Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Department of Biochemistry, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 04/2006; 17(3):1239-49. DOI: 10.1091/mbc.E05-10-0952
Source: PubMed


Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.


Available from: Kevin Fogarty
Molecular Biology of the Cell
Vol. 17, 1239–1249, March 2006
Rapid, Diffusional Shuttling of Poly(A) RNA between
Nuclear Speckles and the Nucleoplasm
Joan C. Ritland Politz,* Richard A. Tuft,
Kannanganattu V. Prasanth,
Nina Baudendistel,
Kevin E. Fogarty,
Larry M. Lifshitz,
Jo¨rg Langowski,
David L. Spector,
and Thoru Pederson*
*Department of Biochemistry and Molecular Pharmacology and Program in Cell Dynamics and
of Physiology, University of Massachusetts Medical School, Worcester, MA 01605;
Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY 11724; and
German Cancer Research Center, 69120 Heidelberg,
Submitted October 14, 2005; Accepted December 2, 2005
Monitoring Editor: A. Gregory Matera
Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A)
RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is
dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa
cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence
correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both
speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which
caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle
or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged
poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of
whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactiva-
tion-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA
in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require
metabolic energy.
Nuclear speckles are morphologically distinct regions of the
nucleoplasm that contain pre-mRNA splicing components
as well as poly(A) RNA (Carter et al., 1993; Zhang et al., 1994;
Lamond and Spector, 2003). They are operationally defined
by their immunostaining with a variety of pre-mRNA splic-
ing factor antibodies, and they also show in situ hybridiza-
tion signal using probes for poly(A). When these sites are
viewed in the electron microscope, most of them are found
to represent interchromatin granule clusters (Fakan et al.,
1984, Deerinck et al., 1994). However, RNA polymerase II
transcription sites are distributed throughout the nucleus,
indicating that although some of the RNA present in inter-
chromatin granules/speckles is nascent, transcription is not
restricted to this compartment, and much of the poly(A)
RNA there has likely been transcribed previously and/or at
distant sites (Fakan and Nobis, 1978; Wansink et al., 1993,
Zeng et al., 1997, Neugebauer and Roth, 1997, Cmarko et al.,
1999; Guillot et al., 2004). Indeed, although transcripts that
are being rapidly produced or that contain many introns are
sometimes observed in speckles at the light microscopy level
(Johnson et al., 2000; Shopland et al., 2003), the majority of
studies over the years has indicated that most speckles are
not primary sites of pre-mRNA splicing (for reviews, see
Mattaj, 1994; Huang and Spector, 1996a; Neugebauer and
Roth, 1997; Lamond and Spector, 2003). In two cases, a single
species of pre-mRNA has been followed from its transcrip-
tion site to the nuclear pore, and in neither case did the RNA
seem to accumulate in nuclear subcompartments (Singh et
al., 1999; Shav-Tal et al., 2004). Instead, it seems that splicing
components typically move away from speckles to sites of
gene expression where splicing occurs cotranscriptionally
(Huang and Spector, 1996b; Misteli et al., 1997, 1998; Eils et
al., 2000). These findings have therefore made it difficult to
understand why poly(A) RNA is associated with speckles.
One idea has been that perhaps there is a metabolically
stable, nucleus-restricted poly(A) RNA population that
helps to organize and structurally define certain nuclear
bodies such as speckles (Fakan et al., 1984; Huang et al., 1994;
Lamond and Spector, 2003). This idea stems from observa-
tions that as much as 30% of the poly(A) RNA never leaves
the nucleus in growing mammalian cells (Perry et al., 1974,
Herman et al., 1976). At least some of this poly(A) RNA
consists of noncoding sequences that seem to have a variety
of nuclear functions (Morey and Avner, 2004). We earlier
studied the movement of poly(A) RNA in the nucleus of live
This article was published online ahead of print in MBC in Press
on December 21, 2005.
The online version of this article contains supplemental material
at MBC Online (
Address correspondence to: J.C.R. Politz (
Abbreviations used: FCS, fluorescence correlation spectroscopy;
mRFP-SC35, monomeric red fluorescent protein fused to SC35 pro-
tein; oligo, oligodeoxynucleotide.
© 2006 by The American Society for Cell Biology 1239
Page 1
rat myoblasts using the technique of fluorescence correlation
spectroscopy (FCS) (Politz et al., 1998) and found that al-
though there was a class of poly(A) RNA that moved rather
rapidly throughout the nucleoplasm, there also was a sizable
fraction that moved much more slowly. It was therefore
possible that this fraction represented molecules that were
confined within nuclear bodies such as speckles and thus
constrained in their motion.
In the present investigation, we developed methods to
address the possible structural nature of the poly(A) RNA
directly in the nuclear speckles of living cells. A stable cell
line expressing a chimeric SC35 protein coupled to a mono-
meric red fluorescent protein (mRFP-SC35) was generated,
and the chimeric protein was found to behave similarly to
the native SC35 protein (Politz et al., 2003). Endogenous
nuclear poly(A) RNA was tagged in these cells using fluo-
rescently labeled oligo(dT) as a hybridization probe (Peder-
son, 1999; Politz, 1999; Politz et al., 1999). We used both FCS
(Magde et al., 1972; Politz et al., 1998; Wachsmuth et al., 2000)
and photoactivation-tracking techniques (Politz, 1999; Politz
et al., 1999, 2003, 2004) to measure the mobility of the tagged
poly(A) RNA inside speckles and in the nucleoplasm. Time-
lapse digital imaging microscopy was also used to visually
track the movement of photoactivated tagged poly(A) RNA
as it moved between speckles and the nucleoplasm. We
show that poly(A) RNA moves in and out of speckles with
the characteristics of a diffusive process and that there is not
a substantial immobile population of poly(A) RNA within
the speckle as might be expected if it were serving as a
structural scaffolding. In fact, the behavior of poly(A) RNA
within and outside speckles was indistinguishable, and the
rate of movement did not change when the temperature was
lowered from 37 to 22°C. The results indicate that rather
than speckles harboring an actively sequestered, kinetically
distinct population, the poly(A) RNA that is associated with
a speckle at any given time exchanges freely with the nucle-
oplasm and other speckles.
mRFP-SC35 Stable Cell Line and Transfection
PCR was used to generate a restriction site at the stop codon of a human SC35
cDNA (Prasanth et al., 2003) for cloning into a vector encoding the mRFP
(Campbell et al., 2002). A HeLa stable cell line containing the mRFP-SC35 was
generated and maintained in DMEM (low glucose) with 10% fetal bovine
serum (FBS) and 0.5 mg/ml G418 (Invitrogen, Carlsbad, CA).
