close

Вход

Забыли?

вход по аккаунту

?

133

код для вставкиСкачать
Cell Motility and the Cytoskeleton 43:159–166 (1999)
Poly(A) mRNA Is Attached to Insect
Ovarian Microtubules In Vivo in a
Nucleotide-Sensitive Manner
Susan Stephen, Nicholas J. Talbot, and Howard Stebbings*
School of Biological Sciences, Washington Singer Laboratories,
University of Exeter, United Kingdom
In ovarioles of hemipteran insects, RNA passes from anteriorly positioned nurse
cells to the chain of developing oocytes via extended nutritive tubes. These
intercellular connections may reach several millimeters in length. Each nutritive
tube is comprised of many thousands of parallel microtubules. We have extracted
microtubule bundles from isolated nutritive tubes of Notonecta glauca and, using
hybridization techniques, provide evidence of poly(A) mRNA attachment to
microtubules in vivo. We also show this attachment to be nucleotide-sensitive,
which is typical of a motor protein-mediated interaction. The pattern of nucleotide
sensistivity is indicative of a kinesin motor mechanism. We provide evidence that a
kinesin is present in the nutritive tube translocation channels and is a component of
the mRNA/microtubule bundles isolated and extracted from them. Our findings are
consistent with kinesin-driven transport of mRNA along the nutritive tube
microtubules. Cell Motil. Cytoskeleton 43:159–166, 1999. r 1999 Wiley-Liss, Inc.
Key words: insect oogenesis; mRNA transport; microtubule motor proteins
INTRODUCTION
The localization of mRNA to discrete domains has
been reported in a number of polarised somatic cells such
as neurones, fibroblasts, and epithelial cells, and particularly in the oocytes and eggs of different species [Wilhelm
and Vale, 1993]. The distribution of mRNA allows the
spatial control of gene expression and in oocytes asymmetric localization of specific mRNAs has been shown to
underlie the establishment of morphogen protein gradients, which in turn determine cell fate during early
development [for reviews see St Johnston, 1995; Bashirullah et al., 1998]. In Drosophila oogenesis, the example
that has been most extensively studied, a large number of
transcripts, including those encoding polarity determinants [St Johnston and Nusslein-Volhard, 1992] and
cell-cycle regulatory proteins [Dalby and Glover, 1992],
have been shown to possess localization signals in their 38
untranslated regions (UTRs) [see Spradling, 1993] and to
pass selectively in a multistep process from a group of
nurse cells, via intercellular bridges called ring canals, to
precise locations within the adjacent oocyte [Theurkauf
and Hazelrigg, 1998].
r 1999 Wiley-Liss, Inc.
In Drosophila egg chambers, microtubules traverse
the short ring canals between fifteen precisely arranged
nurse cells and the oocyte, and are therefore appropriately
placed to act as substrates for mRNA transport. The
microtubules derive from a centrosome within the oocyte
[Theurkauf et al., 1992], so that transport between the
nurse cells and an oocyte is in the retrograde direction.
The involvement of the microtubules in the passage of
maternal mRNA from nurse cells to oocytes has been
demonstrated, because if flies are fed with the antimicrotubule agent colchicine, the microtubules are disrupted and the mRNAs are incorrectly localized [Pokrywka and Stephenson, 1991; Theurkauf et al., 1993].
Mislocalization of transcripts also occurs in Drosophila
Contract grant sponsor: The Wellcome Trust; Contract grant number:
047511; Contract grant sponsor: University of Exeter Research Fund.
*Correspondence to: Howard Stebbings, School of Biological Sciences, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG,
UK. E-mail: H. Stebbings@exeter.ac.uk.
Received 15 January 1999; accepted 22 March 1999.
160
Stephen et al.
mutants showing abnormal arrangements of microtubules
in their egg chambers [Lane and Kalderon, 1994].
Particles believed to contain maternal meassages have
been tracked in Drosophila egg chambers and shown to
move in a microtubule-dependent fashion [Theurkauf and
Hazelrigg, 1998], and the co-localization of microtubule
motor proteins with different maternal-effect gene products within the system [Clark et al., 1994; Li et al., 1994]
has suggested that they could propel the mRNA along the
microtubules. However, while it is apparent that microtubules are necessary for the localization of mRNAs, no
direct evidence for their role in mRNA transport has been
obtained, and a participating motor protein has not been
identified.
