1 Department of Biology, Indiana University, Bloomington, 1001 East 3rd Street,
IN 47405, USA
2 Program in Cell Dynamics, University of Massachusetts, 55 Lake Avenue, North
Worcester, MA 01655, USA
* Author for correspondence (e-mail: bsaxton{at}bio.indiana.edu)
Accepted 22 June 2005
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SUMMARY |
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Key words: Drosophila, Oocyte, Kinesin-1, Dynein, Streaming, Microtubule, Actin, Oskar
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Introduction |
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Intracellular arrays of cytoskeletal filaments (F-actin or microtubules)
are required for most forms of active transport
(Vale, 2003). The filaments,
which have structural polarity, act as directional tracks for the transport of
organelles or other cargoes by molecular motors; myosins that move on F-actin
or kinesins and dyneins that move on microtubules. Many microtubule motors act
as force-producing crosslinks with a mechanochemical, filament-binding `head'
at one end and a cargo-binding `tail' at the other. Motors of the kinesin-1
subfamily (conventional kinesins) move cargoes towards microtubule plus-ends,
which are usually distal to microtubule organizing centers (MTOCs), while
cytoplasmic dynein moves cargoes toward minus-ends, which are usually at or
near MTOCs. Thus, the positions of MTOCs and the paths of microtubules that
project away from them dictate the directions and paths of microtubule-based
cargo movements.
It is thought that kinesins and cytoplasmic dynein can attach to the same
cargo, which raises questions about conflict between their opposing forces.
Live imaging has shown that cargoes usually move alternately toward
microtubule plus- and minus-ends, in a saltatory manner. Net transport is
accomplished by a bias in favor of one direction
(Mallik and Gross, 2004).
Studies of melanosomes in Xenopus
(Deacon et al., 2003
), and
lipid droplets, peroxisomes and mRNA particles in Drosophila
(Gross et al., 2002b
;
Kural et al., 2005
;
Ling et al., 2004
) suggest
that minus- and plus-end microtubule motors strictly alternate rather than
competing with one another in a tug-of-war. This raises the question of
whether or not coordinated alternation of opposing motors is a universal
feature of microtubule-based transport processes.
Drosophila oocytes provide a good system for investigating
microtubule-dependent transport. Microtubule motors are important both for
targeted localization of polarity determinant mRNAs, and for dispersal of
components delivered to the oocyte anterior from adjoining nurse cells. During
mid-oogenesis, bicoid (bcd), oskar (osk)
and gurken (grk) mRNAs are localized in the oocyte to their
respective anterior, posterior and dorsal positions in a kinesin-1- and
dynein-dependent manner (Brendza et al.,
2000a; Brendza et al.,
2002
; Duncan and Warrior,
2002
; Januschke et al.,
2002
). Throughout that period of targeted localization, slow
microtubule-dependent bulk streaming movements occur
(Gutzeit, 1986b
;
Theurkauf et al., 1992
). After
polarity determinant localizations are well established, microtubule-based
streaming becomes fast and well-ordered just before nurse cells
non-selectively dump their contents into the anterior end of the oocyte
(Gutzeit and Koppa, 1982
).
Coincident with the start of fast streaming, oocyte microtubules align to form
bundles that lie parallel to the cortex
(Theurkauf et al., 1992
).
Although kinesin-1, dynein and microtubules are important for these processes,
how they contribute and how they relate to one another remains poorly
understood.
To address issues about the mechanism of streaming and how it influences targeted localization and dispersal transport processes, we used time-lapse confocal microscopy to study the behavior of endosomes, determinant mRNAs and microtubules during slow and fast streaming. Tests of kinesin-1 and cytoplasmic dynein suggest a novel competitive relationship during slow streaming stages. Suppression of dynein activity allows a transition to robust, fast, plus-end movement by kinesin-1 that aligns microtubules into parallel arrays, which orders and amplifies plus-end cargo motion and thus fast streaming of surrounding cytoplasm. An allelic series of Khc mutations revealed that while posterior oskar mRNA localization did not require streaming, it did require some kinesin-1 activity, supporting the hypothesis that kinesin-1 can form physical links with oskar RNPs that contribute to posterior oskar localization by direct microtubule-based transport.