-Tropomyosin Mini-Gene Transfection and In Situ
Electroporation was performed on trypsinized cells (240 V; 950
F), which
were then resuspended in 250
l of growth medium and incubated with 4
of a rat
-tropomyosin mini-gene construct (Helfman et al., 1988) plus 20
of sheared salmon sperm DNA. Cells were then seeded onto acid-washed
coverslips and processed for in situ hybridization 24 h posttransfection. The
-tropmyosin probe was labeled with Spectrum-Green-dUTP using a nick
translation reagent kit (Vysis, Downer’s Grove, IL), and in situ hybridization
was performed essentially as described previously (Huang and Spector,
1996b). Cells were hybridized with 100 ng of probe in 20
l of hybridization
mixture (50% formamide, 2 SSC, 5% dextran sulfate, and 20
g of yeast
tRNA) at 37°C for 12–14 h. After posthybridization washes, DNA was stained
with 4-6-diamidino-2-phenylindole. Imaging acquisitions for in situ experi-
ments were performed with a Zeiss Axioplan microscope and a 100 oil
immersion objective. Images were processed using Openlab software (Impro-
vision, Lexington, MA).
Immunoblot Analysis
Cell lysates were prepared from the mRFP-SC35 stable cell line and treated for
30 min at 37°C with 500 U/ml calf intestinal phosphatase (CIP; New England
Biolabs, Beverly, MA), and, after gel electrophoresis, were blotted onto a
nitrocellulose membrane and probed by an antibody that recognizes mRFP
(Chemicon International, Temecula, CA).
Oligodeoxynucleotide (Oligo) Synthesis
Oligo(dT) and control oligo(dA) probes were synthesized by Integrated DNA
Technologies (Coralville, IA) as 43mers, with a thymidine containing a C6-
aminohexyl group present at every 10 bases [in both oligo(dT) and oligo(dA)].
The oligos were then labeled with either fluorescein (Molecular Probes,
Eugene, OR) or caged fluorescein (Mitchison et al., 1994) as described previ-
ously (Politz et al., 1999, 2004). Both the aminohexyl linker arms and the
spacing of the labeling groups along the oligo are thought to prevent RNase
H degradation of the RNA target after the oligo is hybridized (Ueno et al.,
Oligo Uptake and Live Cell Imaging
Cells were either plated into two-well dishes (Nalge Nunc, Naperville, IL) or
onto 25-mm coverslips and transfected with oligo(dT) or oligo(dA) as de-
scribed previously (Politz et al., 2004) except that the concentration of oligo
was 0.125
M. After a 1-h incubation without oligo in DMEM (with serum),
cells on coverslips were mounted in a holder and then maintained at 37 or
22°C as described in DMEM buffered with 25 mM HEPES (10% FBS, no
phenol red) (Politz et al., 2004). Cells containing fluorescein-labeled oligo(dT)
growing in two-well dishes were imaged using a Quantix 57 charge-coupled
device camera (Roper Scientific Photometrics, Tucson, AZ) coupled to a Leica
DMIRB microscope equipped with a 100 objective (numerical aperture 1.4)
as described previously (Politz et al., 2000). Signal intensity was quantitatively
scaled using MetaMorph software. All uncaging, rapid acquisition digital
microscopy, and image processing were performed using equipment and
software previously described in detail (Politz et al., 1999, 2004). Some images
were deconvolved using exhaustive photon reassignment (Carrington et al.,
In Situ Reverse Transcription
Cells were allowed to take up oligo as described above (at an oligo concen-
tration of 0.5
M in the medium) and after a 1-h efflux period, fixed and
subjected to in situ reverse transcription as described in detail previously
(Politz et al., 1995; Politz and Singer, 1999), except that the anti-digoxigenin
antibodies were linked to horseradish peroxidase (anti-digoxigenin-POD, Fab
fragments; Roche Diagnostics, Indianapolis, IN), and the blocking step was
performed using 1% normal sheep serum (The Jackson Laboratory, Bar Har-
bor, ME) in SSC. The colorimetric assay was carried out using a diaminoben-
zidine substrate according manufacturer’s instructions (Roche Diagnostics),
and bright field images of the resulting signal were captured using the Leica
microscope system described above. Contrast was quantitatively scaled using
MetaMorph software (Universal Imaging, Downingtown, PA).
Fluorescence Correlation Spectroscopy
FCS (Magde et al., 1972) was carried out using a previously described instru-
ment (Wachsmuth et al., 2003). Cells were grown in eight-well dishes (Nalge
Nunc) in RPMI medium (with 10% serum, no phenol red) and transfected as
described above. The dishes were then mounted on the stage of the FCS
microscope, and either a speckle or a site in the nucleoplasm was aligned in
the laser path using a galvanometer scanner in point-addressable mode.
Measurements were recorded at a laser intensity of 1–5 kW/cm
0.5-mW laser power at the focus). A 485-nm excitation filter (DF 22; Omega
Optical, Brattleboro, VT) was placed in the laser light path to reduce the
intensity of the mRFP for imaging of the fluorescein-labeled oligo. For exper-
iments done at 37°C, a chamber around the microscope and stage was heated
to maintain a temperature of 37°C in a humidified 5% CO
atmosphere. Ten
readings were taken at each site and averaged; readings that showed bleach-
ing were discarded. Best fits for the autocorrelation curves were chosen and
recorded using Quickfit (Press et al., 1992), and in some cases, the MaxEnt
fitting program (Modos et al., 2004) was also used to help determine which fit
was optimal.