In this study, we have focused on oogenesis in the
insect Notonecta glauca. By contrast with Drosophila, in
Notonecta and hemipterans generally the nurse cells are
retained at the anterior while the oocytes are displaced as
a chain down an ovariole (see Fig. 1). As a consequence,
the intercellular bridges between the nurse cell region and
each of the oocytes become greatly extended into nutritive tubes. In Notonecta, the nutritive tubes may reach
several millimeters in length. Each is packed with some
thirty thousand aligned microtubules interspersed with
ribosomes, and bounded by sparsely arranged mitochondria [Macgregor and Stebbings, 1970]. The extensive
microtubule aggregates in nutritive tubes of Notonecta
are long-lived and stable, and are not depolymerised by
anti-microtubule agents or cold [Macgregor and Stebbings, 1970; Lane and Stebbings, 1994]. Importantly,
microtubules are the only cytoskeletal elements present in
nutritive tubes. They have a common polarity, with their
minus ends towards the oocyte [Stebbings and Hunt,
1983], so that nurse cell/oocyte transport is in the
retrograde direction.
Large quantities of RNA pass from the nurse cells
along the nutritive tubes to the oocytes, and autoradiographic studies with Notonecta have shown that the bulk
of this travels at a slow rate (⬃30 µm/h), probably
passively and as monomeric ribosomes [Macgregor and
Stebbings, 1970]. Similar studies reported a comparable
rate of RNA movement along the nutritive tubes of
Pyrrhocoris, a different hemipteran, but also resolved a
smaller faster component travelling at ⬃200 µm/h, believed to be actively transported mRNA [Mays, 1972].
The presence of mRNA in nutritive tubes was subsequently demonstrated in a further species, Oncopeltus, by
in situ hybridization to sections of ovarioles [Capco and
Jeffery, 1979].
Because of their size and stability, microtubule
bundles can be isolated and detergent-extracted from
isolated nutritive tubes of Notonecta [Hyams and Stebbings, 1979; Stebbings and Hunt, 1983] and this presents
a unique opportunity to investigate directly the relation-
Fig. 1. Diagrammatic representation of the disposition of the nurse
cells and a developing oocyte within an ovariole of Notonecta. In
Notonecta, each oocyte is connected to a large number of nurse cells by
a greatly extended nutritive tube (NT). Large numbers of microtubules
(MTS) pack the nutritive tube (plus and minus signs indicate their
polarity). In Drosophila, by comparison, an oocyte is closely connected
to fifteen nurse cells via short ring canals.
ship between microtubules and components that translocate along them. Using hybridization techniques we
confirm that poly(A) mRNA is present in the nutritive
tubes of Notonecta. We provide direct evidence that the
poly(A) mRNA is attached to microtubules extracted
from isolated nutritive tubes. We also show that this
attachment is nucleotide-sensitive, a property consistent
with a motor protein-mediated interaction between poly(A)
mRNA and the microtubules. The pattern of nucleotide
sensitivity predicts kinesin-driven transport of mRNA,
and this is supported by our identification of a kinesin
within the nutritive tubes and, more specifically, mRNA/
microtubule bundles isolated from them.
MATERIALS AND METHODS
Animals and Dissection
Notonecta glauca females were dissected in Locke’s
modified insect Ringer [Hyams and Stebbings, 1979] and
Attachment of mRNA to Microtubules
their ovaries placed immediately into RNAse-free microtubule support buffer (PEM); 0.1 M PIPES pH 6.9,
monosodium salt (Calbiochem, La Jolla, CA), 1 mM
EGTA, 2.5 mM MgSO4 (BDH-Merck, Lutterworth, UK).
All subsequent manipulations were performed in RNasefree PEM. All chemicals were from Sigma Chemical Co.
(St Louis, MO) unless stated otherwise.
Isolation and Detergent Extraction
of Nutritive Tubes
Nutritive tubes were isolated from ovarioles in
polarised light, using tungsten needles [Hyams and
Stebbings, 1979]. They were left attached to the nurse cell
trophic regions for ease of handling. Extraction was
performed with 0.5% Triton X-100 (Boehringer Mannheim UK Ltd., Lewes, UK) for 30–60 sec [Stebbings and
Hunt, 1983].
Oligonucleotide Hybridization and Detection
Isolated and isolated, extracted nutritive tubes were
rinsed briefly in PEM and fixed in 3% paraformaldehyde
in phosphate buffer for 15 min, and rinsed in PBS.
