Chapter 17

Using the mRNA-MS2/MS2CP-FP System to Study

mRNA Transport During Drosophila Oogenesis
Katsiaryna Belaya and Daniel St Johnston
Abstract
Asymmetric mRNA localisation to specific compartments of the cell is a fundamental mechanism of
spatial and temporal regulation of gene expression. It is used by a variety of organisms and cell types to
achieve different cellular functions. However, the mechanisms of mRNA localisation are not well understood. An important advance in this field has been the development of techniques that allow the visualisation of mRNA movements in living cells in real time. In this paper, we describe one approach to visualising
mRNA localisation invivo, in which RNAs containing MS2 binding sites are labelled by the MS2 coat
protein fused to fluorescent reporters. We discuss the use of this mRNA-MS2/MS2CP-FP system to
study mRNA localisation during Drosophila oogenesis, and provide a detailed explanation of the steps
required for this approach, including the design of the mRNA-MS2 and MS2CP-FP constructs, the
preparation of fly oocytes for imaging, the optimal microscope configurations for live cell imaging, and
strategies for image processing and analysis.
Key words: MS2 coat protein, mRNA localization, Live cell imaging, Widefield deconvolution
microscopy

1. Introduction
mRNA localisation is a widespread phenomenon that occurs in
organisms as diverse as yeast and humans. It contributes to many
cellular processes including the establishment of intracellular
asymmetry, directed cell motility, asymmetric cell division, and
possibly also synaptic plasticity (1).
The molecular mechanisms of mRNA localisation have been
intensively studied in Drosophila, because the formation of the
body axes of this organism depends on the correct localisation of
several maternal mRNA species to specific regions of the oocyte.
Localisation of oskar (osk) mRNA to the posterior pole of the
Jeffrey E. Gerst (ed.), RNA Detection and Visualization: Methods and Protocols, Methods in Molecular Biology, vol. 714,
DOI 10.1007/978-1-61779-005-8_17, Springer Science+Business Media, LLC 2011

265

266

Belaya and St Johnston

oocyte specifies the site of the pole plasm assembly, and thus
where the abdomen and germ cells develop in the embryo (24).
nanos (nos) mRNA is recruited to the pole plasm downstream of
oskar mRNA and functions as the posterior determinant to specify
the formation of the abdomen (5). Localisation of bicoid (bcd)
mRNA to the anterior pole is essential for proper patterning of
the head and the thorax (6, 7). Finally, the localisation of gurken
(grk) mRNA to the anterior-dorsal corner of the oocyte specifies
the dorso-ventral axis of the future embryo (8).
Initially, the localisation of different mRNAs was studied using
in situ hybridisation techniques. This method visualises mRNA
localisation in fixed oocytes, and therefore provides a picture of
the steady-state distribution of mRNA molecules, but suffers from
several limitations. Firstly, the hybridisation technique is not ideal
for studying the localisation of mRNA during late stages of oogenesis, since the deposition of the vitelline membrane makes the
oocyte impermeable to in situ probes. Secondly, the method does
not provide any information about the dynamics of mRNA movement, and this information is crucial for a complete understanding
of the mechanisms of mRNA transport. A great deal of effort has
therefore been directed towards developing a system that allows
visualisation of mRNA movements in living oocytes.
1.1. Methods
for Visualisation
of mRNA Localisation
In Vivo

There are currently four major methods for visualising mRNA

localisation in vivo (Fig. 1). The first is the injection of the
fluorescently labelled mRNA molecules into the cell (Fig.1a). This
method has been successfully used to study the movement of bcd
and grk mRNAs during oogenesis (912). However, it has two
major drawbacks. Firstly, labelling of the RNA molecule can affect
its structure, and thus disrupt the natural localisation process.
Second, localisation of many mRNAs ultimately depends on nuclear
events such as splicing and processing, during which the RNAs
start to assemble with different trans-acting factors into higherorder particles (1315). Thus, this technique may be not applicable
to the analysis of certain mRNAs (for example, osk mRNA).
The second method is the injection of molecular beacons
(Fig. 1b, (16)). A molecular beacon is a small oligonucleotide,
which is complementary to the RNA of interest. This oligonucleotide has a fluorescent molecule attached to one end and a
fluorescence quencher attached to the opposite end. The secondary structure of the oligonucleotide is such that when it is not
bound to its target mRNA, it is folded in a way that quenches the
fluorescence. Once it hybridises to its target RNA, it unfolds,
leading to the appearance of the fluorescence. The technique has
been developed and first applied for the study of the osk mRNA
localisation (16). The major advantage of this technique is that it
can be used to label endogenous mRNAs. However, the sensitivity of this technique is quite low and the oligonucleotides tend to
bind non-specifically to other structures.

Using the mRNA-MS2/MS2CP-FP System

267

Fig.1. Methods for visualising mRNAs invivo. (a) Injection of fluorescently labelled mRNA. The mRNA is transcribed invitro
in the presence of a fluorescently labelled nucleotides. These labelled transcripts are injected into the living cells. (b) Injection
of molecular beacons. Beacons are small oligonucleotides with a fluorophore attached to one end and a fluorescence
quencher attached to the other. Upon hybridisation to the target mRNA, the beacon unfolds, thereby separating the quencher
from the fluorophore which can then fluorescence. (c) GFP-tagging of an RNA-binding protein. An endogenous RNA-binding
protein that is known to be specific for the mRNA of interest is tagged with GFP. (d) RNA-MS2/MS2CP-GFP tagging system.
The 19-nucleotide MS2-binding sequence is inserted into the mRNA of interest. This mRNA is co-expressed with the NLSMS2CP-GFP construct. MS2CP-GFP-NLS binds to the MS2-binding sequence and allows visualisation of the target mRNA.
Unbound MS2CP-GFP-NLS is confined to the nuclei due to the presence of the NLS signal. (e) Secondary structure of the
19-nucleotide MS2-binding RNA sequence. The structure was predicted using the mfold program (44).

