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First published online 16 May 2007
doi: 10.1242/dev.004952
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1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA 98109, USA.
2 Molecular and Cellular Biology Program and Department of Biology, University
of Washington, Seattle, WA 98195, USA.
3 Howard Hughes Medical Institute, Seattle, WA 98109, USA.
* Author for correspondence (e-mail: jpriess{at}fred.fhcrc.org)
Accepted 6 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Actin, Cytoskeleton, C. elegans, Cytoplasmic streaming, Oogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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23 minutes, a rate of biosynthesis that is equivalent to
the entire gonad doubling in size every 6.5 hours
(Hirsh et al., 1976
;
McCarter et al., 1999). Most
of the growth of the oocyte occurs during stages when its nucleus appears to
be transcriptionally quiescent, raising the issue of where oocyte materials
originate (Starck, 1977). In
many animals with large oocytes, much of the oocyte volume consists of yolk
lipoproteins. The yolk lipoproteins are typically secreted by non-gonadal
tissues, such as the liver in vertebrates or the fat body in
Drosophila, and then taken up into oocytes by endocytosis
(Valle, 1993). Similarly,
C. elegans oocytes use receptor-mediated endocytosis to take up yolk
proteins that are secreted into the body cavity by intestinal cells
(Grant and Hirsh, 1999;
Kimble and Sharrock, 1983).
However, fully grown C. elegans oocytes that lack the yolk receptor
RME-2 are only slightly smaller than wild-type oocytes, indicating that yolk
does not constitute most of the oocyte volume
(Grant and Hirsh, 1999).
C. elegans oocytes are incompletely cellularized until late stages
of oocyte development; the growing oocytes are linked through cytoplasmic
bridges to a shared anucleate region called the gonad core (see
Fig. 1A). The core extends
through most of the adult gonad, interconnecting hundreds of germ cells at
various stages of mitosis, meiosis and oogenesis. Similar cytoplasmic bridges
link small groups of germ cells, called cysts, in Drosophila, Xenopus
and mice (Pepling et al.,
1999). Although the bridges may have different functions in male
and female germlines, or at different times in germline development, at least
one important function of the bridges during Drosophila oocyte
development is in cytoplasmic transport (reviewed by
Cooley and Theurkauf, 1994;
Mahajan-Miklos and Cooley,
1994). Only one cell in the 16-cell Drosophila cyst
becomes an oocyte; the other 15 cells become highly polyploid `nurse' cells
that contribute protein and RNA to the oocyte, and eventually die by apoptosis
(Cavaliere et al., 1998).
C. elegans gonads do not contain morphologically distinct nurse
cells, and none of the germline nuclei appear to be highly polyploid when
stained for DNA (Hirsh et al.,
1976) (our unpublished data). Prior to oogenesis, most germ cells
appear to be highly active transcriptionally, producing RNA that accumulates
in the gonad core (Gibert et al.,
1984; Starck,
1977). As the transcriptionally quiescent oocytes enlarge, they
presumably incorporate core cytoplasm that they, or other germ cells,
synthesized during earlier developmental stages and deposited in the core.
Thus, most germ cells could function transiently as nurse cells before
becoming gametes. It has been estimated that about half of all C.
elegans germ cells die by apoptosis around the end of the pachytene stage
(Gumienny et al., 1999); if
these germ cells contribute material to the core before dying, they might
function solely as nurse cells. However, all C. elegans germ cells
appear to have the potential to differentiate into functional gametes if the
apoptotic pathway is blocked (Gumienny et
al., 1999).
The mechanism through which core cytoplasm is incorporated into C.
elegans oocytes is not understood. The oocyte plasma membranes could
simply extend across the core, slicing off a section of adjacent cytoplasm
(Hirsh et al., 1976). For
example, in the transition from syncytial blastoderm to cellular blastoderm in
Drosophila embryos, cytoplasm is engulfed as the plasma membrane
invaginates between nuclei and progressively furrows into the egg
(Foe and Alberts, 1983).
Alternatively, core cytoplasm could be transported into C. elegans
oocytes, analogous to the movement of nurse cell cytoplasm into
Drosophila oocytes. In the early stages of Drosophila
oogenesis, there is a steady movement of nurse cell cytoplasm into the
enlarging oocyte through cytoplasmic bridges, here termed ring canals
(Mahajan-Miklos and Cooley,
1994). Inhibitor studies have suggested that movement of particles
such as mitochondria and lipid droplets through the ring canals is dependent
on microfilaments, but not microtubules, although large numbers of
microtubules are present in the ring canals
(Bohrmann and Biber, 1994;
Theurkauf et al., 1992).