Digoxygenin-labelled 24-mer oligo (dT) probe (R & D
Systems Inc., Minneapolis, MN) was applied to the tubes
at a concentration of 1.5 µg ml⫺1 in hybridization solution
[Alison et al., 1994] in a humidified chamber at 37°C for
at least 4 h [Sambrook et al., 1989]. After hybridization,
tissues were washed to high stringency in 1 ⫻ SSC, 37°C
for 30 min [Sambrook et al., 1989] and rinsed 3 ⫻ 3 min
in Tris buffered detergent (TBD); 50 mM Tris, 150 mM
sodium chloride, 0.1% Triton X-100, pH 7.6. Tissues
were blocked in TBDB (TBD with 5% non-fat dried
milk) for 10 min prior to incubation with 1:200 dilution
alkaline phosphatase-conjugated sheep anti-digoxygenin
fab fragments (Boehringer Mannheim) in TBDB for 30
min at 37°C. Tissues were rinsed for 2 ⫻ 3 min in TBD
and 2 ⫻ 3 min in deionised water. Detection of hybridization was carried out in 100 µl Western Blue Stabilized
Substrate (Promega, Madison, WI) for 30 min in the dark
at room temperature (RT), and the reaction stopped by
washing 3 ⫻ 5 min in deionised water.
In some control preparations, extracted nutritive
tubes were treated for 30 min at 37°C with 0.1 Uµl⫺1
RNase One (Promega UK Ltd., Southampton, UK) prior
to hybridization. In others, either the oligo (dT)digoxygenin probe or the antidigoxygenin-alkaline phosphatase detection conjugate were omitted. In still further
preparations, 24-mer oligo (dA) replaced the oligo (dT).
Following the hybridization procedures, isolated
nutritive tubes or isolated, extracted tubes were located
by differential interference contrast (DIC) microscopy
using a Zeiss (Oberkochen, Germany) Axiophot photomicroscope with ⫻20 and ⫻40 objectives, and then the
presence or absence of hybridization label in such
161
preparations was observed using bright field (BF) microscopy.
Application of Nucleotides
Isolated extracted nutritive tubes were incubated in
10 mM ADP, 2–10 mM ATP, or 2–10 mM GTP for 2 min,
and then rinsed briefly 3 times in PEM prior to fixation
and hybridization with oligo (dT), as previously described. Other tubes were pre-incubated in 0.5 mM
AMP-PNP or 2 Uml⫺1 apyrase for 30 min.
Electrophoresis and Western Blot Analysis
Notonecta ovarian clarified supernatant was prepared as per Anastasi and coworkers [1990]. Ovaries
from 25 Notonecta were dissected and placed in 0.1 M
PIPES. Following homogenization on ice in the presence
of 1.5 mM CaCl2 to depolymerise the microtubules, 2
mM EGTA was added to chelate the calcium and the
suspension was spun at 33,000g for 30 min at 4°C. The
supernatant was removed and ultracentrifuged at 132,000g
for a further hour at 4°C. The clarified supernatant was
mixed with Laemmli buffer [Laemmli, 1970] and separated by one-dimensional SDS-PAGE on 5–10% polyacrylamide gels under non-reducing conditions. Proteins
were either detected using 5% Coomassie Blue R-250 or
transferred to Immobilon-P membrane (Millipore, Bedford, MA) using a semi-dry blotting system (Schleider
and Schuell Ltd., Dassel, Germany) for Western blot
analysis. Membranes were washed in PBS and incubated
in 5% Marvel (Nestle, Basel, Switzerland) in PBS for 1 h
at RT. After rinsing in PBS, blots were treated with 1:100
dilution rabbit anti-kinesin (a gift from Dr. P.J. Hollenbeck, Purdue University, W. Lafayette, IN) for 1 h at RT
in the presence of 0.1% Nonidet P-40 (NP-40). Blots
were washed three times for 5 min with PBS/0.1% NP-40
and then incubated for 1 h at RT with 1:300 dilution horse
radish peroxidase-conjugated goat anti-rabbit IgG (DAKO
Laboratories, High Wycombe, UK) followed by further
washings in PBS/NP-40 buffer. Antibody binding was
detected using L-chloronaphthanol/H2O2 reaction [LaRochelle, 1996].