Another method of mRNA visualisation involves tagging with

GFP an endogenous protein that binds to the RNA of interest
(Fig.1c). This technique has been used to study bcd (with Exu-GFP,
(17)) and osk (with Stau-GFP, (18)) mRNAs. The major disadvantage of the method is that most RNA-binding proteins bind to several different RNA molecules. This method therefore requires
additional controls to prove that the observed GFP-labelled particles
contain the RNA of interest, and this is hard to implement.

268

Belaya and St Johnston

A solution to many of the problems described above came

with the advent of the mRNA-MS2/MS2CP-FP tagging system
(Fig.1d, (19)). This fourth method relies on the use of two constructs. The first construct is a fusion between a fluorescent protein (FP), the MS2 bacteriophage coat protein (MS2CP) and a
nuclear localisation sequence (NLS). The second construct is the
mRNA of interest containing a number of copies of the MS2CP
binding site (a 19-nucleotide RNA sequence; Fig.1e). When both
of these constructs are expressed in the same cell, the NLSMS2CP-FP protein binds to the mRNA containing the MS2CP
binding sites, and the localisation of the RNA is therefore revealed
by the FP fluorescence signal. The unbound NLS-MS2CP-FP
protein is retained in the nucleus due to the presence of the NLS.
The MS2CP-FP protein binds mRNAs containing its binding
sites (mRNA-MS2) with high specificity and high sensitivity.
Since mRNA-MS2 molecules are transcribed in the cell nucleus,
this technique allows one to follow endogenous mRNA molecules, which have undergone all of the required pre-mRNA processing events and have associated with the appropriate trans-acting
factors. This latter property is especially important for studying
such mRNAs as osk mRNA, whose proper localisation is known to
ultimately depend on pre-mRNA splicing (13).
The technique was developed for the visualisation of Ash1
mRNA in yeast, but has since been applied to study other mRNAs
in a variety of organisms (20). During Drosophila oogenesis, this
technique has now been developed for the study of the localisation of four different mRNAs: bcd, grk, nos and osk.
1.2. Possible
Applications
of mRNA-MS2/
MS2CP-FP Labelling
System

mRNA-MS2/MS2CP-FP system can be used to study different

steps of mRNA localisation process. And the use of different microscopy approaches can provide complimentary information which,
taken together, may provide a full picture of mRNA transport.
Slow time-lapse imaging can provide information about the
gross changes in mRNA localisation during different stages of
oogenesis. For example, Weil et al. (21), used this approach to
describe different steps in the localisation of bcd mRNA to the
anterior pole of the oocyte (21). Additionally, similar low resolution imaging approaches have been used to study the effect of
pharmacological disruption of the actin and microtubule cytoskeletons on the process of bcd, grk and nos mRNA localisation
(2224).
Imaging at high temporal and spatial resolution may allow
visualisation of movement of individual mRNA particles in real
time, provided that the particles are large enough to be resolved
by light microscopy (see for example Fig. 2jm). Subsequent
analysis of the parameters of particle movement can reveal whether
the transport is active or not, whether it is unidirectional or
bidirectional, and whether the RNA switches between different

Using the mRNA-MS2/MS2CP-FP System

269

Fig.2. Visualisation of osk mRNA with the RNA-MS2/MS2CP-GFP system. (a, d, g) Egg chambers expressing both oskMS2
and MS2CP-GFP. (a) oskMS2 is localised to the middle of the stage 8 oocyte. (d, g) oskMS2 mRNA localises to the posterior of the oocyte from stage 9 onwards. The pattern of localisation of oskMS2 mRNA is indistinguishable from that of
the endogenous osk mRNA at all stages of oogenesis (b, e, h). (c, f, i) When MS2CP-GFP is expressed on its own, the GFP
signal remains confined to the nuclei of the nurse and follicle cells. Scale bars are 50mm. (j) Overlay of six frames from
a high magnification time-lapse movie of an oskMS2/MS2CP-GFP expressing oocyte to show the movements of osk
mRNA particles. Examples of individual tracks are highlighted by coloured rectangles, and are shown in separate boxes
with higher magnification (l, m). (k) Co-visualisation of oskMS2/MS2CP-GFP particles and Tral-RFP in a Drosophila
oocyte. The red and green channels were imaged sequentially. (l) Example of the fast-moving particle track highlighted
by the green rectangle in (j). (m) Example of a stationary particle highlighted by the orange rectangle in (j).

modes of movement (e.g. movement and pauses). This analysis

can be extended to the analysis of particle behaviour in different
mutant backgrounds that disrupt mRNA localisation. This provides information about the roles of these proteins in mRNA targeting. Zimyanin etal. (18) have used this approach to characterise
the process of osk mRNA transport in stage 9 oocytes (18).
Co-localisation studies using labelled RNA and labelled transacting factors have been used to provide information about
whether the trans-acting factor is a component of the transport
complex. This approach can also elucidate the timing of association of the trans-acting factor with the mRNA. This has been
performed for the characterisation of the role of Staufen in the
process of bcd and osk mRNA localisation (18, 24).
FRAP, FLIP, photoactivation and photoswitching (when
MS2CP is fused to photoactivatable or photoswitchable FP)
experiments are useful in elucidating how the mRNAs are maintained at their final destination (e.g. whether the molecules are
anchored or continuously transported). This approach has
revealed that bcd mRNA is continuously transported to the

270

Belaya and St Johnston

a nterior of the oocyte during stages 1013 of oogenesis, whereas

grk mRNA is more stably anchored at the dorsal/anterior of the
oocyte at stage 9 (21, 23).
Introduction of affinity tags into the MS2CP-FP protein may
allow the biochemical purification of RNP complexes and thus
provide information about the composition of the RNP transport
particles. This approach has not yet been implemented in fly
oocytes, but has been successfully used in other systems (25).
Purified cytoplasmic fractions containing labelled mRNPs could
also be added to invitro motility assays containing preassembled
cytoskeletal filaments to analyse the dynamics and directionality
of mRNP particle movement in greater detail.
Finally, the mRNA-MS2/MS2CP-FP system can be used in
conjunction with another RNA labelling technique, the mRNABoxB/lN-FP system, to co-visualise two different mRNA molecules in the same cell and thus to determine whether they share
any step of the transport process (see for example (26)). In yeast,
this approach has been used to show that different mRNAs coassemble into the same localisation complex and are transported
to the target localisation site together.
Thus, the mRNA-MS2/MS2CP-FP technique can be used
to study the mechanisms of mRNA localisation from many different angles. In this chapter, we will describe the development and
use of the mRNA-MS2/MS2CP-FP system to study of mRNA
transport during Drosophila oogenesis.