However, a GFP-tagged Exuperantia fusion protein appears to move through the
ring canals independently of microfilaments
(Theurkauf and Hazelrigg,
1998). Late in oogenesis, the nurse cells undergo apoptosis and
expel their remaining contents into the oocyte through a process called
dumping. Dumping is thought to be mediated by the contraction of the
actin/myosin-rich cortex of the nurse cells (reviewed by
Robinson and Cooley,
1997).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Unless
otherwise indicated, experiments were performed on the wild-type N2 strain
(var. Bristol), maintained at room temperature (22-23°C) and analyzed as
soon as they were fully gravid (12-24 hours after last molt). The following
strains and alleles were used: fem-1(hc17ts), fem-3(q20gf)
(Barton et al., 1987),
fog-1(q241) (Barton and Kimble,
1990), VIT-2::GFP (YP-170::GFP; strain DH1033)
and rme-2(b1008) (Grant and
Hirsh, 1999). fem-1(hc17ts) worms were shifted to the
restrictive temperature (25°C) at the L1 stage. fem-3(q20gf)
hermaphrodites were raised at semi-permissive temperature (15°C), and
analyzed 1 day after the final molt. fog-1(q241)/hT2::pharyngeal GFP
was kindly provided by Tim Schedl (Washington University School of Medicine,
St Louis, MO). Homozygous fog-1(q241) `males' (XO animals with
feminized germline that produce oocytes) were grown at 17°C and analyzed
when they produced oocytes.
Plasmid construction and generation of transgenic strains
Standard techniques were used to amplify and manipulate DNA. The Advantage
High Fidelity PCR Kit (Clontech) was used for all PCR procedures. pUW005
(nmy-2::PGL-1::GFP; unc-119): the nmy-2 coding region
present in the nmy-2::GFP; unc-119 plasmid
(Nance et al., 2003) was
replaced with PCR-amplified genomic pgl-1 coding sequence. pUW024
(nmy-2::PGL-1::mRFP-1; unc-119): the gfp coding sequence in
pUW005 was replaced with mRFP-1 amplified from the plasmid
[mRFP-1in pRSETB]
(Campbell et al., 2002).
Transgenic strains expressing these plasmids were obtained by microparticle
bombardment of unc-119 worms
(Praitis et al., 2001).
Time-lapse imaging, image analysis and oocyte volume measurements
Individual adult worms were anaesthetized by incubation in M9 buffer
(Brenner, 1974) containing
0.01% levamisole (Sigma) for about 5 minutes. Gonads were imaged within intact
worms, unless stated otherwise. Worms were transferred onto agarose pads (4%
agarose in M9), a coverslip was added and the resulting chamber sealed with
petroleum jelly.
Differential interference contrast (DIC) 4D time-lapse movies were acquired
as described (Thomas et al.,
1996) using 4D Grabber (v. 1.32) software (C. Thomas, Integrated
Microscopy Resource, University of Wisconsin, Madison, WI). Frames were
recorded at 15-second intervals. Movies were analyzed using ImageJ (v. 1.33)
software
(http://rsb.info.nih.gov/ij/).
Particle tracks were generated using the ImageJ `manual tracking' plug-in.
Individual particles were tracked for 2-minute intervals, unless indicated
otherwise. Particle speeds were then calculated for each time point and
averaged over the 2-minute time interval using Microsoft Excel (v.
11.1.1).
Area and length measurements of oocytes were performed with ImageJ (v.
1.33) software
(http://rsb.info.nih.gov/ij/)
and Microsoft Excel (v. 11.1.1). Oocyte volumes before and after influx were
5682±1000 and 13729±3000 µm3, respectively,
corresponding to a 2.4-fold increase in size (n=19 oocytes; six
movies). Fully mature oocytes that have taken up yolk are 21700±4000
µm3 (n=8 oocytes), suggesting that 37% of the final
volume originates from streaming. Flux measurements generate a similar
estimate of 29%: oocytes are ovulated every 23 minutes
(McCarter et al., 1999) and
the cytoplasmic flux through the late pachytene zone is [core diameter (7.6
µm)/2]2 x 
x flow velocity (6 µm/minute at
22°C)=272 µm3/minute (n=6 animals).
Confocal time-lapse movies were obtained using a Leica TCS SP microscope and Leica Confocal software, and a Zeiss LSM 510 and Zeiss software. We used argon (488 nm) and helium/neon (543 nm) lasers. Laser powers at the sample were in the range 0.01-0.07 mW (exception: Movie 1, 0.5 mW for red channel). Pinhole size was between 0.9 and 3.4 Airy units. To label mitochondria, worms were fed overnight on bacterial lawns soaked with 1 ml of DiOC6(3) solution (2 µg/ml).