Immunohistochemistry
Ovarioles were processed for immunofluorescence
according to the methods of Anastasi and co-workers
[1991]. Ovarioles were fixed overnight in 4% paraformaldehyde in PBS buffer, washed three times in PBS,
transferred to 10% sucrose in PBS for 1 h and then
mounted and frozen in Oct compound (Gurr, BDH, Poole,
UK). Transverse sections of ovarioles (10 µm) were cut
using a Reichart-Jung (Vienna, Austria) Cryocut E, and
collected on glass slides coated with 0.01% poly-l-lysine.
Before labelling, sections were permeabilized with 0.1%
Triton X-100 in PBS buffer, pH 7.4, for 10 min, washed
162
Stephen et al.
Fig. 2. a–f: Isolated nutritive tubes after hybridization with digoxygeninconjugated oligo (dT), detected with alkaline phosphatase. Top: Bright
field (BF) images. Bottom: Equivalent differential interference contrast (DIC) images for positional reference of the unlabelled tubes.
a: Isolated nutritive tube showing hybridization signal throughout its
length. b: After detergent extraction, hybridization signal labelled the
extracted microtubule bundle. c: No signal was seen when oligo (dT)
was substituted by oligo (dA). d: Pretreatment of the extracted
microtubule bundle with RNase resulted in no hybridization signal. e:
No signal was seen when oligo (dT) was omitted, or (f) when alkaline
phosphatase-conjugated antibody was absent from the detection procedure. Bar ⫽ 50 µm.
three times with PBS and then incubated with 5%
BSA/5% normal goat serum in PBS buffer for 1 hour at
RT to block non-specific binding, before rinsing with
PBS containing 0.1% BSA. Sections were incubated with
1:100 dilution rabbit anti-kinesin polyclonal antibody, or
mouse anti-kinesin heavy chain monoclonal antibody
(Sigma Chemical Co.) for 1 h at RT. Sections were then
washed three times in PBS/BSA buffer and incubated
with 1:100 dilution of either rhodamine-conjugated goat
anti-rabbit IgG (DAKO) or fluorescein isothiocyanateconjugated goat anti-mouse IgG (DAKO) as appropriate,
for 1 h at RT. After rinsing three times in PBS/BSA,
preparations were mounted in Vectashield (Vector Labs.
Inc., Burlingame, CA) and viewed using a Zeiss Axiophot
fluorescence microscope.
Nutritive tubes were isolated and their microtubule
bundles extracted and fixed, before being processed as
described above for sections.
RESULTS
Electron Microscopy
Nutritive tubes were isolated and extracted as above
and prepared for electron microscopy [Stebbings and
Hunt, 1987].
In ovarioles of N. glauca, nurse cells are retained
anteriorly and supply a chain of developing oocytes via
extended intercellular connections called nutritive tubes.
Nutritive tubes are approximately 30 µm in diameter,
reach several millimeters in length, and are strongly
birefringent. Stripping away of the oocytes freed the
nutritive tubes from the ovarioles, and isolated tubes
remained birefringent for many hours. Electron micrographs of nutritive tubes revealed many thousands of
aligned microtubules interspersed with ribosomes, with a
few mitochondria at the tube peripheries. As seen in
previous studies [Stebbings and Hunt, 1983], isolated
nutritive tubes remained birefringent on extraction with
non-ionic detergents, indicating that the microtubules
persist as stable bundles following this procedure. This
was confirmed in electron micrographs that showed that
on removal of the membrane surrounding the tube, the
ribosomes had been solubilised, and the microtubules,
which packed more closely together, were the only
structures remaining.
After the incubation of isolated nutritive tubes with
oligo (dT) followed by alkaline phosphatase detection of
Attachment of mRNA to Microtubules
163
Fig. 3. a–f: Microtubule bundles from isolated, extracted nutritive
tubes exposed to different nucleotide regimes prior to hybridization
with oligo (dT). Top: Bright field. Bottom: Differential interference
contrast. a: Nutritive tube extracted in the presence of apyrase, which
appeared to have little effect on the hybridization signal. b: In the
presence of AMP-PNP, the signal was consistently more intense. c:
Pretreatment with 10 mM ADP had no effect on the hybridization
signal. d: 5 mM ATP resulted in greatly reduced, although detectable,
signal, while (e) no signal was seen after 10 mM ATP or (f) 5 mM GTP.
Bar ⫽ 50 µm.
the annealed probe, hybridization precipitation signal was
seen throughout lengths of all the tubes examined (Fig. 2a).