2. Materials
2.1. Preparation
of Drosophila Oocytes
for Live Cell Imaging

1. Drosophila females and males of correct genotype, 23 days

old.
2. Vials or bottles for growing Drosophila. Standard D. melanogaster food (27). Yeast paste for fattening flies overnight prior
to dissection.
3. Colcemid (Sigma) for microtubule depolymerisation.
4. Latrunculin A (Sigma) for actin depolymerisation.
5. Dissection tools: fine tweezers (DUMOSTAR from Dumont;
style No. 5 thickness) and tungsten needles.
6. Dissection microscope.
7. Voltalef PCTFE 10S oil (ARKEMA).
8. Coverslips borosilicate cover glass thickness No. 1 (VWR).

2.2. Imaging and

Image Analysis

1. A Microscope system optimised for imaging FP, for example

a DeltaVision Core (Applied Precision) with the following
configuration: Olympus inverted IX71 microscope, xenon or

Using the mRNA-MS2/MS2CP-FP System

271

mercury arc light source, highly sensitive Cascade II EMCCD

camera (Photometrics), objectives for imaging (UPlanSApo
20 0.75 NA air [Olympus]; UPlanSApo 100, 1.40 NA oil
[Olympus]), specifically chosen filter sets, DeltaVision softWoRx software for image acquisition and processing,
DualView or QuadView system for simultaneous imaging of
several channels (optional), TIRF module (optional).
Immersion oil kit for microscope objective (Applied Precision).
2. Deconvolution software, for example softWoRx (Applied
Precision) for images obtained on a DeltaVision microscope.
3. Image analysis software, for example Metamorph (Molecular
Devices).
4. Quantification software, e.g. Microsoft Excel or MatLab
(MathWorks).

3. Methods
The major steps in the development of mRNA-MS2/MS2-FP
labelling system for the visualisation of mRNA localisation in
Drosophila oocytes are outlined in Fig.3.
3.1. C
 loning
3.1.1. Generation
of mRNA-MS2 Construct

To create the mRNA-MS2 construct, one needs to insert multiple

copies of the MS2 binding site into the mRNA of interest. The
MS2 binding site is a 19nt sequence that forms a stemloop secondary structure. Biochemical experiments have shown that a
single nucleotide mutation (U at the -5 position to C) in the wildtype MS2 stemloop sequence increases its affinity for the MS2CP
protein about 50-fold (28, 29). Thus, this mutated C-variant version of MS2 stem-loop is recommended for use in creating the
mRNA-MS2 construct (ACAUGAGGAUCACCCAUGU the
underlined nucleotide shows the U-5C mutation; [a construct with
ten copies of MS2 binding sites is available from the St Johnston
lab upon request]).
In creating this construct, it is important to consider several
factors. Firstly, it is necessary to decide which form of the gene to
use for cloning the genomic sequence including all exons and
introns, the cDNA with the introns spliced out, or a hybrid which
includes a reporter of interest (fluorescent proteins, LacZ) fused
to the mRNA localisation sequence. In deciding between these
possibilities, it is essential to know the location of the mRNA
localisation elements, as these sequences must be included in the
construct without perturbation. Such elements are often entirely
contained within the 3UTR of the mRNA (for example bcd and
nos mRNAs (30, 31)), and in these cases the use of 3UTR alone
is sufficient to create a functional localisation construct. In some

272

Belaya and St Johnston

Fig.3. Major steps in the development of mRNA-MS2/MS2CP-FP labelling system.

cases, such as gurken mRNA, targeting elements are located in

the coding region of the mRNA, and the use of the entire cDNA
is required (12, 32). Moreover, for certain mRNAs it has been
shown that the nuclear events, such as splicing, are essential for
proper localisation (for example, splicing of the first intron of osk
mRNA (13)). In these cases, it is essential to use the genomic

Using the mRNA-MS2/MS2CP-FP System

273

region of the gene that includes the required introns. In cases

where the targeting elements have not been well described, it is
advisable to use a genomic region containing all of the regulatory
elements of the gene.
The expression level of the construct is important as well. For
example, over-expression of certain mRNAs may lead to production of an excess of mRNA molecules, which overload the localisation pathway and prevent proper localisation (for example,
overexpression of osk mRNA driven by the UAS promoter leads to
ectopic accumulation of the mRNA in the middle of the oocyte
(33)). In such cases, it is important to place the construct under the
control of its endogenous promoter or a promoter that provides
similar levels of expression. In cases where the endogenous promoter of the gene is used, the mRNA-MS2 construct can be
inserted into the pCaSpeR vector (DGRC). pCaSpeR is a Drosophila
P-element transformation vector, and contains a white gene selectable marker for selecting transgenic flies and Amp marker for growing the plasmid in bacteria, but lacks any regulatory sequences for
construct expression (34). In cases where an exogenous promoter
is to be used, the mRNA-MS2 construct can be put into such vectors as pUASp or pUMAT, which already contain the UAS and
maternal a tubulin promoters respectively (35).
Secondly, it is crucial to choose an appropriate position to
insert the MS2-binding sites into the mRNA of interest. Insertion
of this element should not disrupt the sequences responsible for
the post-transcriptional control of mRNA (mRNA transport, stability and translation). Such sequences are often located in the
5- and 3UTR regions of the mRNAs. In cases where the location of such sequences is unknown, it is often safe to insert MS2
binding sites immediately after the STOP codon of the coding
region and before the start of the 3UTR. Since there is unlikely
to be a convenient restriction site in this position, it is usually
necessary to introduce a restriction site here by site-directed
mutagenesis. Another potential insertion site may be just before
the poly-adenylation signal of the mRNA. Alternatively, one can
perform a sequence alignment of the 3UTRs of the orthologues
from related species to identify the least conserved regions, and
then insert the MS2 sites into one of these regions, as they are less
likely to have important regulatory functions.
Finally, one needs to decide how many copies of MS2 binding site to introduce into the mRNA of interest. Each MS2binding RNA motif can associate with two MS2CP proteins
simultaneously. However, two MS2CP proteins (and hence two
FP molecules) are probably insufficient to detect a single mRNA
molecule and to distinguish it from the unbound cytoplasmic
MS2CP-FP. Thus, it is advisable to introduce several tandem copies of the MS2-binding site, and mRNA reporters containing
624 copies of MS2 binding sites have been described. The exact