Imaging of extruded gonads and drug treatments
Worms were picked onto a coverslip, into a drop of freshly made embryonic
culture medium [ECM; modified from Park and Priess
(Park and Priess, 2003): 84%
L-15 (Leibovitz L15 with L-glutamine, without Phenol Red; Gibco),
9.3% fetal calf serum (Gibco), 4.7% sucrose, 0.01% levamisole, 2 mM EGTA].
Worms were cut behind the pharynx to extrude gonads. Many gonads appear
damaged after extrusion and do not display streaming (data not shown); these
gonads were removed. For drug treatments, ECM containing latrunculin A
(Sigma), ML-7 (Calbiochem) or DMSO (for controls) was then added. The
coverslip was inverted onto an agarose pad (0.8% agarose in water, soaked in
ECM and inhibitor drugs). The pad was surrounded by petroleum jelly spacers
and, after applying gentle pressure to the coverslip, the chamber was sealed
as above. DIC time-lapse movies (see above) were recorded starting no later
than 10 minutes after extrusion. Some movies, of both experimental and control
gonads, were discarded based on abnormal morphology such as loops and
constrictions that formed during the movie.
Visualization of microtubules by immunofluorescence
Cut worms with extruded gonads were fixed in 2% paraformaldehyde in 75 mM
PIPES pH 7.0, 10 mM EGTA, 10 mM MgCl2 for 15 minutes, then rinsed
once with PBS and three times with 75 mM PIPES pH 7.0. Gonads were dissected
free from body tissues and transferred by mouth pipette to a Teflon-coated
slide (Erie Scientific) treated with 0.1% polylysine (Sigma). Gonads were
gently compressed by drawing across an eyelash glued to a toothpick. Nearly
all of the fluid surrounding the gonads was removed by mouth pipette, and the
slides immersed in -20°C methanol for 10 minutes. Slides were rinsed in
PBS containing 0.1% Tween 20 (Sigma) and immunostained with anti-tubulin
antibody YL1/2 (Boehringer Mannheim); imaging was performed with a Leica TCS
SP scanning confocal microscope.
Phalloidin staining
In method I, Gonads were dissected in culture buffer (1.6% sucrose, 73 mM
HEPES pH 6.9, 40 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM EGTA)
containing 0.5 mM levamisole, then fixed in 3% formaldehyde, 73 mM HEPES (pH
6.9), 40 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM EGTA for 5-10
minutes. Gonads were rinsed briefly in PBS, permeabilized in 0.025% Triton
X-100 in PBS for 5 minutes, then rinsed in PBS and stained with Alexa Fluor
488 phalloidin (Invitrogen) for 20 minutes. Fixation method II is identical to
method I except that 15 mM sodium azide (Sigma) was substituted for levamisole
in the culture buffer. In a typical experiment, no more than 1.5 minutes
elapsed between placing the worms in the buffer, dissecting and adding
fixative. Alternatively, worms could be dissected in buffer without azide, and
then sodium azide was added for 20 seconds before fixation. Live gonads
expressing the actin-binding protein GFP-moesin
(Motegi et al., 2006) that
were exposed to sodium azide at this concentration showed no evidence of de
novo formation or aggregation of actin filaments, suggesting that the azide
treatment stabilized an existing actin population.
RNA interference
RNAi against tubulin genes: wild-type L4-stage worms were fed with
tba-2, tbb-1 and tbb-2 feeding strains from the Ahringer
laboratory RNAi feeding library at 25°C
(Kamath and Ahringer, 2003).
As a control, we used an empty-vector feeding strain.
Dynein heavy chain (dhc-1) RNAi: a 603 bp fragment of the
dhc-1 gene was amplified from worm genomic DNA (N2 strain) using
T7-tagged primers 5'-ATCGATAATACGACTCACTATAGGGTCTTCATCCGCCCTCGT-3'
and 5'-ATCGATAATACGACTCACTATAGGGGCTGATGGACGCATCTGA-3'.
Double-stranded RNA (dsRNA) was then synthesized using standard techniques and
injected into adult wild-type gonads (Fire
et al., 1998). Worms were analyzed 24-30 hours after
injection.