No signal was seen after RNase One pretreatment or with
oligo (dA), suggesting specific hybridization of the oligo
(dT) probe to polyadenylated RNA species within the
nutritive tubes. To investigate the relationship between
poly(A) mRNA and the microtubules within nutritive
tubes, further hybridizations were carried out using
microtubule bundles extracted from isolated tubes. These
microtubule bundles consistently hybridized with oligo
(dT) (Fig. 2b), demonstrating that poly(A) mRNA is
attached to the microtubules. No hybridization occurred
with oligo (dA) (Fig. 2c). In an additional series of
control experiments, the application of RNase One to the
microtubules prevented the hybridization with oligo (dT)
(Fig. 2d). No hybridization signal was seen when the
digoxygenin-conjugated oligo (dT) probe was omitted
(Fig. 2e), thereby removing the antigen and showing that
the antibody did not bind non-specifically to the microtubules. Similarly, no signal was observed when the
alkaline phosphatase-conjugated antibody was absent
from the detection procedure (Fig. 2f), showing that the
signal was not due to endogenous alkaline phosphatase
activity.
To investigate the nature of mRNA attachment to
the microtubules, the mRNA-microtubule complexes
extracted from isolated nutritive tubes were subjected to
different nucleotide regimes prior to the application of
oligo (dT) probe. Extraction under conditions of ATP
depletion using apyrase appeared to have little or no
effect and resulted in the same levels of oligo (dT)
hybridization to the microtubule bundles (Fig. 3a). Microtubule bundles extracted in the presence of AMP-PNP
were strongly labelled with oligo (dT) hybridization
signal (Fig. 3b). Signal was also present after the
application of ADP (Fig. 3c). ATP (5 mM) resulted in
greatly reduced hybridization signal (Fig. 3d), and no
signal was detected in preparations treated with 10 mM
ATP (Fig. 3e). The results were similar with GTP (Fig.
3f). Nucleotide sensitivity provides strong evidence that
the attachment of poly(A) mRNA to nutritive tube
microtubules is mediated by a microtubule motor-protein,
and the pattern of sensitivity indicates involvement of a
kinesin.
Anti-kinesin antibodies identified a single protein
band of ⬃120 kDa in Western blots of Notonecta ovarian
proteins (Fig. 4a,b) and immunofluorescence studies of
transverse sections of ovarioles of Notonecta, using two
different anti-kinesin antibodies, showed strong labelling
in the nutritive tubes (Fig. 4c). Similar microtubule
bundles to those extracted from isolated nutritive tubes
164
Stephen et al.
Fig. 4. a: Notonecta ovarian proteins stained with Coomassie Blue and
(b) probed with anti-kinesin antibody in a corresponding Western blot,
which detected a protein of ⬃120 kDa. c: Transverse section of an
ovariole of Notonecta probed with anti-kinesin antibody showing
bright fluorescent labelling in the nutritive tubes (arrowheads) Bar ⫽
30 µm. d: Microtubule bundles extracted from isolated nutritive tubes
also label strongly with anti-kinesin antibodies, while (e) no fluorescence is seen in controls when the primary antibody is omitted. Bar ⫽
30 µm.
for hybridizations studies also labelled strongly with the
anti-kinesin antibodies (Fig. 4d,e).
DISCUSSION
A number of mechanisms could underlie the localization of mRNAs during oogenesis in Drosophila [St
Johnston, 1995; Glotzer and Ephrussi, 1996], but transport by microtubule-dependent motors is most frequently
proposed to play a part in the passage of mRNAs from the
nurse cells to the oocytes. However, direct evidence for
microtubule-mediated mRNA transport in this and other
systems has been difficult to obtain, and the motor
molecules involved have not been identified.
Although mRNA and polysomes have been shown
to associate with microtubules reassembled in vitro
[Hamill et al., 1994; Han et al., 1995] a specific cellular
attachment between mRNA and microtubules has not
previously been demonstrated. In situ hybridization at the
light and ultrastructural levels has, however, revealed the
colocalization of mRNA with microtubules, as well as
with microfilaments and intermediate filaments, in cultured neurones and fibroblasts [Taneja et al., 1992;
Bassell et al., 1994a,b]. Also, following the biochemical
detergent-extraction of many cell types, including Drosophila oocytes [Pokrywka and Stephenson, 1994], mRNA
and polysomes have been found to be associated with the
insoluble cytoskeletal fraction, with monomeric ribosomes in the soluble fraction [Lenk et al., 1977; Jeffery,
1982; Hesketh and Pryme, 1991]. Attempts have then
been made to distinguish the relative involvement of
microtubules and microfilaments by perturbing these
cytoskeletal organelles with either colchicine and or
cytochalasin.