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Materials and methods |
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In situ hybridization and immunolabeling
For osk fluorescent in situ hybridization, flies were dissected in
Robb's medium (in under 4 minutes), then fixed, rinsed and probed as described
previously (Cha et al., 2002).
Anti-
-tubulin staining was carried out as described previously
(Brendza et al., 2002
).
Specimens were imaged with either a BioRad MRC600 scanning confocal or a
PerkinElmer Ultraview spinning disk confocal fluorescence microscope.
Movies
For most endosome movies, 1 mg/ml Trypan Blue dye was injected into female
fly abdomens and allowed to incubate for 2-7 hours. The dye, endocytosed with
yolk by the oocyte, served as a bright fluorescence marker to make yolk
granules more visible (B.-J.C., unpublished)
(Danilchik and Denegre, 1991;
Gutzeit and Arendt, 1994
).
Ovaries were dissected under halocarbon oil as described
(Theurkauf, 1994b
;
Theurkauf and Hazelrigg,
1998
). Antibody injections were made for a rabbit
anti-Drosophila Khc (Cytoskeleton), a mouse monoclonal anti-Dhc
(PIH4) from Tom Hays (McGrail and Hays,
1997
), or a mouse monoclonal anti-DIC (74.1, Santa Cruz
Biotechnology). All antibodies were dialyzed against PBS for at least 4 hours
before injection at 2 mg/ml. Movies of Khc mutants were recorded with
a BioRad MRC600 confocal, movies of antibody injections were made with a Leica
TCS-SP confocal, and movies of GFP::
-tubulin were captured with a
PerkinElmer Ultraview spinning disk confocal. Although acquisition rates
varied between some sets of movies, all were compressed to 225x real
time using QuickTime. Thus, 4 seconds of movie playback represents 15 minutes
of real time in all videos.
Tracking
Digital organelle tracking was carried out with software created by Aaron
Pilling (A. Pilling, PhD thesis, Indiana University, 2005). A grid was
superimposed over the first frame of each movie. Grid line intersection
coordinates were selected by a random choice generator, either restricted to
the anterior half of stage 8-9 oocytes or throughout stage 10B-11 oocytes. The
center of the endosome nearest each selected coordinate in the first frame was
marked in succeeding frames with a cursor until it left the focal plane or
until 95 frames had elapsed. During slow streaming in stage 8-9, images were
collected every 15 seconds. During fast streaming, images were collected every
2.67 seconds. Endosomes that left the focal plane within five frames were not
tracked. Ten endosomes were tracked per oocyte, and three to 13 oocytes were
analyzed for each test.
To compare the effects of various experimental conditions on streaming velocities, we computed the distance between initial and final positions for each endosome, then divided by total time. This strategy produced slight underestimates of mean velocities, because the paths of some moving endosomes were curved. The advantage over the alternative approach, calculating distance from frame-frame position changes, was in suppressing the `noise' of random saltation, which was substantial during slow streaming.
The amount of error caused by mis-marking of organelle centers and specimen drift was estimated by tracking non-saltating endosomes in dead oocytes. The calculated mean `tracking-error velocity' in stages 8-9 was 1.1 nm/second±0.28 (s.e.m.) (n=20 endosomes). In stages 10B-11, that error increased to 6.4 nm/second±1.6 (n=20 endosomes) primarily because of the 5.6-fold decrease in time between images in the movies used to track fast streaming stages. Tracking-error velocity was subtracted from the raw velocity of each endosome tracked in live oocytes. Those with zero or negative corrected velocities were not included in calculation of the means for `moving organelles'. This was particularly important for analysis of stages 8-9, because many endosomes were stationary during slow streaming. After the error correction, endosomes with positive velocities were used to calculate means for each test, using SPSS 10.0.7 (SPSS). To determine peak velocities, means were calculated for the fastest 10% of endosomes tracked in each genotype.