A 100-
m aperture placed in the UV illumination path during photoactiva-
tion produced an 1-
m uncaging spot at the focal plane. This spot is the
convolution of the three-dimensional point-spread-function (3-D PSF) of the
microscope with the two-dimensional (2-D) illumination aperture. In wide-
field imaging systems, the captured images of fluorescence are the result of
the convolution of the 3-D fluorescent oligo distribution with this 3-D PSF. To
generate computer simulations of the uncaging and imaging processes, the
3-D PSF of the microscope system was measured using subresolution fluo-
rescent beads (Carrington et al., 1995). Images spanning 10
m of focus
about the bead were acquired at 0.25-
m intervals, with a pixel size of 0.15
m to match the experimental data. Custom software designed for modeling
reaction and diffusion events inside cells in 3-D (Zou et al., 1999; Fogarty et al.,
2000; ZhuGe et al., 2000) was then used to simulate both the uncaging and
diffusion of the fluorescent oligos. The nucleus was modeled as a cylinder
with its central axis along the z-axis (axis of focus) of the microscope (x,y 15
m; z 10
m; pixel size 0.15
m). The initial 3-D distribution of uncaged
oligo was calculated by digitally convolving a computer generated image of
m, 2-D aperture with the empirically measured 3-D PSF and was used as
J.C.R. Politz et al.
Molecular Biology of the Cell1240
Page 2
the starting distribution for the diffusion simulations. The 3-D uncaged spot
was centered within the cylinder (as necessitated by the imposed symmetry)
and focused 4
m from the bottom. The edges of the nucleus were a barrier
to diffusion. Diffusion was simulated using a single diffusion constant, D
(units of square micrometers per second), for up to 30 s, and images were
saved at times corresponding to the experimental data. The saved simulation
images were converted from cylindrical coordinates to Cartesian coordinates
(x,y,z) using interpolation to produce 3-D images having pixel sizes of 0.15
minx and y, and 0.25
minz. Each 3-D image time point was then
“blurred” by convolving with the 3-D PSF and the infocus image was ex-
tracted, producing a temporal sequence of 2-D images of diffusion modeling
the experimental data.
Simulations were conducted using a range of diffusion constants. Because
the microscope system can be approximated as a linear system (Carrington et
al., 1995), mixes of two or more different Ds were created by simulating each
D separately. The resulting image sequences for each D were digitally added
in the desired proportions. In the case of modeling oligo confined to a
“speckle,” the initial uncaged distribution was masked using a 2-
ter sphere center at the infocus position of the spot, and D was set to zero. For
the simulation shown in Figure 6, D–F, the step convolving the simulation
with the 3-D PSF was omitted.
The spatial profile of the UV uncaging spot intensity at the focal plane,
before diffusion, can be well fit with a single Gaussian:
is the e
half-width of the spot. The equation for the spatial profile
produced by diffusion of this initial spot is
Ix, t
2 Dt兲兴
where D is the diffusion constant, t is time,
is as for Eq. 1, and * denotes
convolution. The convolution of two Gaussians is another Gaussian:
Ix, t
2 Dt
We can well fit the spatial profile of the image of the initial uncaged fluores-
cence, before diffusion, with the sum of two Gaussian spatial profiles, roughly
corresponding to the infocus and out-of-focus components of the 3-D fluo-
rescence distribution:
Ix A
and the corresponding diffusion profile:
Ix, t
2 Dt
2 Dt
The 2-D spatial and temporal intensity profile of both simulated images and
experimental images were fit to either Eq. 3 or Eq. 5, using custom software
implementing a multiparameter least-squares curve fit approach, to derive
estimates of both D and
Characterization of Oligo(dT) Uptake by a Stable HeLa
Cell Line Expressing mRFP-SC35
A stable HeLa cell line expressing the splicing factor SC35
fused to monomeric red fluorescent protein (mRFP-SC35)
was generated using standard procedures (see Materials and
Methods). Greater than 95% of the cell population showed
expression of the red SC35 protein, which was observed to
be most concentrated at multiple nucleoplasmic sites (Figure
1A). Signal was also observed in nucleoplasmic regions out-
side the speckles. A similar distribution pattern has also
been observed in stable cell lines expressing the splicing
protein SF2/ASF linked to green fluorescent protein (GFP)
(Phair and Misteli, 2000). The transformed cell population
maintained nearly 100% expression of mRFP-SC35 over sev-
eral generations.
We examined the phosphorylation status of the chimeric
mRFP-SC35 protein and found it to be present in its active,
phosphorylated form in the stably transfected cell line (Fig-
ure 1B), demonstrating that this mRFP fusion protein was
behaving similarly to its endogenous SC35 counterpart
(Misteli and Spector, 1996). To further test the degree to
which the behavior of the mRFP-SC35 fusion protein resem-
bled that of the native SC35 protein, we investigated its
dynamic association with a specific polymerase II transcrip-
tion site. As shown in Figure 1C, the mRFP-SC35 chimeric
protein was recruited to the sites of active transcription of a
-tropomyosin gene, similar to the behavior of
native SC35 (Huang and Spector, 1991; Jimenez-Garcia and
Spector, 1993; Huang and Spector, 1996b; Misteli et al., 1997;
Misteli and Spector, 1999). The cells also seemed morpho-
logically normal and grew with a doubling time similar to
the parental cell line. Therefore, by all these criteria the
mRFP-SC35 fusion protein seemed to be mirroring the be-
havior of the native SC35 protein and therefore was judged
to be a valid marker for speckles in these cells.
Introduction of fluorescein-labeled oligo(dT) into this sta-
bly transfected HeLa cell line (see Materials and Methods) was
optimized to give detectable signal in greater than 50% of
the cells, with minimal effect on cell viability (as judged by
degree of spreading and population size 24 h later; Politz
1999; Politz et al., 2004). As previously found in L6 myoblasts
(Politz et al., 1995, 1999), oligo(dT) signal was distributed
throughout the nucleus and within speckles but did not
seem concentrated in speckles (Figure 2, A and B). An in situ
reverse transcription assay (Eberwine et al., 1992; Politz and
Figure 1. Characteristics of HeLa cell line
stably expressing mRFP-SC35. (A) Raw and
restored (exhaustive photon reassignment;
see Materials and Methods) midplanes of an
optical stack showing a live HeLa cell stably
expressing mRFP-SC35. Bars, 4.2
m. (B) Im-
munoblot of total cellular proteins probed
with RFP antibody before () and after ()
treatment with CIP in wild-type HeLa cells
(left lane) and the stable SC35 HeLa cell line
(middle and right lanes). (C) Fluorescent in
situ hybridization to fixed SC35 stable HeLa
cell line transiently transfected with a plas-
mid that expresses
-tropomyosin mRNA at
high levels (see Materials and Methods).
pomyosin mRNA was detected with a Spec-
trum-Green-labeled probe (green) and over-
lap with mRFP-SC35 (red) is shown as
yellow. Bar, 5
Poly(A) RNA Diffuses through Speckles
Vol. 17, March 2006 1241
Page 3
Singer, 1999; Politz et al., 1999) was used to detect hybrid-
ization of the oligo to endogenous RNA. In this assay, only
oligo(dT) that is hybridized to poly(A) can act as a primer
for reverse transcription. As can be seen in Figure 2C, re-
verse transcription products were observed in both the nu-
cleus and the cytoplasm of cells that took up oligo(dT), as
expected for hybridization to poly(A) RNA. Cells that had
taken up the control oligo(dA) showed no signal (Figure
2D), indicating that the oligo(dA) does not hybridize appre-
ciably to RNA in the cell. (HeLa cell nuclear RNA contains
some oligo(U) tracts, but these are present at a much lower
concentration than poly(A); Kish and Pederson, 1977).