Additional microtubule associated protein genes depleted by RNAi (soaking or injection): F42A6.3, C36A4.5, F32A7.5, lgg-1, lgg-2, F54A3.1, T08D2.8, F53F4.3, dnc-1, Y79H2A.11, W0761.5, dhc-1, che-3, B0365.7, W05B2.4, C17H12.1, dli-1, dlc-1, Y10G11A.2, Y10G11A.3, M18.2, F41G4.1, F13G3.4, T05C12.5, D1009.5.
Injection of mineral oil drops and microspheres
Young adult worms were injected with drops of mineral oil (heavy white oil,
Sigma) into one or both gonad arms, using techniques as described
(Mello et al., 1991)
(http://www.wormbook.org/).
The worms spent a maximum of 5 minutes on the injection pads, and were then
transferred in M9 buffer to bacterial lawns. Only worms that moved normally
were analyzed further. Time-lapse movies (see above) were started 20 minutes
after the injection was completed.
Fluorescent polystyrene microspheres [FluoSpheres, carboxylate-modified microspheres, 0.2 microns, red fluorescent (580/605 nm), Molecular Probes] for injection were washed four times in water, incubated in 1% bovine serum albumin in 50 mM phosphate buffer (pH 7.4) for 1 hour, washed twice and then resuspended in phosphate buffer (pH 7.4).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Worm Book online
(http://www.wormbook.org/).
The gonad is composed almost entirely of germ cells; a few somatic cells
called sheath cells partially surround the gonad. Throughout most of the
gonad, germ nuclei are located at the periphery of the gonad, encircling a
large anucleate zone called the core of the gonad (or rachis). The plasma
membrane around each germ nucleus is incomplete, creating an intercellular
bridge with the core. We refer to these circular openings between the germ
cells and the core as ring channels. Although most C. elegans germ
nuclei are syncytial, it is conventional to refer to a nucleus, the associated
cytoplasm and membranes as a `germ cell'. Mitosis in the germline occurs at
the distal end of each gonad arm (away from the uterus and vulva), eventually
producing about 1000 germ cells that enter successive stages of meiosis as
they move proximally (towards the uterus). Most of the distal half of the
gonad consists of nuclei at the pachytene stage of meiotic prophase I; for
convenience, we subdivide this region into equally sized early, mid, and late
pachytene regions. In fourth-larval-stage hermaphrodites, the first set of
about 150 germ cells differentiate as sperm in the proximal region of the
gonad; subsequent adult germ cells differentiate as oocytes. The oocytes begin
to enlarge in the loop area, and finally sever their connection to the core
(cellularization) after leaving the loop. Oocyte meiotic maturation is
signaled by extracellular major sperm protein (MSP) from sperm in the
spermatheca; mature oocytes are ovulated into the spermatheca and fertilized
(for a review, see Greenstein,
2005).
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). We
found that there was a general, proximal flow of mitochondria along the core
(see Movie 1 in the supplementary material), similar to the movement of the
DIC particles. In addition, we observed several examples of mitochondria that
first moved radially away from positions near pachytene nuclei, and then moved
proximally (data not shown). We next examined the germ cell-specific P
granules, which are associated with nuclear pore clusters on germ cell nuclei
from mitosis to late pachytene stages of meiosis, but detach from nuclei
during diakinesis as oocytes differentiate
(Pitt et al., 2000;
Strome and Wood, 1982).
Transgenic worm strains were constructed expressing the P-granule component
PGL-1 fused to either GFP or mRFP1. The tagged proteins localized correctly to
nuclear-associated P granules, and to the detached cytoplasmic P granules
(Fig. 1D, arrow and arrowhead,
respectively). In addition, we found that the tagged proteins were present in
much smaller cytoplasmic particles throughout the gonad
(Fig. 1E,F, green square and
yellow triangle; average diameter of cytoplasmic particles was
0.29±0.06 µm, n=86 particles). We observed similar small
particles after immunostaining wild-type gonads for PGL-1, and found that
additional P-granule components called GLH-1 and GLH-2 colocalized to the same
particles (data not shown) (Gruidl et al.,
1996; Roussell and Bennett,
1993). These small particles could arise from fragments of
nuclear-associated P granules, or form de novo in the cytoplasm. In 18 out of
21 time-lapse movies, the small PGL-1::GFP or PGL-1::mRFP particles moved
proximally along the core (Fig. 1D-G and
H-L, arrowhead; see Movie 1 in the supplementary material). In the
loop region, we observed several examples of fluorescently labeled P granules
that moved proximally in the core after detaching from nuclei; some of these P
granules were eventually incorporated into oocytes several cells distant from
the oocyte nucleus of origin (see Movie 2-2 in the supplementary
material). We observed similar movement rates in pairwise recordings of DIC particles, mitochondria and PGL-1::GFP particles within the same gonads (data not shown), suggesting that there was a general streaming of core cytoplasm. We thus asked whether presumably inert materials could be transported by this flow. Fluorescently labeled polystyrene microspheres or small droplets of mineral oil (Table 2; Fig. 2; see Movies 2-1 and 2-2 in the supplementary material) were injected into gonads. Microspheres and oil droplets appeared to move at the same rate, and in the same pattern, as cytoplasmic P granules and DIC particles, respectively. We conclude that there is a general flow of core cytoplasm towards the proximal end of the gonad where oocytes enlarge, and refer to this flow as proximal streaming. Based on the average speed of DIC particles, we calculate that the flux in the core is about 270 µm3/minute at 22°C. We estimate that oocytes increase in volume about 2.4-fold from influx of cytoplasm, and that roughly 30-40% of the final egg volume originates from cytoplasmic streaming (see calculations in Materials and methods).