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Results |
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Determinant mRNA localization in the absence of slow streaming
Slow streaming currents may be important for random movements of
determinant mRNA particles that facilitate contact with anchorage sites in
specific regions of the oocyte cortex (Cha
et al., 2002; Glotzer et al.,
1997
). Consistent with this, the null Khc27
allele, which eliminates slow streaming currents, prevents oskar mRNA
accumulation at the posterior pole and hinders gurken mRNA
accumulation at the anterodorsal corner
(Brendza et al., 2000a
;
Brendza et al., 2002
;
Ephrussi et al., 1991
;
Kim-Ha et al., 1991
;
Neuman-Silberberg and Schupbach,
1993
). A recent model suggests that, by walking on microtubules
that have minus-ends at the cortex and plus-ends away from the cortex,
kinesin-1 drives oskar mRNPs towards the center of the oocyte. Then
random movements such as diffusion and/or slow streaming deliver those mRNPs
to cortical anchorage sites at the posterior pole where microtubule density is
lowest (Cha et al., 2002
). To
address the question of whether or not slow streaming is required for mRNP
localization, we used fluorescence in situ hybridization to study effects of
the hypomorphic Khc alleles (Khc23 and
Khc17) on gurken and oskar mRNA
localization. Despite the absence of slow streaming currents in the mutant
oocytes, gurken localization often appeared normal (14 out of 24
Khc23 and seven out of seven Khc17
oocytes; see Fig. S1 in the supplementary material). Likewise, oskar
mRNA localized to the posterior pole in all hypomorphic Khc oocytes
examined (Fig. 2B,C; see Fig.
S2 in the supplementary material), although its concentration may have been
somewhat reduced. Thus, slow streaming is not an essential element of the
gurken or oskar mRNA localization mechanisms.
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Disordered microtubules in slow streaming versus ordered microtubules in fast streaming
Based on staining of fixed egg chambers (e.g.
Fig. 6C), it has been suggested
that formation of subcortical microtubule bundles that lie parallel to the
oocyte surface is an important element of the fast streaming mechanism
(Theurkauf et al., 1992), and
that bundling may trigger the conversion from slow to fast streaming
(Manseau et al., 1996
). To
investigate the relationship of microtubule organization to the slow/fast
streaming modes, tubulin antibodies and GFP::
-tubulin were imaged by
confocal fluorescence microscopy. During slow streaming (stages 8-10A),
microtubules were more abundant at the oocyte anterior than at the posterior,
but they had the appearance of a random network lacking detectable order
(Fig. 6A,B). This was seen most
clearly in time-lapse movies focused near the cortex, where image contrast was
highest. Surprisingly, microtubules near the anterior were in a constant state
of motion and dynamic reorientation (see Movie 12 in the supplementary
material). This adds to arguments that, although minus-ends are embedded in
the oocyte cortex (Theurkauf et al.,
1992
), most microtubules are not otherwise well-ordered during
stages 8-10A (Cha et al.,
2002
). We saw no evidence supporting the hypothesis that plus ends
are uniformly directed towards the posterior pole.
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Discussion |
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Ooplasmic streaming and development
It is reasonable to assume that the purpose of active but random transport
processes like streaming is to facilitate the dispersal of cytoplasmic
components that do not diffuse fast enough to support cellular and
developmental demands. However, it could also be important for asymmetric
localization processes by facilitating encounters of cytoplasmic components
with localized anchors (Cheeks et al.,
2004; Glotzer et al.,
1997
). More specific insights into how microtubule-based streaming
contributes to particular processes have been elusive, in part because the
only means to prevent it was to eliminate microtubules, which are needed for
many fundamental cellular processes. Identification of kinesin-1 as the motor
for streaming in Drosophila (this report)
(Palacios and St Johnston,
2002
) provides the opportunity for more focused studies, because
it has a narrower range of functions and is not essential for early oocyte
development (Brendza et al.,
2000b
).