FCS Used to Measure Mobility of Poly(A) RNA in
Speckles and the Nucleoplasm
We first used the technique of FCS (Magde et al., 1972;
Krichevsky and Bonnet, 2002; Kim and Schwille, 2003) to
compare the mobility of poly(A) RNA in speckles and the
nucleoplasm. This method quantitatively measures the
amount of fluorescence in a very small volume and rapidly
monitors the fluctuation of that signal intensity over time.
The more the signal fluctuates, the more rapidly the mole-
cules are moving in and out of the interrogated volume. This
is manifest as an increased rate of decay in the autocorrela-
tion function. Using curve-fitting algorithms, the number of
differently diffusing components within the sample area can
be determined, and their respective diffusion coefficients
were estimated (Politz et al., 1998; Wachsmuth et al., 2000).
The confocal assay volume of FCS is less than a femtoliter; in
our experiments, the radial diameter of the confocal volume
was 0.44
m and the z-axis height was 1.6
m when
exciting fluorescein (and 0.5 and 2
m, respectively, when
exciting mRFP). These dimensions are similar to or smaller
than that of a speckle; deconvolved images of the stable
SC35 cell line used to estimate speckle size gave radial
diameters of 0.5–2.5
m and z-axis heights of 1.5–2.5
(our unpublished data). Therefore, FCS measurements could
be taken inside a speckle without substantial excitation of
the surrounding nucleoplasm.
We used the galvanometer scanner of the fluorescence
fluctuation microscope in the point-addressable mode to
direct a laser beam into a confocal volume inside the nucleus
of live SC35-expressing HeLa cells containing fluorescent
oligo(dT). The beam was focused either within a speckle or
into an area of the nucleoplasm devoid of a speckle. Fluo-
rescence fluctuations in each interrogated volume were then
measured over time and recorded as an autocorrelation
curve. Figure 3A shows representative autocorrelation
curves obtained within (blue) and outside (red) a speckle,
with the best fit curves (smooth lines) shown for each in the
same color. It can be seen that very similar autocorrelation
curves were obtained from both speckle and nucleoplasmic
sites, indicating that the oligo(dT) had similar molecular
dynamics at both sites. Additionally, the inverse particle
number (the y-intercept), which is proportional to the
total concentration of oligo(dT), is very similar in both
cases. The best fit curves most often represented two or
three components of differing mobilities. The relative frac-
tion of each of these components is shown in Figure 3B
(dT on and dT off).
One of these kinetic components was not observed when
measurements were made in cells containing the nonhybrid-
izing control probe, oligo(dA), and is therefore highly likely
to represent oligo(dT) hybridized to poly(A) RNA (Figure
3B, compare red bars in dT columns to the zero-level red
bars in the dA columns). No significant difference in the
mobility of this fraction was observed when measurements
were made either within a speckle or in the nucleoplasm
(Figure 3B, compare red fraction in dT off to dT on). It took
the oligo(dT):poly(A) RNA hybrids an average of 22 5ms
(D 0.65 0.19
/s) to traverse the confocal volume
within a speckle and 20 4ms(D 0.70 0.19
/s) to
traverse the same distance in the nucleoplasm. Thus, we
observed no significant difference in mobility of the poly(A)
RNA populations inside or outside a speckle.
The fastest moving component present in the oligo(dT)-
containing cells (Figure 3B, dT black bars) closely corre-
sponded to the most prevalent (80% of signal) mobility
fraction obtained when measurements were made in control
oligo(dA)-containing cells (Figure 3B, dA black bars) and
therefore probably represents unhybridized oligo(dT) in the
nucleus (average dT on, 1.1 0.2 ms; dT off, 1.1 0.3 ms; dA
on, 1.8 0.2 ms; and dA off, 2.0 0.3 ms).
Interestingly, when the mobility of the mRFP-SC35 pro-
tein itself was measured in speckles, the same two mobility
fractions were observed (Figure 3C), although the faster
moving fraction was a somewhat higher percentage of the
total. A similar distribution of SC35 mobility classes was
observed when measurements were taken outside a speckle
(Figure 3C). Therefore, the behavior of the mRFP-SC35 pro-
tein as judged by FCS was similar to the behavior of oli-
go(dT), both with respect to the presence of two mobility
categories, presumably one bound and the other free, and in
Figure 2. In vivo signal distribution and in situ reverse transcrip-
tion in the mRFP-SC35 HeLa cell line after uptake of fl-oligo(dT) or
fl-oligo(dA). (A and B) HeLa cells stably transfected with SC35-
mRFP were allowed to take up fluorescently labeled oligo(dT) and
the distribution of signal was visualized as described in Materials
and Methods. (A) SC35-mRFP. (B) fl-oligo(dT). Bars, 3
m. (C and D)
After uptake of either fl-oligo(dT) or fl-oligo(dA), cells were fixed
and subjected to in situ reverse transcription as described in Mate-
rials and Methods. Incorporation of biotin-labeled deoxynucleotides
was detected using anti-biotin antibody coupled to horseradish
peroxidase as described in text. (C) Bright field image of cells
containing oligo(dT). (D) Bright field image of cells containing oli-
go(dA). Bars, 9.4
J.C.R. Politz et al.
Molecular Biology of the Cell1242
Page 4
the fact that there was no difference in the mobility of these
fractions on or off speckles.