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).
Streaming, and oocyte production, resumed in old wild-type hermaphrodites
after mating to males (Table
2). We considered the possibility that ovulation itself might
contribute to proximal streaming. Approximately every 23 minutes, contractions
of somatic sheath cells that surround the proximal end of a gonad arm force
the terminal, mature oocyte into the spermatheca
(Hall et al., 1999;
McCarter et al., 1999). C.
elegans has a high internal hydrostatic pressure that maintains body
shape, and this pressure might force core cytoplasm proximally into the space
vacated by the oocyte. Although we observed a rapid and local redistribution
of gonad cytoplasm coincident with each ovulation (see Movie 1 in the
supplementary material), these discrete events appeared to be superimposed on
otherwise continuous proximal streaming. Indeed, we observed proximal
streaming under conditions in which ovulation does not occur: (1) very young
adult wild-type hermaphrodites before the first ovulation; (2)
fem-3(q20gf) mutant worms in which abnormal spermatogenesis prevents
ovulation (Barton et al.,
1987); and (3) fog-1(q241) `males'
(Table 2; for fog-1,
see also below). We conclude that proximal streaming is associated with
oogenesis, but is not caused by ovulation.
Although endocytosis of yolk lipoproteins does not account for most of the
size increase of enlarging oocytes (see Introduction), we considered the
possibility that yolk uptake might trigger streaming of other cytoplasmic
materials; the yolk receptor RME-2 is expressed throughout the loop region of
the gonad where oocytes enlarge (Grant and
Hirsh, 1999). However, we observed that a GFP fusion of the yolk
protein VIT-2 (Grant and Hirsh,
1999) begins to enter oocytes right after they have ceased
receiving cytoplasm (data not shown). In addition, proximal streaming appears
normal in the gonads of rme-2(b1008) mutants lacking the receptor
(Table 2)
(Grant and Hirsh, 1999).
Finally, we asked if germline apoptosis is required for streaming.
ced-3(n717); ced-1(e1735) double mutants lacking apoptosis showed
normal streaming, although at a slightly reduced average particle speed
compared with wild-type worms (Table
2) (Gumienny et al.,
1999).
Proximal streaming does not require sheath cell contraction
Because the gonad sheath cells undergo frequent small contractions between
ovulation events (McCarter et al.,
1999), we considered the possibility that peristaltic waves of
sheath contractions might contribute to proximal streaming. Contractions of
wild-type sheath cells can be reduced or eliminated by depleting calcium with
EGTA (Yin et al., 2004). We
found that extruded gonads treated with 2 mM EGTA had few or no sheath
contractions, but most appeared to have normal proximal streaming for at least
30 minutes (50 out of 53 gonads; Fig.
4E,F; Table 2; see
Movie 4-3 in the supplementary material). Previous studies have shown that
sheath contractions are highly reduced in feminized mutants such as
fem-1 (McCarter et al.,
1999). Although we found that sheath cell contractions were often
not detectable in time-lapse recordings of fem-1(hc17) young adults,
these animals showed normal proximal streaming
(Table 2). We next examined
fog-1(q241) mutant `males' that produce oocytes, but lack sheath
cells and other female somatic gonad structures
(Barton and Kimble, 1990;
Jin et al., 2001). Gonad
morphology in these mutants was highly aberrant and oogenesis very slow. Many
animals showed very slow, or no, streaming. However, 7 of the 21 animals
examined showed streaming, at a reduced average speed of 2.5±0.8
µm/minute, into individual oocytes
(Table 2). Taken together,
these results suggest that sheath contractions are not essential for
streaming, and that at least some streaming can occur in the absence of sheath
cells.