274

Belaya and St Johnston

number of the sites will depend on the fluorescence intensity

levels required and the copy number of mRNA molecules in each
localisation complex. For mammalian cells, it was found that
insertion of 24 copies of the MS2 sequence is sufficient to visualise single mRNA molecules (36). Although insertion of multiple
copies of MS2-binding sites will increase the fluorescence intensity of the labelled mRNA, one also needs to bear in mind that the
presence of an excessive number of MS2 sites may disrupt the
secondary or tertiary structure of the mRNA, destabilise the molecule, disrupt the assembly of the functional localisation complex,
affect the movement of the RNP complex through an increase in
the size of the transport particle, or lead to aggregation of several
mRNAs together. Thus, it is advisable to insert only as many MS2
repeats as are sufficient for the detection of mRNA particles. The
exact number might need to be determined empirically.
Cloning of constructs that have multiple tandem repeats of
MS2-binding sequence can lead to recombination in bacteria and
to loss of some repeats. It is therefore advisable to grow such
plasmids in bacteria that have decreased recombination rates (for
example SURE cells [Stratagene] or Stbl2 cells [Invitrogen]).
3.1.2. Generation of
MS2CP-FP Construct

The MS2CP coat protein of the RNA bacteriophage MS2 has

two functions: it binds to the MS2 binding site in the RNA
genome of the virus and acts as a translational repressor, and it
associates with other MS2CP proteins to form the icosahedral
shell of the virus. It is therefore best not to use the wildtype
MS2CP protein for the mRNA-MS2/MS2CP-FP labelling of
mRNA, since it will multimerise in the cytoplasm of the cell to
form aggregates. Instead, one should use the dlFG deletion
mutant, which lacks the FG loop involved in the intersubunit
interaction during capsid formation, and is therefore unable to
multimerise (37). In addition, a V29I mutant of MS2CP that
binds to its RNA recognition motif with higher affinity has been
described (37). Thus, the best choice is to use the V29I, dlFG
double mutant for the MS2CP-FP (construct available from the
St Johnston lab upon request).
The MS2 needs to be fused to a fluorescent protein (FP), and
experience in our and other labs has shown that fusing of GFP to
the C-terminus of MS2CP does not compromise its ability to bind
to the MS2 motif. A large number of fluorescent proteins are now
available, and the choice of which one to use will largely depend
on the intended application. Some important considerations when
choosing the fluorescent protein include its brightness, its photostability and whether the protein is monomeric or forms dimers.
In previous work in our lab, the constructs containing eGFP, eYFP
(Venus) and Tomato have been successfully used to visualise
mRNAs in Drosophila ovary. However, new improved versions of
the fluorescent proteins have been described recently and might

Using the mRNA-MS2/MS2CP-FP System

275

provide certain advantages. Additionally, photoswitchable and

photoactivatable fluorescent proteins provide new opportunities
for the analysis of the dynamics of mRNA localisation.
In order to increase the signal-to-noise ratio in labelling
experiments, it is important to maximally reduce the levels of the
unbound MS2CP-GFP in the cytoplasm. This can be achieved by
fusing the MS2CP-FP to a nuclear localisation signal (for example
the SV40 NLS), which will target unbound MS2CP-FP to the
nucleus. We have found that one copy of the SV40 NLS is insufficient to target all of the free MS2CP-FP to the nuclei in the
Drosophila female germ line, and it might therefore be worth
including a second NLS in the construct.
Another important consideration when designing the construct is to decide which promoter to use. Different promoters
will express the construct in different tissues, and the strength of
expression can vary widely. The use of a ubiquitous promoter can
be very convenient, since the same construct can then be used to
analyse mRNA localisation in many different tissues. In some
cases, however, expression in one tissue may make imaging the
neighbouring tissue quite challenging. For example, use of
the hsp83 promoter to express MS2CP-FP will lead to accumulation of GFP in the nuclei of follicle cells, which makes it more
difficult to image mRNA molecules in the underlying oocyte. In
these cases, the use of a germline specific promoter (for example
the osk promoter) helps overcome this issue.
The strength of MS2CP-FP expression is equally important.
The amount of MS2CP-FP produced should be sufficient to
occupy all of the MS2-binding sites in the mRNA-MS2 molecule
and thus to ensure the RNA has maximum fluorescence intensity.
On the other hand, its important to avoid over-expression of the
MS2CP-FP construct, as an excess of these molecules in the cytoplasm will lead to a low signal-to-noise ratio and will make it difficult to identify the specific mRNA-MS2/MS2CP-FP complexes.
For Drosophila egg chambers, the osk promoter (germline specific) and hsp83 (ubiquitous) promoters are expressed at low but
sufficient levels, and both have been successfully used to study the
localisation of different mRNAs in the oocyte ((18, 21, 24, 38),
our unpublished results). At the same time, our experience with
using UAS-GAL4 system to express NLS-MS2CP-FP showed
that this promoter drives over-expression of the protein that leads
to a high background of cytoplasmic MS2CP fluorescence, aggregation of the MS2CP-FP protein in the cytoplasm, and an excessively high intensity of fluorescence in the nuclei, which makes it
difficult to image the cytoplasm.
The MS2-FP construct can also incorporate other tags, if
required. For example, it is possible to introduce protein tags for
affinity purification into the construct, and then use these to pull
down the RNP complexes for biochemical analysis.