A low abundance, very slow third component was also
identified using FCS in some oligo(dT)-containing cells (Fig-
ure 3B, dT white bars). This minor, low-mobility fraction
was not thought to contain hybridized oligo(dT) because it
was also present in some control oligo(dA)-containing cells
(Figure 3B, dA white bars). The nature of this slow fraction
of oligo(dT) and oligo(dA) is not understood at present, but
it should be noted that a slow component with similar
characteristics is also detected in some cells in which the
intranuclear mobility of GFP is measured (Wachsmuth et al.,
2000; Baudendistel and Langowski, unpublished observa-
tions). Another observation, perhaps related, was that in
some FCS measurements an initial bleaching of signal oc-
curred. We were not able to characterize this very slow
moving or immobile fraction in detail, but it was detected in
both speckles and in the nucleoplasm at approximately
equal frequencies.
We also used FCS to measure the mobility of the hybrid-
ized oligo(dT) in cells at 22°C compared with 37°C. Our
previous studies had shown no difference in poly(A) RNA
mobility in L6 myoblasts as a function of temperature (Politz
et al., 1999) or under conditions that inhibited ATP produc-
tion (Politz et al., 1998). However, in these previous studies
it was not possible to distinguish speckles from other areas
within the nucleoplasm. Using the stable mRFP-SC35 HeLa
cell line, where oligo(dT) mobility within and outside speck-
les could easily be compared at 22 and 37°C, no significant
difference was observed in the shape of the autocorrelation
curves obtained at each temperature (our unpublished data),
and the best fit mobility distributions showed no significant
difference in the behavior of the poly(A) RNA at either
temperature (Figure 3D, red bars). As described above, at
37°C it took poly(A) RNA an average of 22 5msto
traverse a confocal volume within a speckle and 20 4ms
to traverse the same distance in the nucleoplasm proper.
When the measurements were made at 22°C instead, it took
poly(A) RNA an average of 27 6 ms to traverse the
confocal volume within a speckle and 28 8msinthe
nucleoplasm. Additional details of the FCS measurements
are provided in the Supplemental Material.
Visualization and Tracking of Poly(A) RNA by
Photoactivation in Speckles and Nucleoplasm
To visually track the movement of poly(A) RNA in and out
of speckles and within the nucleoplasm, we next introduced
oligo(dT) labeled with photoactivatable fluorescein (Mitchi-
son et al., 1994; Politz, 1999; Politz et al., 1999) to the SC35-
expressing HeLa cell line. The coverslips containing the
growing cells were mounted in a temperature-controlled
chamber on an inverted microscope, and the oligo present in
a particular speckle was photoactivated using a laser di-
rected through a pinhole (Politz et al., 1999, 2004). The un-
caging spot within the nucleus was typically 1.0 –1.5
diameter (see Figure 6 for image), similar to or smaller than
the speckles. The movement of the photoactivated signal
away from the speckle was tracked over time by capturing
sequential digital images (Figure 4). The signal was ob-
served to move out in all directions from the uncaging site
into the surrounding nucleoplasm but did not enter nucleoli.
Signal moved to remote speckles in a manner indistinguish-
able from the movement through the nucleoplasm, but with
no accumulation of signal in other speckles (see red circled
speckles in Figure 4). There was no evidence of a concentra-
tion of signal moving in a directed way toward another
speckle or toward the nuclear envelope. Instead, the pattern
Figure 3. Mobility of oligo(dT) and oligo(dA) on and off speckles
in mRFP-SC35 HeLa cells measured using FCS. (A) Autocorrelation
curves of cells containing oligo(dT), based on FCS measured either
within a speckle (blue) or in the nucleoplasm (off a speckle, red). (B)
Fraction of oligo(dT) and oligo(dA) present in different mobility
classes measured within speckles (on) and in the nucleoplasm (off).
Each kinetic component is indicated by black, red, or open bars in
the histogram. The 10- to 100-ms component (red) was undetectable
in oligo(dA) containing-cells. (C) Fraction of mRFP-SC35 protein
present in different mobility classes measured within speckles (on)
and in the nucleoplasm (off). (D) Average fraction of oligo(dT)
present in different mobility classes measured using FCS within
speckles (on) and in the nucleoplasm (off) at 22 and 37°C.
Poly(A) RNA Diffuses through Speckles
Vol. 17, March 2006 1243
Page 5
of signal dispersion reproducibly approximated a Gaussian
The oligo(dT) moved away from the speckle much more
slowly than a control nonhybridizing oligo(dA). Over half of
the oligo(dA) had left the uncaging site by 2 s, whereas
90% of the oligo(dT) remained and then moved away
more slowly (Figure 5A). This is the expected result because,
as observed in the FCS experiments, the oligo(dT) that is
hybridized to poly(A) RNA moves much more slowly than
does free oligo in the nucleus. The signal intensity along a
line drawn through the nucleus and the center of the uncag-
ing site generally described a Gaussian distribution (typical
example in Figure 5B, broken curves), although the curves
were never completely smooth. The small fluctuations in
signal distribution along the curve represent regions where
the signal did not spread in a completely uniform way and
thus may reflect the presence of obstacles in the intranuclear
When the rate of movement away from the uncaging site
was plotted as a function of the width (at e
) of the Gauss
ian distribution at increasing times (Cardullo et al., 1991), the
mean square displacement of the signal was found to be
linearly proportional to time (Figure 5C), as expected of a
diffusive process. The average estimated diffusion coefficient
using this method was 0.67 0.05
/s, which was very
similar to that estimated earlier in the nucleoplasm of L6
cells using the same method (Politz et al., 1999). Because this
method of estimating a diffusion coefficient is dependent on
the position at which the width of the Gaussian is measured,
and also does not account for any potential optical blurring,
we also used a more refined algorithm to estimate the dif-
fusion coefficient. This algorithm fit the relative shapes of the
entire Gaussian distributions over multiple time points with
a two-component model (Figure 5B, smooth lines) to more
accurately estimate a diffusion coefficient (Fogarty et al.,
2000; Figure 6). Using this method, an average diffusion
coefficient of 0.32 0.04
/s was estimated.
When a site in the nucleoplasm outside the speckle was
uncaged, the signal was observed to behave in a virtually
identical manner to signal uncaged in a speckle: it still left
the site at a similar rate (Figure 5, D and E), and the square
of the displacement again varied linearly with time (Figure
5F). The average diffusion coefficient was estimated to be
0.39 0.04
/s using the global algorithm (Figure 5E,
smooth lines show fit), very similar to the diffusion coeffi-
cient estimated within speckles. This result is consistent with
the FCS experiments described above in which no significant
difference was observed in the mobility of poly(A) RNA
within speckles or in the nucleoplasm.