|
). We
developed a protocol that better preserves long microtubules (see Materials
and methods). In the distal mitotic zone of the gonad, microtubules are
present in the spindles of dividing cells or in apparently random interphase
arrays (data not shown). Beginning with the pachytene zone, each germ nucleus
is surrounded by a basket of microtubules that extend through the ring channel
into the core of the gonad, and then continue proximally for long distances
(Fig. 3A,B). Similar
microtubules run through the ring channels of enlarging oocytes, bridging each
oocyte with the core until cellularization
(Fig. 3C). To investigate
whether microtubules were required for proximal streaming, we depleted alpha-
or beta-tubulin by dsRNA-mediated interference (RNAi). RNAi against single
isoforms can result in depletion of multiple alpha- or beta-tubulin isoforms
(Sonneville and Gonczy, 2004;
Wright and Hunter, 2003). We
found that dsRNA corresponding to tba-2 (alpha-tubulin) and/or
tbb-1 and tbb-2 (beta-tubulin) genes caused a marked
depletion in microtubules detectable by immunostaining. For example, worms
exposed to tba-2 dsRNA for 24 hours had only short microtubules
associated with the pachytene nuclei, and essentially no microtubules in the
gonad core or in oocytes (Fig. 3, compare E
with F). The treated animals produced only inviable embryos with
severe cellular abnormalities (data not shown), and had variable but mild
defects in gonad morphology. Nevertheless, proximal streaming appeared normal
in the microtubule-depleted gonads (Fig.
3G,H; Table 2).
After treatment with tba-2 dsRNA for 48 hours, the gonads showed more
pronounced morphological defects; germ nuclei were present in the normally
anucleate gonad core, and many oocytes had abnormal shapes and sizes. However,
most of these gonads continued to show proximal streaming. Remarkably, germ
nuclei that were present inappropriately in the core also travelled proximally
(see Movie 3-1 in the supplementary material). In a parallel series of
experiments, we were unable to prevent streaming by depleting the gene
products of 23 predicted microtubule motors and other microtubule-associated
proteins (MAPs) by RNAi (see Materials and methods; data not shown). Depletion
of some gene products, such as the dynein proteins DHC-1 and DLC-1 and the MAP
DNC-1, resulted in gross morphological defects in the gonad. However, we
observed proximal streaming in most of the treated animals (see Movie 3-2 in
the supplementary material). Together, our results argue that microtubules are
not essential for proximal streaming.
Previous studies have shown that actin and non-muscle myosin are highly
enriched in the cortex of germ cells and oocytes
(Maddox et al., 2005;
Strome, 1986), but have not
described localization throughout the cytoplasm. We used two staining
procedures to visualize actin with fluorescent phalloidin (see Materials and
methods). Using method I, stained filaments were significantly enriched around
oocyte nuclei, with relatively few filaments visible in the core region of
most gonads (Fig. 4A). A small
fraction of these gonads showed fine filaments aligned in the direction of
streaming in the core and ring channels, and this appearance was observed
consistently using fixation method II (Fig.
4B; see Movies 4-1 and 4-2 in the supplementary material). With
method II, most of the filaments within oocytes appeared to be randomly
oriented, except for groups of filaments that extended between the oocyte
nucleus and the plasma membrane. By contrast, numerous filaments in the ring
channels of enlarging oocytes were oriented in the direction of streaming.
Many of these filaments appeared to extend from the core plasma membrane
toward and into enlarging oocytes (Fig.
4B, arrowheads). The staining intensity of these oriented
filaments was comparable to that of the apparently randomly oriented filaments
in the core region of the pachytene area
(Fig. 4C).
To determine whether microfilaments are required for proximal streaming, we
analyzed gonads extruded in culture medium with or without inhibitors of actin
polymerization or myosin light chain kinase; EGTA was added to the medium in
all experiments to prevent sheath cell contractions. In gonads treated with
the actin depolymerizing drug latrunculin A (LatA)
(Severson and Bowerman, 2003;
Spector et al., 1983) at 0.5-5
µm, the actin-rich lateral membranes of germ cells and enlarging oocytes
appeared to collapse. Although a few gonads treated with LatA showed normal
proximal streaming (2 of 13), most lacked streaming (9 of 13) or had aberrant,
reverse flow in the distal direction (2 of 13;
Fig. 4G,H); 50 of 53 control
gonads without LatA treatment showed normal proximal streaming
(Fig. 4E,F; see Movie 4-3 in
the supplementary material).