Unlike most cells, the nutritive tube transport
channels linking the nurse cells to the oocytes in Notonecta have an uncomplicated cytoskeleton comprised
solely of large bundles of stable microtubules. Moreover,
the microtubule bundles are sufficiently large to be
dissected manually from ovarioles, while viewed in
polarised light. They have, therefore, proved to be an
ideal system for investigating the relationship between
microtubules and transported RNA. In this study, we have
found that oligo (dT) probes hybridized evenly throughout the lengths of isolated nutritive tubes, confirming the
presence of mRNA. As found previously [Stebbings and
Hunt, 1983], detergent-extraction of isolated nutritive
tubes resulted in the release of the massive microtubule
bundles, and the solubilisation of the ribosomes. The
latter are, therefore, free within the nutritive tubes and
probably translocate passively along them, as previously
proposed [Macgregor and Stebbings, 1970]. Hybridization of oligo (dT) to the extracted microtubule bundles
showed that, by contrast, poly(A) mRNA remained
attached to the microtubules, suggesting that its transport
is microtubule-based. These findings provide evidence
that mRNA is attached to microtubules isolated directly
from cells, and therefore in vivo.
We set out to investigate the nature of the mRNA/
microtubule attachment and found it to be disrupted by
the addition of ATP and also GTP, but unaffected by ADP
and possibly even enhanced by the nonhydrolyzable ATP
analogue, AMP-PNP. The attachment was also apparently
unaffected by apyrase, which results in ATP depletion.
Nucleotide-sensitive binding to microtubules is a property of microtubule-based motor molecules [Vallee and
Shpetner, 1990], and our results indicate that mRNA may
Attachment of mRNA to Microtubules
be attached to the nutritive tube microtubules via a motor
protein.
Kinesin and dynein motors have both been identified in hemipteran ovaries [Anastasi et al., 1990] and,
while suggesting that motor proteins drive mRNA transport along the microtubules in the ovaries, our results do
not resolve the precise identity of the motor involved.
However, the ‘‘nucleotide fingerprint’’ of the mRNA
attachment to microtubules is more consistent with the
involvement of a kinesin motor, since the latter is
removed from microtubules by both ATP and GTP, while
dynein is only removed by ATP [Paschal et al., 1987].
This is supported by our immunolocalization of a kinesin
within the nutritive tubes and more specifically as a
component of mRNA/microtubule complexes isolated
and extracted from them.
The polarity of microtubules in nutritive tubes of
Notonecta [see Stebbings and Hunt, 1983] means that a
minus end-directed kinesin would be required for mRNA
transport to the oocytes. It would appear, therefore, to be
similar to Drosophila, where dynein is not involved in
morphogen localization [McGrail and Hays, 1997] and a
minus end-directed kinesin has been shown to co-position
with localized mRNAs [Clark et al., 1997]. Genetic and
molecular biological screens have resulted in the discovery of large numbers of kinesin-like motor proteins, with
more than thirty genes encoding members of the kinesin
superfamily in Drosophila alone [see Hirokawa, 1998].
Each kinesin, with some redundancy, is believed to have
its own cargo with many being involved in cell division
and others in transporting specific membranous organelles, and also DNA. Kinesins for transporting mRNA
have been predicted [Moore and Endow, 1996].
The molecular mechanism for mRNA/motor protein associations also remains unclear. It has been shown
that the non-motor domains of plus and putative minus
end-directed chromokinesins bind directly to DNA [Afshar et al., 1995; Wang and Adler, 1995]. It appears,
however, that additional components are involved in
mRNA transport and localization, since a number of
RNA-binding proteins have been found—the most studied being Drosophila staufen [Ferrandon et al., 1994]—
which bind to localization signals in the 38 UTRs of
mRNAs and mediate interactions with the cytoskeleton
[Ainger et al., 1993]. When the 38 UTR of bicoid mRNA
was injected into Drosophila embryos, it was found to
associate with staufen and form large and easily visible
particles that moved in a microtubule-dependent manner
[Ferrandon et al., 1994]. However, these macromolecular
particles may be artefactual because they were not seen
with endogenous bicoid mRNA or injected full-length
message, and we did not observe such particles in our
preparations.