We next followed the movement of poly(A) RNA after
photoactivation inside a speckle at 22°C, rather than at 37°C.
No change in the characteristics of movement of the tagged
poly(A) RNA was observed (Figure 5, G and H), and the
average diffusion coefficient of 0.27 0.08
/s estimated
from the global fit algorithm was not significantly different
from that estimated at 37°C (0.32 0.04
/s; Figure 5B).
No difference was observed in the rate of movement at the
two temperatures in the nucleoplasm either (our unpub-
lished data), as has been reported previously for poly(A)
RNA in L6 myoblasts (Politz et al., 1999; Politz and Pederson,
2000). Therefore, using either FCS or photoactivation tech-
niques, poly(A) RNA showed similar dynamics in the nu-
cleoplasm and the speckle, and no evidence was obtained
for the presence of a distinct slow-moving poly(A) RNA
population within the speckle.
Although the results presented up to now indicated that
most poly(A) in speckles was dynamically exchanging with
the nucleoplasm, we wanted to address the degree to which
our methods could have detected a minor population of
immobile RNA in the speckle because this was the key
hypothesis being tested. Additionally, we wanted to deter-
mine whether uncaged signal above and below the plane of
focus was affecting our diffusion coefficient estimates. We
therefore carried out a series of quantitative uncaging sim-
ulations. Figure 6 shows simulations (see Materials and Meth-
ods) of a population of molecules moving away from a
m-diameter uncaging site with a diffusion coefficient of
/s within a nucleus that is 15
s in diameter under
our standard widefield imaging conditions (blurred, Figure
6, A and B) versus a simulated ideal case where no optical
blurring of the signal takes place (i.e., contributions from
uncaged molecules above and below the plane of focus were
omitted from the simulation; Figure 6, D and E). When the
global fit algorithm was used to fit the Gaussian curves for
the blurred simulation at each time point (Figure 6C, smooth
lines), the best fit was obtained when a two-component
Gaussian spatial profile was assumed. The second compo-
nent thus very likely represents the blurred, or out-of-focus
Figure 4. Movement of oligo(dT):poly(A)
RNA hybrids away from a speckle after pho-
toactivation. mRFP-SC35 HeLa cells were al-
lowed to take up caged-fl oligo(dT) and then
the probe was photoactivated using 360-nm
light directed at a speckle in a live cell nu-
cleus as described in Materials and Methods.
The uncaging site is marked with a white
circle in the “caged” panel, and two speckles
are circled in red in both the SC35 panel and
in the final panel. High-speed time-lapse dig-
ital image microscopy was used to capture
2D images of the signal as it moved away
from the uncaging site. (The brighter dots
visualized here sometimes occur in cells that
have taken up oligodeoxynucleotides and do
not correspond to hybridization sites (Politz
and Pederson, unpublished data; also see
Lorenz et al., 1998). Bar, 2.6
J.C.R. Politz et al.
Molecular Biology of the Cell1244
Page 6
light, because it is not present in the fits to the nonblurred
image (Figure 6, E and F). It therefore can be concluded that
the contribution of the out-of-focus signal above and below
the plane of focus in our experiments inflates our estimate
for the diffusion coefficient of poly(A) RNA by at most a
factor of 2, and we can account for this using the two
component global fit algorithm. As mentioned, we used this
two-component global fit algorithm to calculate the Ds for
all the uncaging data described above, so that the contribu-
tion of the optical blurring was removed.
We noted that the global fits to the real data Gaussian
distributions shown in Figure 5 do not fit the early time
points as well as the global fits to the simulations shown in
Figure 6 (compare Figure 5C to 6C). We think this is because
of the presence of free oligo near the uncaging site at these
early times points. Indeed, the FCS studies described above
predict that about half of the oligo(dT) in the nucleus in
unhybridized, but this fraction cannot be tracked in the time
frame of the uncaging studies because free oligo would
move away from the site within the first few time points
Figure 5. Characterization of signal move-
ment away from photoactivation site. (A)
Probe was uncaged in speckles in cells con-
taining either oligo(dT) (red curve) or oli-
go(dA) (black curve) and the average signal/
pixel remaining at the uncaging site (bleach
adjusted) was calculated for each time point
and plotted. The bar on each time point rep-
resents the SE of the mean. (B) The Gaussian
distributions of the signal intensity across a
line across the nucleus and through the cen-
ter of the uncaging site were digitally re-
corded at successive times after photoactiva-
tion (broken curves, top to bottom, 65, 450,
900, 1350, 1800, 3150, and 3500 ms), and a
global algorithm was used to determine the
best fit diffusion coefficient for the curves and
time simultaneously (see Figure 6; see Mate-
rials and Methods). A representative distribu-
tion and the simulated fit (smooth lines) for
an uncaging on a speckle is shown here; the
diffusion coefficient estimated from this un-
caging was 0.346
/s. (C) The mean
square displacement (at e
) versus time
plotted for the same uncaging site as shown
in B. The line through the points is based on
a linear least squares regression analysis
0.92) and predicts a diffusion coefficient
of 0.7
/s. (D) Average signal remaining
at uncaging sites over time after uncaging on
(red curve) or off (blue curve) a speckle in
cells containing oligo(dT). Error bars for each
point have been omitted so that the two
curves can be seen clearly. For each point the
SE of the mean was less than or equal to
3%. (E) Same as in B except the uncaging
site was nucleoplasmic (off a speckle). The
diffusion coefficient calculated from this
global fit is 0.285
/s. (F) The mean square
displacement (at e
) versus time plotted for
the same uncaging site as shown in E. (D
/s; R
0.98). (G) Average fraction of
signal remaining at uncaging site after pho-
toactivation of caged-fl oligo(dT) in speckles
at 37°C (pink) and 22°C (blue). The bar on
each point is the SE of the mean. (H) Same as
B except at 22°C. The diffusion coefficient
estimated from this global fit is 0.351
Poly(A) RNA Diffuses through Speckles
Vol. 17, March 2006 1245
Page 7
(seconds). This interpretation is supported by the fact that if
the global fits are done only to the first two time points after
uncaging, a faster diffusion coefficient is obtained.
