out in situ
hybridization. Endogenous grk mRNA was weakly localized in the oocyte
of early (stage 2-7) dhc mutant egg chambers
(Fig. 5E-H). To test whether
the MT distribution was affected in dhc mutants, we imaged the
distribution of MTs in living nurse cells of
dhc6-6/dhc6-12 mutant egg chambers, using
Tau-GFP, and found that it was indistinguishable from wild-type controls (see
Fig. S2 in the supplementary material), consistent with our previous work in
the oocyte (MacDougall et al.,
2001). We conclude that the transport of grk RNA within
the nurse cells towards ring canals and from the nurse cell to the oocyte is
Dynein dependent.
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These results could be explained by either a defect in nurse cell-to-oocyte transport or by a defect in RNA localization within the oocyte. To distinguish between these possibilities and determine directly whether the movement of grk RNA towards the ring canals is Dynein dependent, we studied the movement of RNA particles at high resolution in dhc6-6/dhc6-12 mutant egg chambers. We found that the speed of movement of grk RNA particles within the nurse cells towards the ring canals was substantially reduced (Fig. 6A,B). We conclude that the transport of grk RNA within the nurse cells towards the ring canals joining them to the oocyte occurs by Dynein along MTs and that Dynein is also required for the efficient transport of bcd and osk RNA from the nurse cell to the oocyte.
grk and bcd RNA recruits BicD and Egl within the nurse cells, and BicD is required for the accumulation of grk RNA in front of the ring canal
To investigate the basis of the specificity of grk transport
compared with that of control lacZ, we first determined whether these
RNAs recruit the Dynein co-factors BicD and Egl upon injection into the nurse
cells. BicD and Egl have previously been shown to be required for oocyte
specification (Mach and Lehmann,
1997) and for the efficient Dynein-dependent transport of mRNA in
the embryo (Bullock and Ish-Horowicz,
2001). We injected biotinylated grk and bcd RNA
into wild-type egg chambers, fixed them after 20 minutes and detected BicD and
Egl protein. Our results show that both BicD and Egl were recruited by
injected grk and bcd RNA at the site of injection in the
nurse cell cytoplasm and at the ring canals, whereas injected lacZ
RNA failed to recruit BicD and Egl (Fig.
7A-D and data not shown, respectively). To test whether BicD is
required for grk RNA transport to the ring canals, we injected
grk RNA into nurse cells of BicD mutant egg chambers. We
used the BicDmom mutant combination, in which the early
lethality of a strong allele of BicD is rescued with the early
expression of BicD from the heat shock promoter
(Swan and Suter, 1996). In
this way, later-stage egg chambers that lack BicD protein are obtained
(Fig. 7E,H). We injected
fluorescent grk RNA into the nurse cells of
BicDmom egg chambers and found that the RNA failed to
accumulate in front of the ring canals, as it does in controls
(Fig. 7F,G,I,J)
(n=10). We conclude that BicD, and probably also Egl, is required for
grk RNA to move along directed paths within the nurse cells towards
the ring canals and that lacZ RNA is not able to move along these
paths because it fails to recruit BicD and Egl.
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), and
plotted trails of nurse cell cytoplasmic movements. Our results show that the
path and direction of cytoplasmic flow in the nurse cells is significantly
different to that of the injected grk RNA (see Fig. S4 in the
supplementary material). We found that cytoplasmic particles did not move in
directed paths towards the ring canals. By contrast, grk, and
probably bcd and osk, RNAs are specifically transported by
Dynein along direct paths that lead to ring canals, and are therefore
transported more efficiently into the oocyte. We propose that these distinct
routes to the ring canals are along a subset of MTs that we observe as a
particularly high concentration of MTs near the ring canals
(Fig. 4A and see Fig. S2 in the
supplementary material).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The movement of macromolecules, including RNA, within and between cells is
an important process in the biology of most organisms. Although diffusion is,
in principle, fast enough to distribute macromolecules over relatively large
cellular distances, active transport can provide more-efficient, directed
movements to specific locations as well as overcome effective diffusion
barriers provided by cytoskeletal networks in the cell
(Luby-Phelps, 2000). Moreover,
diffusion of mRNA within a dense cytoplasm is very slow, because transcripts
are decorated with many proteins to form very large ribonucleoprotein
particles with a large hydration volume
(Shav-Tal et al., 2004). It
has therefore been proposed that all mRNAs, both localized and unlocalized,
are associated with molecular motors to varying degrees to allow them to be
rapidly distributed within the cytoplasm, and the presence of a localization
signal increases the efficiency of association with the motors
(Fusco et al., 2003) and
perhaps even influences the activity of the motors and their co-factors
(Bullock et al., 2003). Our
study highlights that the presence of localization signals and recruitment of
specific motor co-factors affects the choice of intracellular routes adopted
by such cargo in the oocyte.
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), and why pair-rule transcripts, which are transported in the
blastoderm embryo in a MT-dependent manner
(Lall et al., 1999), by Dynein
(Wilkie and Davis, 2001), are
also transported into the oocyte when exogenously expressed in the nurse cells
(Bullock and Ish-Horowicz,
2001). We suggest 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 (Dubowy and Macdonald,
1998) could first be transported by the same Dynein-dependent
mechanism into the oocyte.
The Dynein-dependent transport route we have 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
(Chekulaeva et al., 2006;
Ferrandon et al., 1997;
Tekotte and Davis, 2006;
Wagner et al., 2001) 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 (also known as Arrest - FlyBase)
(Castagnetti et al., 2000;
Filardo and Ephrussi, 2003;
Kim-Ha et al., 1995) and Hrp48
(also known as Hrb27C - FlyBase) (Goodrich
et al., 2004; Huynh et al.,
2004; Norvell et al.,
2005; Yano et al.,
2004); however, it is unclear whether they are transported within
the same complexes into the oocyte. At stage 10B, the mechanism we have
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.
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),
it is not required for the general growth of the oocyte or for the presence of
mRNAs in the oocyte (Brendza et al.,
2000). These observations suggest that Kinesin 1 is not important
for cytoplasmic transport or for specific mRNA transport into the oocyte.
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
(Cha et al., 2001).
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 (Van De Bor et
al., 2005), but is not shared with bcd and K10
transcripts (MacDougall et al.,
2003), 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
(Setou et al., 2002). 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
(Bullock et al., 2003), raising
the interesting possibility that cargos could also influence the choice of MT
route adopted by their motors. Our 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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/10/1955/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Bohrmann, J. and Biber, K. (1994).
Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic
to mid-vitellogenic ovarian follicles of Drosophila: time-lapse
analysis using video-enhanced contrast microscopy. J. Cell
Sci. 107,849
-858.[Abstract]
Brendza, R. P., Serbus, L. R., Duffy, J. B. and Saxton, W.
M. (2000). A function for kinesin I in the posterior
transport of oskar mRNA and Staufen protein.
Science 289,2120
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