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Extruded gonads treated with the myosin light chain kinase inhibitor ML-7
(Lee and Goldstein, 2003;
Saitoh et al., 1987) at 250
µm showed proximal streaming defects and membrane defects similar to those
induced by 0.5-5 µm LatA. The treated gonads either lacked proximal
streaming (4 of 8), showed only brief streaming (1 of 8), or had aberrant flow
in the distal direction (3 of 8). Gonads treated with 60 µm ML-7 usually
showed normal proximal streaming for several minutes, but then stopped
streaming either just before, or coincident with, a collapse in plasma
membrane structure (3 of 9 and 6 of 9 gonads, respectively). Finally, we
observed rare cases of reversed streaming in gonads that appeared to have
intact plasma membranes (see Movie 4-4 in the supplementary material).
Together, these results indicate that proximal streaming requires the
actomyosin cytoskeleton.
Streaming forces are generated in or near oocytes
To determine where the forces involved in streaming are generated, we
attempted to block the gonad core at different positions along the
distal-proximal axis by injecting large drops of mineral oil into the gonads
of intact worms. We found that cytoplasmic particles were unable to travel
through or around oil drops injected into the core, indicating that the gonad
was effectively partitioned into separate zones. Blocking the core within the
pachytene region had no apparent effect on proximal streaming (n=2;
Fig. 5A-C). We next injected
oil drops between the pachytene region and the loop. In the pachytene region,
particles either did not move or moved slowly in a reversed, distal direction
(Fig. 5D-F; see Movie 5-1 in
the supplementary material). Surprisingly, proximal streaming appeared
relatively normal in the loop and enlarging oocytes, despite the fact that
only a small segment of gonad core separated the oil drop from the enlarging
oocytes. Moreover, several of these truncated proximal gonads showed abnormal
patterns of flow between adjacent oocytes: large proximal oocytes continued to
expand, apparently by pulling cytoplasm from neighboring smaller oocytes that
simultaneously decreased in size (see Movie 5-1 in the supplementary
material). When we blocked all enlarging oocytes with a large drop of oil,
streaming in the gonad ceased entirely (n=6; see Movie 5-2 in the
supplementary material). Finally, injecting oil into already cellularized
oocytes did not affect streaming (n=3). Together, these results
suggest that proximal streaming is caused by forces generated within or near
the enlarging oocytes, rather than in the pachytene area.
If the oocytes indeed generated their own streaming force, damage of individual oocytes might prevent influx of cytoplasm. Conversely, if cytoplasm was pushed, rather than pulled, through the ring channel into an enlarging oocyte, damaged oocytes might remain competent to receive streaming cytoplasm. We attempted to mechanically damage individual enlarging oocytes by penetrating their apical cell membrane with a microneedle, and marking the damaged cells with a small oil droplet (Fig. 5G-I; see Movie 5-3 in the supplementary material). In almost all cases (6 of 7), cytoplasm immediately ceased streaming into the injected oocyte. In 2 of 7 cases, cytoplasm and the small oil droplet flowed out of the damaged oocyte and into neighboring oocytes on both sides (Fig. 5G-I; see Movie 5-3 in the supplementary material). Cytoplasm flowing from a damaged oocyte into the distal neighbor moved opposite to the general, proximal direction of cytoplasmic streaming, supporting the model that forces in or around individual oocytes generate flow.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Schisa et al., 2001;
Starck, 1977). Because
materials are transported from numerous pachytene-stage nuclei into single,
enlarging oocytes, the pachytene-stage nuclei can be considered a nurse cell
population. Thus, C. elegans appears to use a strategy in which
hundreds of syncytial germ cells contribute materials to an oocyte, in
contrast to the 15 highly polyploid nurse cells in Drosophila
(Cooley and Theurkauf,
1994).
|
;
Mahajan-Miklos and Cooley,
1994).
Cytoplasmic streaming and the cytoskeleton
The rate of cytoplasmic streaming in C. elegans gonads is
comparable to the microtubule-dependent `fast' streaming that occurs in
Drosophila oocytes prior to the dumping of nurse cell contents
(Serbus et al., 2005).
Although gonads contain large numbers of long microtubules that are oriented
in the direction of streaming, our RNAi studies suggest that microtubules do
not play a major role in proximal streaming. These results do not exclude the
possibility of a minor role in organelle transport. For example, plant cells
that undergo rapid actomyosin-based streaming of their bulk cytoplasm can have
much slower kinesin- and microtubule-based transport of specific organelles
(Shimmen and Yokota, 2004).