Finally, we built differing levels of a putative immobile
RNA population into the simulation as fixed parameters to
determine what fraction of immobile molecules would be
detectable in the photoactivation-tracking experiments.
These simulations revealed that an immobile RNA popula-
tion comprising as little as 5% of the total signal within the
uncaging site would have been easily visualized in both
the tracking images (compare Figure 6G with H) and in the
Gaussian plots of the intensity distribution used to estimate
the diffusion coefficient (compare Figure 6I with B).
In this work, we have found that the mobility of poly(A)
RNA in HeLa cells is essentially the same both in speckles
and in the nucleoplasm. Therefore, although this RNA pop-
ulation includes not only processed mRNA destined for
cytoplasmic export but also a substantial fraction of nucleus-
retained poly(A) RNA molecules (Perry et al., 1974, Herman
et al., 1976; Huang et al., 1994; Morey and Avner, 2004) that
might have been thought to be involved in maintaining
nuclear substructure in some way, we did not find any
poly(A) RNA to be specifically tethered within speckles. Our
results do not rule out the presence of a very small slow-
moving or immobile class of poly(A) RNA in the nucleus,
but if it exists, our results indicate that it is 5% of the total
poly(A) RNA and is present in similar amounts in both
speckles and the nucleoplasm. Therefore, it is unlikely that
poly(A) RNA serves as an immobile scaffolding to form the
speckle; rather, it can move freely in and out of speckles in
all directions and visit other speckles.
These findings are consistent with numerous studies on
the mobilities of nuclear proteins that have demonstrated
rapid exchange between the nucleoplasm and speckles
(Kruhlak et al., 2000; Phair and Misteli, 2000; Molenaar et
al., 2004), the nucleolus (Phair and Misteli, 2000; Snaar et al.,
2000; Chen and Huang, 2001), and Cajal bodies (Snaar et al.,
2000; Handwerger et al., 2003; Dundr et al., 2004). We also
show here that the SC35 protein is extremely dynamic in
both within and outside speckles. It is therefore becoming
increasingly clear that nuclear proteins that were once
thought to perhaps serve static structural roles because they
seemed concentrated in a certain structure, actually move in
and out of these structures very rapidly (Misteli, 2001; Bubu-
lya and Spector, 2004). Our results demonstrate that poly(A)
RNA behaves in a similar manner and exchanges rapidly
between speckles and the nucleoplasm.
We looked for temperature-dependent behavior of the
poly(A) RNA movement in speckles, but we observed no
difference in the rate of movement of poly(A) RNA at 22
versus 37°C within speckles or in the nucleoplasm. If move-
ment into or out of speckles were dependent on enzymatic
activity (perhaps requiring a motor protein driven by ATP
Figure 6. Simulations of a population of
molecules moving away from a one micron
diameter uncaging site with a diffusion coef-
ficient of 0.3
/s. (A) Simulated (see Mate
rials and Methods) wide-field microscope im-
age of the uncaged spot before any diffusion
has occurred. The uncaged profile is reim-
aged with both in-focus (the aperture) and
out-of-focus components. (B) Plot of intensi-
ties along a line through the center of the spot
in A (circles), are well fit by a two component
Gaussian model (black line) compared with a
single Gaussian model (red line). The two
Gaussian components capture the in-focus
(narrower) and out-of-focus (broader) contri-
butions (see Materials and Methods). (C) Plot of
intensities along the same line over time, after
allowing the initial uncaged distribution
shown in A and B to diffuse in 3-D with D
/s. Colors (black, red, green, cyan,
blue, magenta, violet, and orange) correspond
to data from images at times 0.15, 0.6, 1.05,
1.5, 1.95 (2.4 and 2.85 not shown), 3.3, 3.75,
and 8.25 s, respectively. The intensity line
data from the images up to 3.75 s were jointly
fit with the equation for a single (Gaussian)
diffusion component convolved with a two
Gaussian component initial uncaged distribu-
tion (solid lines). The diffusion coefficient
from the fit was D 0.38
/s. (D) Same as
in A except before being reimaged (blurred)
by the PSF of the microscope. This yields only
the infocus portion of the 3-D uncaged spot.
(E) Plot of intensities along a line through the
center of D (circles) are well fit by a one-component gaussian model (black line). (F) Same as C except the lines were through D, and the
equation fit to the data is a single diffusion component convolved with a single Gaussian component for the initial distribution. From the fit,
D 0.34
/s. (G) From the same simulation as in A, after diffusing for 8.25 s (orange circles in C). (H) Same as in G except for this
simulation, 5% of the uncaged molecules at the uncaging spot were assumed to be immobile. (I) Same as C, but the line was drawn through
the center of the uncaging site on H. The presence of the 5% fixed molecules within a 2
m speckle is quite evident by 8 s (orange circles,
compare with C). Bars (A, C, F and G), 4
J.C.R. Politz et al.
Molecular Biology of the Cell1246
Page 8
hydrolysis, for example), one would expect to see a three- to
fivefold slowing of the rate at the lower temperature because
enzymatic reactions have Q
s (change in reaction rate with
every 10 K change in temperature) between 2 and 3. Fur-
thermore, if poly(A) were being directed specifically to the
nuclear pores by a metabolic energy-requiring process, one
would expect to see a change in the distribution of the
poly(A) RNA as it moved out into the nucleoplasm from the
speckle at the different temperatures. However, we did not
see this. Thus, the simplest interpretation is that poly(A)
RNA can move freely between speckles and the nucleo-
plasm of HeLa cells without a direct input of metabolic
Calapez et al. (2002) reported an 1.5- to 2-fold (estimated
from Figure 8 in Calapez et al., 2002) reduction in the rate of
movement (which gives a threefold reduction in diffusion
coefficient) of two different GFP-labeled proteins bound to
poly(A) RNA at 37 versus 22°C and suggested that this
implies metabolic energy-dependent mobility. Similarly,
Molenaar et al. (2004) have reported an 1.5-fold (estimated
from their Figure 7B) decrease in the rate of recovery after
photobleaching of a tagged poly(A) RNA at 37 versus 22°C
and suggested on this basis that metabolic energy is neces-
sary for poly(A) RNA mobility. However, reductions of this
magnitude are not easily explained by rate decreases in
enzymatic reactions because, as explained above, one would
expect to see a much larger effect upon a 15°C temperature
shift (see also Shav-Tal et al., 2004 for a discussion of this
point). In addition, a recent study of mRNA movement in
the nucleus of mammalian cells has somewhat clarified the
situation by revealing that ATP is required for the resump-
tion of movement when RNA becomes corralled within tight
confinements, rather than the nucleotide being involved in
the movement itself (Vargas et al., 2005).