165
The number and identity of the proteins involved in
transporting mRNA along nutritive tubes of Notonecta is
not presently known. Detailed characterization of the
mRNA/microtubule complexes from nutritive tubes, which
clearly possess the essential components for transporting
mRNA, will resolve this question.
ACKNOWLEDGMENTS
N.J.T. is a Nuffield Science Research Fellow. S.S. is
in receipt of a BBSRC studentship.
REFERENCES
Afshar K, Barton NR, Hawley RS, Goldstein SLB. 1995. DNA binding
and meiotic chromosomal localization of the Drosophila Nod
kinesin-like protein. Cell 81:129–138.
Ainger K, Avossa D, Morgan F, Hill SJ, Barry C, Barbarese E, Carson
JH. 1993. Transport and localization of exogenous myelin basic
protein mRNA microinjected into oligodendrocytes. J Cell Biol
123:431–441.
Alison M, Chaudry Z, Baker J, Lauder I, Pringle JH. 1994. Liver
regeneration: a comparison of in situ hybridization for histone
mRNA with bromodeoxyuridene labelling for the detection of
S-phase cells. J Histochem Cytochem 42:1603–1608.
Anastasi A, Hunt C, Stebbings H. 1990. Isolation of micotubule motors
from an insect ovarian system: characterization using a novel
motility substratum. J Cell Sci 96:63–69.
Anastasi A, Hunt C, Stebbings H. 1991. Characterization of a
nucleotide-sensitive high molecular weight microtubuleassociated protein in the ovary of a hemipteran insect. Cell
Motil Cytoskeleton 19:37–48.
Bashirullah A, Cooperstock RL, Lipshitz HD. 1998. RNA localization
in development. Annu Rev Biochem 67:335–394.
Bassell GJ, Powers CM, Taneja KL, Singer RH. 1994a. Single mRNAs
visualized by ultrastructural in situ hybridization are principally
localized at actin filament intersections in fibroblasts. J Cell Biol
126:863–876.
Bassell GJ, Singer RH, Kosik KS. 1994b. Association of poly(A)
mRNA with microtubules in cultured neurons. Neuron 12:571–
582.
Capco DG, Jeffery WR. 1979. Origin and spatial distribution of
maternal RNA during oogenesis of an insect, Oncopeltus
fasciatus. J Cell Sci 39:63–76.
Clark IE, Giniger E, Ruohola-Baker H, Jan LY, Jan YN. 1994.
Transient posterior localization of a kinesin fusion protein
reflects anteroposterior polarity of the Drosophila oocyte. Curr
Biol 4:289–300.
Clark IE, Jan LY, and Jan YN. 1997. Reciprocal localization of Nod and
kinesin fusion proteins indicates microtubule polarity in the
Drosophila oocyte, epithelium, neuron and muscle. Development 124:461–470.
Dalby B, Glover DM. 1992. 38 non-translated sequences in Drosophila
cyclin B transcripts direct posterior pole accumulation in
oogenesis and peri-nuclear association in syncitial embryos.
Development 115:989–997.
Ferrandon D, Elphick L, Nusslein-Volhard C, and St Johnston D. 1994.
Staufen protein associates with the 38UTR of bicoid mRNA to
form particles that move in a microtubule-dependent manner.
Cell 79:1221–1232.
Glotzer JB, Ephrussi A. 1996. mRNA localization and the cytoskeleton.
Semin Cell Dev Biol 7:357–365.
166
Stephen et al.
Hamill D, Davis J, Drawbridge J, Suprenant KA. 1994. Polyribosome
targeting to microtubules: enrichment of specific mRNAs in a
reconstituted microtubule preparation from sea urchin embryos.
J Cell Biol 127:973–984.
Han JR, Yiu GK, Hecht NB. 1995. Testis/brain RNA-binding protein
attaches translationally repressed and transported mRNAs to
microtubules. Proc Natl Acad Sci USA 92:9550–9554.
Hesketh JE, Pryme IF. 1991. Interaction between mRNA, ribosomes
and the cytoskeleton. Biochem J 277:1–10.
Hirokawa N. 1998. Kinesin and dynein superfamily proteins and the
mechanism of organelle transport. Science 279:519–526.
Hyams JS, Stebbings H. 1979. The mechanism of microtubule
associated cytoplasmic transport: isolation and preliminary
characterization of a microtubule transport system. Cell Tissue
Res 196:103–116.
Jeffery WR. 1982. Messenger RNA in the cytoskeletal framework:
analysis by in situ hybridization. J Cell Biol 95:1–7.