Our Khc allelic series allowed investigation of the significance of nurse cell/ooplasm mixing. Khc-null oocytes, with no streaming, usually showed yolk stratification as evidence of mixing failure. Embryos developing from those oocytes arrested in early stages, suggesting that mixing may be important for subsequent development. However, hypomorphic Khc17 oocytes, which supported weak fast streaming in only one-third of oocytes, allowed three-fourths of the derived embryos to develop to adulthood (Table 1). Yolk stratification was not seen in Khc17 oocytes, suggesting that some mixing can occur without ordered streaming. Although these observations are consistent with the hypothesis that vigorous ooplasmic mixing helps optimize development, it is likely that fast streaming is not absolutely essential.
The Khc allelic series also allowed exploration of a role for slow
ooplasmic streaming in determinant mRNA localization. The null allele
Khc27 prevented streaming, it blocked oskar mRNA
accumulation at the posterior pole and it blocked gurken mRNA
localization to the anterodorsal corner
(Brendza et al., 2002;
Cha et al., 2002
;
Duncan and Warrior, 2002
;
Januschke et al., 2002
).
However, the hypomorphic alleles Khc17 and
Khc23, which prevented most slow streaming, supported both
oskar and gurken localization (Figs
1,
2; see Figs S1, S2 in the
supplementary material). Thus, although localization of both determinants
requires Khc, it does not require slow streaming.
Kinesin-1 and the mechanism of oskar mRNA localization
It has been suggested that posterior oskar localization during
stages 7-10a proceeds via two phases (Cha
et al., 2002). First, oskar RNPs are driven by kinesin-1
away from microtubule minus ends at the anterior and lateral cortex, which
leads to a transient concentration of oskar in the central region of
the oocyte. Then diffusion or other random forces, coupled with a dearth of
minus ends at the posterior cortex, facilitates encounters of oskar
RNPs with posterior anchors. Our tests of Khc17 and
Khc23, which slow the ATPase activity and velocity of Khc
in vitro, showed a delay in the central accumulation of oskar,
consistent with slowed kinesin-1-driven transport away from the anterolateral
cortex. Strikingly, Khc17 and Khc23
allow that central accumulation to persist through later stages, as if the
shift to posterior anchors is also slowed (see Fig. S2 in the supplementary
material). This correlation between slowed motor mechanochemistry and slowed
oskar localization supports the hypothesis that kinesin-1 links to
and transports oskar RNPs in both phases of localization.
If microtubules are poorly ordered during oskar localization, as
suggested by our GFP-tubulin imaging and by previous studies of fixed oocytes
(Brendza et al., 2002;
Cha et al., 2002
;
Theurkauf et al., 1992
), how
could kinesin-1 accomplish such directed posterior transport? There may be a
special subset of microtubules, with plus-ends oriented directly toward the
posterior pole, that are difficult to distinguish amongst a mass of randomly
oriented microtubules. However, given that the period of oskar
localization spans at least 10 hours, and that the distance from the oocyte
center to the posterior pole is only 25-40 µm, such perfectly oriented
transport tracks should not be necessary. With microtubule minus ends most
abundant at the anterior cortex and least abundant at the posterior cortex,
plus ends should be somewhat biased toward the posterior. If kinesin-1 binds
an oskar RNP and transports it to a plus end, then binds a
neighboring microtubule and runs to its plus end, and so forth, it would
accomplish a biased random walk away from the anterolateral cortex that would
concentrate oskar RNPs near posterior anchors. This highlights a
central question about the mechanism of localization. What is the degree of
directional bias for oskar RNP transport? Advances in osk
RNP imaging that allow single particle tracking will be needed to obtain clear
answers to that question.
Kinesin-1/dynein competition in ooplasmic streaming
Regarding the mechanism of streaming, we suggest a model in which kinesin-1
drives plus-end-directed motion of cargoes that act as impellers, exerting
force on ooplasm that surrounds them (Fig.
7). Concerted movement of multiple impellers along neighboring
microtubules that are oriented in the same general direction creates streams
of ooplasm. Prior to stage 10B, small streams occur, but are slow and not
well-ordered because dynein resists both plus-end-directed transport and
parallel ordering of microtubules. This resistance may be accomplished via:
(1) a tug-of-war between opposing motors co-attached to individual impellers
(Theurkauf, 1994b); (2) by
movement of different impellers in opposite directions, imparting conflicting
forces on cytoplasm; or (3) competition by dynein and kinesin for the same
binding site on microtubules (Mizuno et
al., 2004
). Regardless of how dynein interferes with kinesin-1,
just before nurse cell cytoplasm is dumped into the oocyte, dynein is
suppressed. This allows kinesin-1 to generate fast plus-end-directed impeller
transport that sweeps microtubules into parallel arrays that then enhance more
robust currents that enhance larger arrays, and so forth, in a self-amplifying
loop.