276

Belaya and St Johnston

3.1.3. Creation
of Transgenic Flies
Expressing mRNA-MS2
and MS2CP-FP Constructs

In order to visualise the mRNA molecules, one needs to co-express

the MS2CP-FP protein and mRNA-MS2 construct in the same
cell.
Once the constructs are ready they have to be injected into fly
embryos in order to create transgenic fly stocks (39, 40). The flies
expressing each construct on its own can be crossed together in
order to express both constructs in the same fly. This will label the
mRNA of interest with FP. To simplify the analysis of mRNA
localisation in different genetic backgrounds, it is advisable to
create recombinants that carry both transgenes on the same chromosome (41). In cases where the fluorescence level of the construct is too low, it is possible to improve the signal by increasing
the number of copies of the mRNA-MS2 transgene (for example
through recombination of several constructs onto the same chromosome or through mobilisation of an existing insert).
Before embarking on the analysis of mRNA transport in these
flies, it is important to perform several crucial controls. For the fly
stock expressing MS2CP-FP on its own, it is important to check
that the fluorescence signal is restricted to the nuclei of the cells (if
the NLS was included into the construct) and that the fluorescent
signal outside of the nuclei is minimal (see for example Fig.2ai).
Importantly, MS2CP-FP should not be enriched in any region of
the cell where the mRNA-MS2 is expected to localise.
For the mRNA-MS2 construct it is important to confirm that
the insertion of the MS2 sites has not disrupted the regulatory
regions of the transcript. First of all, one should check whether the
mRNA-MS2 localises to the correct region of the cell. The pattern
of mRNA-MS2 distribution should be identical to that of the
endogenous mRNA visualised by in situ hybridisation (see for
example Fig.2ai). The most rigorous test of whether the RNA-MS2
is functional is to check whether this transgene can rescue the phenotype of an RNA null mutant of the gene under investigation. This
experiment will only be possible if an RNA null mutant is available
and if the RNA-MS2 construct was designed to include all the
essential elements of the gene (both coding and regulatory).

3.2. Preparation
of Samples for Live
Imaging

It is important to observe the transport of mRNA molecules in

conditions as close to physiological as possible.
To study the mRNA transport in Drosophila oocytes, one
needs to have young healthy females expressing the correct constructs. Fly oogenesis is very sensitive to the outside environment
and can be arrested if the conditions are unfavourable. Additionally,
it has been shown that suboptimal conditions can cause the
mRNAs to be targeted to stress granules (our own observations;
see also ref. 42). Thus, it is important to use young healthy
well-fed females for analysis.
Usually, we collect 23 days old females of the correct genotype and feed them overnight with yeast paste in uncrowded vials

Using the mRNA-MS2/MS2CP-FP System

277

at 25C. To increase the number of oocytes produced it is

advisable to add several males to the vial. In cases where one wants
to study the effects of microtubule or actin depolymerising drugs
on mRNA localisation, the flies should be starved for 24h before
allowing to feed overnight on yeast paste containing either
100mg/ml colcemid (Sigma) for microtubule depolymerisation
or 200mg/ml latrunculin A (Sigma) for actin depolymerisation.
To dissect the flies, a small drop of Voltalef 10S oil is placed
on a thin coverslip (thickness No. 1, VWR) and one fly briefly
anaesthetised with CO2 is placed directly into this drop of oil. The
ovaries are hand-dissected with sharp tweezers directly onto the
coverslip, and the single ovarioles are separated using tungsten
needles (making sure to remove the muscle sheath surrounding
the ovarioles). One needs to be careful to cause the minimum
amount of damage to the oocytes. Once dissected, the oocytes
should be imaged immediately. Oocytes of young stages can survive in Voltalef oil for 11.5h, and should be imaged within this
time frame.
3.3. Imaging

Dissected oocytes should be imaged on an inverted microscope.

The choice of the microscope system depends on the experiment.
If one wants to examine the steady state distribution of the
mRNA-MS2/MS2CP-FP molecules, it is possible to image on
laser scanning confocal or multiphoton microscopes. These
microscopes are relatively slow, but are efficient in eliminating the
noise caused by the out-of-focus light. Thus, they provide images
with a good signal-to-noise ratio and are ideal for imaging deep
into the sample. One can therefore image throughout the entire
depth of the oocyte and recreate a 3-D image of the cell. These
microscopes are also good for the photobleaching/photoswitching experiments in which one examines the dynamics of pre-localised mRNA molecules.
For the experiments where one wishes to image the movement of individual mRNA molecules in real time, one needs to
use microscope configurations that allow fast image acquisition
rates. It has been shown that the speed of mRNA movement can
reach up to 2mm/s, and in order to record such movements, one
must image at least several frames per second.
In general, confocal scanning microscopes are too slow for
such applications, and the use of wide-field microscopy or spinning disk laser microscopy is recommended. It is has also been
found that wide-field deconvolution microscopy combined with
image deconvolution is usually superior to laser-scanning confocal
microscopy for the imaging of faint signals, which is often the case
when observing single mRNA molecules labelled with mRNAMS2/MS2CP-FP approach (43). In our lab, we successfully use a
DeltaVision microscope (Applied Precision; with an inverted
Olympus IX71 microscope) in order to visualise movement of