One interesting possibility is that microtubules in C. elegans
function in moving materials radially, away from the pachytene nuclei and
towards the midline of the gonad core. Previous studies have shown that RNA
accumulates along the midline of the core in old adults
(Schisa et al., 2001), and we
have shown here that old adults lack proximal streaming.
Whereas microtubule depletion did not cause major defects in proximal
streaming, we observed a lack of streaming or aberrant, reverse streaming when
gonads were treated with inhibitors of the actomyosin cytoskeleton. We do not
yet understand the basis for the reversed flow, and it is possible that this
flow has no mechanistic relationship to normal streaming. For example, treated
gonads might expand radially as the cytoskeleton breaks down. Gonads treated
with high concentrations of actomyosin inhibitors showed a collapse of the
lateral plasma membranes, similar to the regression of the contractile ring in
dividing embryonic cells that are treated with microfilament inhibitors
(Strome and Wood, 1983). When
we used lower concentrations of actomyosin inhibitors, proximal streaming
often terminated before any apparent collapse of the lateral membranes. This
result suggests that proximal streaming involves an actin-mediated event that
is at least partially separate from the role of actin in membrane
integrity.
Enlarging oocytes are associated with diverse actin populations that might
contribute to streaming forces. The cortex is highly enriched in actin, there
is a meshwork of filaments throughout the oocyte, and filaments that are
aligned in the direction of flow extend into the oocyte through the ring
channels. Several models for actin-dependent streaming can be considered
(Fig. 6A-D). Newly fertilized
C. elegans embryos exhibit cytoplasmic streaming towards their
posterior pole that is thought to be driven by a reciprocal contraction of
cortical actin toward the anterior pole
(Hird and White, 1993;
Munro et al., 2004); a similar
process could operate in enlarging oocytes
(Fig. 6A). The rate of
cytoplasmic streaming in the embryo is about 4-5 µm/min, comparable to the
rate we observe for streaming into oocytes. However, because expanding oocytes
are not closed systems like embryos, it is difficult to imagine how cortical
contraction alone would result in influx of cytoplasm. In addition, we did not
observe sustained movements of cortical granules, or asymmetries in the
distribution of cortical actin.
|
;
Nagai and Rebhun, 1966;
Shimmen and Yokota, 2004). We
have described actin-containing filaments that extend from the outer plasma
membrane of the gonad core and into the enlarging oocytes. Thus, it is
possible that these filaments have a common polarity, an important element of
a cable model for streaming (Fig.
6B). Streaming rates can be 150 µm/min in Arabidopsis
to approaching 600 µm/min in Chara
(Holweg and Nick, 2004;
Shimmen and Yokota, 2004). In
yeast, ASH1 mRNA and Kar9p are transported by myosins along actin
cables from mother to daughter cells at rates of 30 µm/min and 90
µm/min, respectively (Beach et al.,
2000; Bertrand et al.,
1998). By comparison, the rate of streaming into C.
elegans oocytes is relatively slow, at about only 8 µm/min.
The expansion of the plasma membrane of an enlarging oocyte is reminiscent
of the expanding leading edges of motile cells
(Fig. 6C). Leading edge
extension is thought to be driven by the expansion of an underlying branched
network of actin filaments dependent on the Arp2/3 complex (reviewed by
Pollard and Borisy, 2003).
Similarly, expansion of cellular lobes in Arabidopsis pavement cells
is dependent on actin and regulators of the Arp2/3 complex
(Fu et al., 2005). However,
none of the known C. elegans Arp2/3 genes and regulators have been
reported to be required for normal oocyte size or ovulation rate.
A striking feature of streaming in the C. elegans gonad is the
relative absence of particle movement in the oocyte interior compared with
particle movement through the gonad core and ring channels. Our attempts to
fix actin filaments in the gonad suggest that filaments in the core and ring
channels are much harder to preserve, and therefore possibly more dynamic,
than actin in the oocyte interior. If a dynamic, contractile actomyosin
network in the core and ring channels (Fig.
6D, green arrows) is linked mechanically to a relatively stable
network within the oocyte (Fig.
6D, black lines), this asymmetry could pull particles into the
oocyte. Analogous models of asymmetric actomyosin dynamics have been proposed
for cytoplasmic streaming into the leading edges of amoeboid cells and for
fountain streaming in the Drosophila syncytial blastoderm
(Odell, 1977;
von Dassow and Schubiger,
1994). In future studies, it should be possible to evaluate these
and other models for streaming, as tools become available to visualize
actomyosin dynamics within the gonad.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2227/DC1
|
ACKNOWLEDGMENTS |
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