Laemmli UK. 1970. Cleavage of structural proteins during the asembly
of the head of bacteriophage T4. Nature 227:680–685.
Lane JD, Stebbings H. 1994. Independent regulation of microtubule
spacing and microtubule stability following redundancy of
nutritive tubes in telotrophic ovarioles of Hemiptera (Insecta).
Int J Morphol Embryol 23:297–309.
Lane ME, Kalderon D. 1994. RNA localization along the anterioposterior axis of the Drosophila oocyte requires PKA-mediated
signal transduction to direct normal microtubule organization.
Genes & Devel 8:2986–2995.
LaRochelle WJ. 1996. Detection of proteins on blots using avidin- or
streptoavidin-biotin. In: Walker JM, editor. The protein protocols handbook. Totowa: Humana Press Inc. p 323–327.
Lenk R, Ransom L, Kaufmann Y, Penman S. 1977. A cytoskeletal
structure with associated polyribosomes obtained from HeLa
cells. Cell 10:67–78.
Li M-G, McGrail M, Serr M, Hays TS. 1994. Drosophila cytoplasmic
dynein, a microtubule motor that is asymmetrically localized in
the oocyte. J Cell Biol 126:1475–1494.
Macgregor HC, Stebbings H. 1970. A massive system of microtubules
associated with cytoplasmic movement in telotrophic ovarioles.
J Cell Sci 6:431–449.
Mays U. 1972. Stofftransport in ovar von Pyrrhocoris apterus. Z
Zellforsch Mikrosk Anat. 123:395–410.
McGrail M, Hays TS. 1997. The microtubule motor cytoplasmic
dynein is required for spindle orientation during germ line cell
divisions and oocyte differentiation in Drosophila. Development 124:2409–2419.
Moore JD, Endow SA. 1996. Kinesin proteins: a phylum of motors for
microtubule-based motility. Bioessays 18:207–219.
Paschal BM, Shpetner HS, Vallee RB. 1987. MAP 1C is a microtubuleactivated ATPase that translocates microtubules in vitro and has
dynein-like properties. J Cell Biol 105:1273–1282.
Pokrywka NJ, Stephenson EC. 1991. Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis. Development 113:55–66.
Pokrywka NJ, Stephenson EC. 1994. Localized RNAs are enriched in
cytoskeletal extracts of Drosophila oocytes. Dev Biol 166:210–
219.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold
Spring Harbour Laboratory Press.
Spradling AC. 1993. Developmental genetics of oogenesis. In: Bate M,
Martinez-Arias A, editors. The development of Drosophila
melanogaster. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press. p 1–70.
St Johnston D. 1995. The intracellular localization of messenger
RNAs. Cell 81:161–170.
St Johnston D, Nusslein-Volhard C. 1992. The origin of pattern and
polarity in the Drosophila embryo. Cell 68:201–219.
Stebbings H, Hunt C. 1983. Microtubule polarity in nutritive tubes of
insect ovarioles. Cell Tissue Res 233:133–141.
Stebbings H, Hunt C. 1987. The translocation of mitochondria along
insect ovarian microtubules from isolated nutritive tubes: a
simple reactivated model. Cell Tissue Res 233:133–141.
Taneja KL, Lifshitz LM, Fay FS, Singer RH. 1992. Poly(A) RNA
codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J Cell Biol
119:1245–1260.
Theurkauf WE, Hazelrigg TI. 1998. In vivo analyses of cytoplasmic
transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway. Development 125:3655–3666.
Theurkauf WE, Smiley S, Wong ML, Alberts BM. 1992. Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport. Development 115:923–936.
Theurkauf WE, Alberts BM, Jan YN, Jongens TA. 1993. A central role
for microtubules in the differentiation of Drosophila oocytes.
Development 118:1169–1180.
Vallee RB, Shpetner HS. 1990. Motor proteins of cytoplasmic microtubules. Annu Rev Biochem 59:909–932.
Wang S-Z, Adler R. 1995. Chromokinesin: a DNA-binding, kinesinlike nuclear protein. J Cell Biol 128:761–768.
Wilhelm JE, Vale RD. 1993. RNA on the move: the mRNA localization
pathway. J Cell Biol 123:269–274.
Документ
Категория
Без категории
Просмотров
2
Размер файла
233 Кб
Теги
133
1/--страниц
Пожаловаться на содержимое документа