278

Belaya and St Johnston

oskMS2/MS2CP-GFP and bcdMS2/MS2CP-GFP RNPs. Another

microscope setup that can be used for these experiments is a
spinning disk confocal microscope, as it also allows imaging at
relatively fast frame rates. However, we have found that this system
causes faster bleaching of the MS2CP-FP fluorescence.
When selecting the microscope configuration it is important
to consider the detector camera, the objectives, the filter cubes,
and the ability to switch rapidly between wavelengths for imaging
several channels simultaneously.
In order to visualise movement of single molecules in real
time, the detector camera should collect as much of the emission
signal as possible. This can be achieved with one of the modern
high quality cameras such as a cooled CCD (charge coupled
device) or EMCCD (electron multiplying charge coupled device)
camera. These cameras are characterised by high sensitivity and
are capable of fast image acquisition rates.
The next consideration is the objective. In order to detect
small RNA-MS2/MS2CP-FP particles, it is essential to image at
the highest magnification possible. For example, fast-moving
oskMS2 and bcdMS2 RNP particles can only be detected with a
100 objective. The numerical aperture of the objective is an
important attribute. The higher the numerical aperture, the better the resolution that one can achieve. For our imaging, we use
UPlanSApo 100, 1.4 NA immersion oil objective (Olympus). In
cases where the molecules that need to be imaged are in close
proximity to the coverslip (less than 100 nm into the sample),
one can image with a total internal reflection (TIRF) system using
special TIRF objectives.
Selection of the immersion oil for the objective is crucial for
optimal imaging. It is useful to obtain an immersion oil kit (for
example a kit from Applied Precision) that contains a series of oils
of different refractive indices, and use it to determine which oil is
best for the sample under investigation. Comparison of different
immersion oils in our lab showed that the best oil for imaging
oocytes dissected in Voltalef 10S oil is that with a refractive index
of 1.534. If the imaging medium were changed from Voltalef oil
to a water-based medium, the optimal oil would be different.
The choice of the filter sets is very important and depends on
the properties of the FP used. The excitation and emission filters
should be optimised to transmit only the relevant wavelengths of
light while blocking the rest. The selection is based on the excitation
emission spectra of the FP and on the transmission curves of the
filter set supplied by the manufacturer. The correct choice of filter
will maximise the brightness of the RNA-MS2/MS2CP-FP particle signal and reduce the background. In the experiments where
imaging of several channels is required, the speed of switching
between filters becomes important. Multicolour imaging can be
performed with the use of either a filter wheel based system or a

Using the mRNA-MS2/MS2CP-FP System

279

device that allows simultaneous imaging of several channels (for

example DualView or QuadView). The filter wheel allows the use
of a wide range of different filter sets, but it is only possible to
image the different wavelengths sequentially, which reduces the
speed of imaging. In experiments that require simultaneous
image acquisition or where the rate of image acquisition is a
priority, the system of choice is either the DualView or the
QuadView. These devices split the emitted light into two/four
channels, which are recorded simultaneously on separate halves/
quadrants of the CCD camera chip. These devices can be used to
co-visualise mRNA-MS2/MS2CP-FP molecules and other fluorescently-labelled proteins.
The sample can be Illuminated with either mercury or xenon
arc lamps. Xenon lamps provide radiation across the visible spectrum and are not subject to the flicker characteristic of mercury
lamps. Thus, they can be used to image a wide range of fluorophores, and ensure uniform illumination of the sample. At the
same time, certain mercury lamps can provide a stronger intensity
of illumination at discrete wavelengths and will perform better for
imaging in that specific part of spectrum. Thus, the choice
between lamps will depend on the fluorophores used. In our lab
we have successfully used both types of lamps for imaging MS2CPeGFP and MS2CP-eYFP proteins.
3.3.1. Imaging of
Drosophila Oocytes

Prior to imaging, it is important to locate several healthy-looking

oocytes of the desired stage of oogenesis using a low magnification
objective (for example 20NA=0.75 air objective, Olympus), and
then record the stage coordinates of these oocytes (softWoRx software from Deltavision has a special function that automatically
records the coordinates). In order to minimise bleaching of the
samples, the oocytes can be located under bright field illumination
to minimise the exposure of the sample to fluorescence. Once the
oocytes have been located, one switches to the high-magnification
objective for high-resolution fluorescence imaging.
Once at high magnification, one has to manually focus on the
focal plane of the cytoplasm containing mRNA particles and then
choose the best fluorescence exposure time and fluorescence intensity (see Note 1). There is always a compromise between achieving
the maximum speed of image acquisition and minimising the intensity of the fluorescence illumination to avoid photobleaching and
photodamage to the cell. Before acquiring images, it is important
to make sure that the softWoRx software is set up correctly and
records the right pixel size, objective lens type, and fluorescence
filter sets. This information will be crucial at later steps during
image deconvolution. It is also important to ensure that the speed
of image acquisition is constant throughout the entire time-lapse
movie, as this will greatly ease the subsequent data analysis (especially
when estimating the speed of particle movement).

280

Belaya and St Johnston

For the visualisation of oskMS2 and bcdMS2 particles on the

Deltavision microscope, we were able to image with image acquisition speeds of up to three to five frames per second for periods
of 12min (Fig.2jm). After that time, the fluorescence starts to
become photobleached. Due to the limit in the rate of image
acquisition, we were unable to image in three dimensions. Thus,
we were not able to record the full path of mRNA molecules from
their site of synthesis to their final destination. To compensate for
this, we acquired many two-dimensional movies at different focal
planes and compared the parameters of particle movement in
different regions of the oocyte. Sufficient sampling at different
focal planes at different time points of oogenesis should provide a
complete picture of mRNA movement during oogenesis. Future
improvements in imaging technologies and the development of
new fluorescent proteins may allow the imaging of the complete
paths of mRNA molecules in three dimensions.
The Deltavision microscope automatically saves the acquired
images in the Deltavision format and also creates a log file, which
contains all data relevant to the properties of the movie created.
The movie in the Deltavision format can be exported to the
DeltaVision stand-alone analysis computer and can be deconvolved using the Applied Precision softWoRx software. This software uses a constrained iterative deconvolution algorithm, which
efficiently reduces noise through the removal of the out-of-focus
light, increasing the contrast of the resulting image. Once the
deconvolution of the image is complete, the movie can be exported
as a series of TIFF files, which can be used for image analysis in
other software packages.
3.3.2. Image Analysis
and Particle Tracking