Our finding that dynein inhibition enhances a kinesin-1-driven transport
process, to our knowledge, provides the first direct indication of a
competitive relationship between opposing microtubule motors. Other studies
have produced convincing evidence of alternating coordination between dynein
and plus-end-directed motors in a number of processes, including transport of
Drosophila embryo lipid droplets
(Gross et al., 2002b),
Drosophila cultured cell RNPs and peroxisomes
(Kural et al., 2005
;
Ling et al., 2004
),
Drosophila axonal mitochondria (A. Pilling, PhD thesis, Indiana
University, 2005), and Xenopus pigment granules
(Deacon et al., 2003
). In
those processes, inhibition of one motor does not enhance transport in the
opposite direction. In fact kinesin-1 inhibition inhibits not only plus-end
transport but also dynein-driven minus-end transport
(Brady et al., 1990
;
Kural et al., 2005
) (A.
Pilling, PhD thesis, Indiana University, 2005). Furthermore, dynein depletion
can inhibit both directions of peroxisome transport
(Kural et al., 2005
),
confirming that kinesin-1 and dynein each can have positive influences on the
other. Our observation of competition between dynein and kinesin-1 suggests
that alternating coordination and positive interactions between microtubule
motors are not a uniform rule, and that some processes have evolved to take
advantage of motor competition.
|
Control of kinesin-1-based fast streaming by the actin cytoskeleton
The observation that actin cytoskeleton depolymerization or mutation of
certain actin-interacting proteins can induce premature kinesin-1-driven fast
streaming is particularly interesting. Actin filaments are most abundant in
the cortex and ring canals of the oocyte and nurse cells
(Gutzeit, 1986a;
Warn et al., 1985
), but
filaments probably also traverse the internal cytoplasm. An intact actin
cytoskeleton could physically assist dynein in resisting kinesin-based
plus-end-directed transport during slow streaming, either passively by
increasing viscosity or actively by generating antagonistic forces. The active
force idea is supported by reports that myosin V can alter the balance between
alternating dynein and kinesin-2-driven runs of melanosomes in
Xenopus (Gross et al.,
2002a
). Drosophila myosin V inhibition tests have not yet
been reported, but a disordered cortical actin cytoskeleton in Moesin
mutant oocytes did not trigger premature fast streaming
(Polesello et al., 2002
),
suggesting that well-ordered actin-based forces may not be important for the
streaming control mechanism. An alternative to such physical resistance is
that dynein inhibitory factors are sequestered by F-actin prior to stage 10B.
Then, just before dumping, those factors are released, dynein is inhibited,
and kinesin-1 is freed to drive fast streaming
(Fig. 7).
Recently, several other factors have been identified that are required for
prevention of premature fast streaming. Mutations in Maelstrom
(Mael), Orb and Spindle-E (Spn-E) allow
premature fast streaming and parallel microtubule arrays during stages 8-10A
(Clegg et al., 1997;
Martin et al., 2003
). Orb, a
CPEB homolog, is required for osk translation
(Chang et al., 1999
), spn-E is
an RNA helicase (Gillespie and Berg,
1995
), and Mael is a modifier of Vasa
(Findley et al., 2003
), which
is another RNA helicase (Hay et al.,
1988
; Liang et al.,
1994
). Perhaps these proteins control expression of actin
regulators or other factors needed to prevent premature activation of a dynein
inhibitory signal. Future work aimed at identifying the regulatory mechanisms
that control kinesin in oocytes should be an important focus in understanding
the slow-fast streaming transition and also for the broader issue of how the
functions of the actin and microtubule cytoskeletons are integrated.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/16/3743/DC1
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