The data from FRAP and FLIP experiments is analysed by measuring the changes in fluorescence intensities of bleached and
unbleached regions of the oocyte over time. There are different
software packages that allow such analyses, such as Metamorph.
The resulting data can be used to estimate the dynamics of mRNAMS2 in the analysed region of the cell. With such experiments, it
is important to correct for the photobleaching due to image acquisition. This can be done by measuring the changes in fluorescence
intensity in a cell that is in the same field of view, but was not subjected to bleaching (for example a follicle cell nucleus or a neighbouring oocyte).
For the analysis of high-resolution movies, it is often desirable to obtain quantitative information about the parameters of
mRNA particle movement, and this requires the tracking of many
individual fluorescent particles over time. It is important to track
a statistically sufficient number of particle trajectories to be able
to draw significant conclusions from these data. In addition, it is
important to analyse a sufficient number of oocytes per genotype
in order to account for the variation between oocytes. The exact

Using the mRNA-MS2/MS2CP-FP System

281

number required for the analysis will depend on the quality of the
data and on the type of conclusions one wishes to draw.
There are a number of different software packages available
for the automatic tracking of fluorescent particles. These programs are worth investigating to see whether they are capable of
detecting all the particles of interest in a particular setting, and
ignore other fluorescent or autofluorescent objects (e.g. yolk and
vesicles). All those we have tested on oskar mRNA failed to track
all of the particles because of the high particle density and the difficulty in distinguishing between fast moving particles that pass
close to each other.
In many cases, the fluorescence of RNA-MS2/MS2CP-GFP
particles is too low, while the background fluorescence of the
ooplasm too high to use these types of software. In such cases,
one has to use manual tracking algorithms. One software package
that can allow this is MetaMorph Premier Offline (using Track
points application; [Molecular Devices]). This software allows
manual tracking of all the particles of interest and then logs the
coordinates of each data point into a separate file. These data can
then be used for the determination of parameters, such as particle
speed, direction of movement, and length of movement. Analysis
of these data can be performed in MatLab or Microsoft Excel.
When performing manual tracking, it is important to track all of
the detectable particles in the cell, in order to avoid introducing
of biases into the data.

4. Notes
1. In cases where no movement of RNA particles is observed,
one should check whether the oocyte is still alive by acquiring
images every 10s to see whether there are any cytoplasmic
movements. In young oocytes, there are noticeable shortrange movements of limited amounts of cytoplasm called
cytoplasmic seething. In older oocytes, the entire ooplasm
moves in circular fashion around the oocyte a phenomenon
called cytoplasmic streaming. The presence of these movements indicates that the oocyte is viable.

Acknowledgements
K.B. was supported by the Darwin Trust of Edinburgh. D. St J. was
supported by a Wellcome Trust Principal Research Fellowship.

282

Belaya and St Johnston

References
1. St Johnston, D. (2005) Moving messages: the
intracellular localization of mRNAs, Nat Rev
Mol Cell Biol 6, 363375.
2. Kim-Ha, J., Smith, J. L., and Macdonald, P.
M. (1991) oskar mRNA is localized to the
posterior pole of the Drosophila oocyte, Cell
66, 2335.
3. Ephrussi, A., Dickinson, L. K., and Lehmann,
R. (1991) Oskar organizes the germ plasm
and directs localization of the posterior determinant nanos, Cell 66, 3750.
4. Ephrussi, A., and Lehmann, R. (1992)
Induction of germ cell formation by oskar,
Nature 358, 387392.
5. Gavis, E. R., and Lehmann, R. (1992)
Localization of nanos RNA controls embryonic polarity, Cell 71, 301313.
6. Berleth, T., Burri, M., Thoma, G., Bopp, D.,
Richstein, S., Frigerio, G., Noll, M., and
Nusslein-Volhard, C. (1988) The role of
localization of bicoid RNA in organizing the
anterior pattern of the Drosophila embryo,
Embo J 7, 17491756.
7. Ephrussi, A., and Johnston, D. (2004) Seeing
is believing the bicoid morphogen gradient
matures, Cell 116, 143152.
8. Neuman-Silberberg, F. S., and Schupbach, T.
(1993) The Drosophila dorsoventral patterning gene gurken produces a dorsally localized
RNA and encodes a TGF alpha-like protein,
Cell 75, 165174.
9. Cha, B., Koppetsch, B., and Theurkauf, W.
(2001) In vivo analysis of Drosophila bicoid
mRNA localization reveals a novel microtubuledependent axis specification pathway, Cell 106,
3546.
10. MacDougall, N., Clark, A., MacDougall, E.,
and Davis, I. (2003) Drosophila gurken
(TGFalpha) mRNA localizes as particles that
move within the oocyte in two dynein-dependent steps, Dev Cell 4, 307319.
11. Manseau, L., Calley, J., and Phan, H. (1996)
Profilin is required for posterior patterning of
the Drosophila oocyte, in Development 122,
21092116.
12. Van De Bor, V., Hartswood, E., Jones, C.,
Finnegan, D., and Davis, I. (2005) gurken
and the I factor retrotransposon RNAs share
common localization signals and machinery,
Dev Cell 9, 5162.
13. Hachet, O., and Ephrussi, A. (2004) Splicing of
oskar RNA in the nucleus is coupled to its cytoplasmic localization, in Nature 428, 959963.
14. Long, R. M., Gu, W., Meng, X., Gonsalvez,
G., Singer, R. H., and Chartrand, P. (2001) An

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

exclusively nuclear RNA-binding protein

affects asymmetric localization of ASH1 mRNA
and Ash1p in yeast, J Cell Biol 153, 307318.
Oleynikov, Y., and Singer, R. H. (2003) Realtime visualization of ZBP1 association with
beta-actin mRNA during transcription and
localization, in Current Biology 13, 199207.
Bratu, D. P., Cha, B. J., Mhlanga, M. M.,
Kramer, F. R., and Tyagi, S. (2003) Visualizing
the distribution and transport of mRNAs in
living cells, in Proc Natl Acad Sci U S A 100,
1330813313.
Wang, S., and Hazelrigg, T. (1994)
Implications for bcd mRNA localization from
spatial distribution of exu protein in Drosophila
oogenesis., in Nature 369, 400403.
Zimyanin, V. L., Belaya, K., Pecreaux, J.,
Gilchrist, M. J., Clark, A., Davis, I., and St
Johnston, D. (2008) In vivo imaging of oskar
mRNA transport reveals the mechanism of
posterior localization, Cell 134, 843853.
Bertrand, E., Chartrand, P., Schaefer, M.,
Shenoy, S., Singer, R., and Long, R. (1998)
Localization of ASH1 mRNA particles in living
yeast, Mol Cell 2, 437445.
Rodriguez, A. J., Condeelis, J., Singer, R. H.,
and Dictenberg, J. B. (2007) Imaging mRNA
movement from transcription sites to translation sites, Seminars in cell & developmental
biology 18, 202208.
Weil, T. T., Forrest, K. M., and Gavis, E. R.
(2006) Localization of bicoid mRNA in late
oocytes is maintained by continual active
transport, in Dev Cell 11, 251262.
Forrest, K., and Gavis, E. (2003) Live imaging of endogenous RNA reveals a diffusion
and entrapment mechanism for nanos mRNA
localization in Drosophila, Current Biology
13, 11591168.
Jaramillo, A. M., Weil, T. T., Goodhouse, J.,
Gavis, E. R., and Schupbach, T. (2008) The
dynamics of fluorescently labeled endogenous
gurken mRNA in Drosophila, J Cell Sci 121,
887894.
Weil, T. T., Parton, R., Davis, I., and Gavis, E.
R. (2008) Changes in bicoid mRNA anchoring highlight conserved mechanisms during
the oocyte-to-embryo transition, Curr Biol
18, 10551061.
Zhang, Z., and Krainer, A. R. (2007) Splicing
remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex
components, Proc Natl Acad Sci U S A 104,
1157411579.

Using the mRNA-MS2/MS2CP-FP System

26. Lange, S., Katayama, Y., Schmid, M.,
Burkacky, O., Brauchle, C., Lamb, D. C., and
Jansen, R. P. (2008) Simultaneous transport
of different localized mRNA species revealed
by live-cell imaging, Traffic (Copenhagen,
Denmark) 9, 12561267.
27. Sullivan, W., Ashburner, M., and Hawley, R.
S. (2000) Drosophila Protocols, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor,
New York.
28. Lowary, P. T., and Uhlenbeck, O. C. (1987)
An RNA mutation that increases the affinity
of an RNA-protein interaction, Nucleic acids
research 15, 1048310493.
29. Valegard, K., Murray, J. B., Stonehouse, N. J.,
van den Worm, S., Stockley, P. G., and Liljas,
L. (1997) The three-dimensional structures
of two complexes between recombinant MS2
capsids and RNA operator fragments reveal
sequence-specific protein-RNA interactions,
J Mol Biol 270, 724738.
30. Gavis, E. R., Curtis, D., and Lehmann, R.
(1996) Identification of cis-acting sequences
that control nanos RNA localization, Dev Biol
176, 3650.
31. Macdonald, P. (1990) bicoid mRNA localization signal: phylogenetic conservation of
function and RNA secondary structure,
Development 110, 161171.
32. Thio, G. L., Ray, R. P., Barcelo, G., and
Schupbach, T. (2000) Localization of gurken
RNA in Drosophila oogenesis requires elements in the 5 and 3 regions of the transcript, Dev Biol 221, 435446.
33. Zimyanin, V., Lowe, N., and St Johnston, D.
(2007) An Oskar-dependent positive feedback
loop maintains the polarity of the Drosophila
oocyte, in Current Biology.
34. Pirrotta, V. (1988) Vectors for P-mediated
transformation in Drosophila, Biotechnology
10, 437456.

283

35. Rorth, P. (1998) Gal4 in the Drosophila female

germline, Mech Dev 78, 113118.
36. Fusco, D., Accornero, N., Lavoie, B., Shenoy,
S. M., Blanchard, J. M., Singer, R. H., and
Bertrand, E. (2003) Single mRNA molecules
demonstrate probabilistic movement in living
Mammalian cells, in Current Biology 13,
161167.
37. Peabody, D. S., and Ely, K. R. (1992) Control
of translational repression by protein-protein
interactions, Nucleic Acids Research 20,
16491655.
38. Wagner, C., Palacios, I., Jaeger, L., and St
Johnston, D. (2001) Dimerization of the 3
UTR of bicoid mRNA involves a two-step
mechanism, Journal of Molecular Biology 313,
511524.
39. Rubin, G. M., and Spradling, A. C. (1982)
Genetic transformation of Drosophila with transposable element vectors, Science 218, 348353.
40. Spradling, A. C., and Rubin, G. M. (1982)
Transposition of cloned P elements into
Drosophila germ line chromosomes, Science
218, 341347.
41. Greenspan, R. J. (2004) Fly Pushing: the theory and practice of Drosophila Genetics, 2nd
ed., Cold Spring Harbor Press, Cold Spring
Harbor, New York.
42. Snee, M. J., and Macdonald, P. M. (2009)
Dynamic organization and plasticity of sponge
bodies, Dev Dyn 238, 918930.
43. Swedlow, J. R., Hu, K., Andrews, P. D., Roos,
D. S., and Murray, J. M. (2002) Measuring
tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and widefield fluorescence microscopy, Proc Natl Acad
Sci U S A 99, 20142019.
44. Zuker, M. (2003) Mfold web server for
nucleic acid folding and hybridization prediction, Nucleic acids research 31, 34063415.