Molenaar et al. (2004) also reported that several poly(A)
binding proteins moved in and out of speckles at rates
similar to those reported here for both bound SC35 and
poly(A) RNA, and by others for RNA-binding proteins
(Phair and Misteli, 2000; Misteli, 2001; Calapez et al., 2002),
but they reported a 5- to 10-fold slower diffusion coefficient
for poly(A) RNA itself. It is unclear, however, whether the
2-O-methyl modified oligo(U) probe used by Molenaar et al.
(2004) was actually hybridized to poly(A). Hybridization
was tested in only one way: U2OS cells were treated with the
cytotoxic drug cordycepin for 16 h, and after this treatment
time, it was observed that newly injected 2-O-methyl oli-
go(U) was more mobile within the nucleus and no longer
accumulated in speckles. Because cordycepin interferes with
polyadenylation, it was concluded that the probe must have
been hybridized in the untreated cells. However, cordycepin
has not been used in previous studies on mammalian cell
nuclear RNA metabolism for more than 1–3 h (Siev et al.,
1969; Penman et al., 1970; Darnell et al., 1971; Mendecki et al.,
1972) because of severe toxicity effects. Moreover, it has been
clearly established that cordycepin inhibits the production
of cytoplasmic mRNA in HeLa cells within 25–40 min (Pen-
man et al., 1970). The mRNA population of HeLa cells con-
sists of at least two kinetic components; 30 40% has an
average half-life of 2–7 h, and the rest has an average half-
life of 24 h (Singer and Penman, 1973; Puckett and Darnell,
1977). A 16-h treatment with cordycepin would therefore
reduce the shorter lived mRNA population to 6–25% of its
original concentration; cells treated in this way may not
reflect the behavior of normal cells.
We observed one more interesting property of poly(A)
RNA in the experiments described here: poly(A) RNA can
visit more than one speckle as it travels throughout the
nucleoplasm. This implies that a visit to a speckle does not
change the characteristics of the poly(A) RNA in such a way
that it is unable to pass through other speckles. A similar
phenomenon was observed in live cell tracking of 28S rRNA,
which was observed to move between nucleoli (Politz et al.,
2003). Certain nuclear proteins have also been observed to
move between nuclear compartments, such as fibrillarin
between nucleoli and SF2/ASF between speckles (Kruhlak et
al., 2000; Phair and Misteli, 2000), but these proteins are not
destined for transport to the cytoplasm, as is the aforemen-
tioned 28S rRNA and polyadenylated mRNA. It therefore
seems that even macromolecules that must be transported to
the cytoplasm can roam freely not only throughout the
nucleoplasm but also enter and exit similar nuclear compart-
ments with no direct requirement of metabolic energy.
We thank Supriya Prasanth for help in Western blot analysis. This work was
supported by National Institutes of Health Grants GM-60551 to J. P., R. T. and
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Grants DB19200027 and DB19724611 to the University of Massachusetts Bio-
medical Imaging Facility; and a grant to J. L. from the Volkswagen Founda-
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Poly(A) RNA Diffuses through Speckles
Vol. 17, March 2006 1249
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  • Source
    • "Directly studying the dynamics of nuclear components, such as mRNAs in the nucleus of a living cell, will help to define the rules that govern the kinetics, locations, and interactions of proteins and nucleic acids relative to nuclear structure. Advanced microscopy techniques have improved image resolution or enabled fast tracking of individual molecules in living cells, allowing the nuclear mobility of different proteins, RNAs, and other molecules to be probed (Görisch et al., 2004; Shav-Tal et al., 2004; Politz et al., 2006; Grünwald et al., 2008). Currently available single-molecule imaging methods share the limitation that they can only image fast enough to accurately track single molecules in one optical plane (2D) or their 3D capability only allows visualization of small numbers of molecules within a limited field of view (Ragan et al., 2006; Huang et al., 2008; Backlund et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization. © 2015 Smith et al.
    Full-text · Article · May 2015 · The Journal of Cell Biology
  • Source
    • "Thus, human Hub1 is able to associate with splicing speckles also independently of Snu66. This finding supports the above observation (Figure 3E) that binding of Hub1 to Snu66 is not essential for Hub1 function, and suggests that other surfaces of Hub1 may contribute to splicing factor association. Splicing speckles are typically highly dynamic as some of their protein and RNA content cycle continuously between speckles, sites of transcription and other nuclear locations (Misteli et al., 1997; Politz et al., 2006; Spector and Lamond, 2011). The splicing Figure 1 Conserved and divergent properties of Hub1. "
    [Show abstract] [Hide abstract] ABSTRACT: Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.
    Full-text · Article · May 2014 · Journal of Molecular Cell Biology
  • Source
    • "Indeed, photobleaching studies in living cells have shown that nuclear speckles concentrate a population of mobile poly(A) RNAs in continuous flux with the nucleoplasm (Molenaar et al., 2004; Politz et al., 2006), suggesting that a fraction of these polyadenylated RNAs are involved in post-transcriptional processing of pre-mRNAs before their nuclear export (reviewed in Hall et al., 2006; Melcák et al., 2000, 2001). In this context, we propose that the dynamic aggregation of poly(A) RNA/PABPN1 complexes into growing INIs gradually depletes the nuclear speckles of these molecular components, thereby interfering with the normal trafficking and post-transcriptional processing of polyadenylated mRNAs in speckles. "
    [Show abstract] [Hide abstract] ABSTRACT: Nuclear speckles are essential nuclear compartments involved in the assembly, delivery and recycling of pre-mRNA processing factors, and in the post-transcriptional processing of pre-mRNAs. Oculopharyngeal muscular dystrophy (OPMD) is caused by a small expansion of the polyalanine tract in the poly(A)-binding protein nuclear 1 (PABPN1). Aggregation of expanded PABPN1 into intranuclear inclusions (INIs) in skeletal muscle fibers is the pathological hallmark of OPMD. In this study what we have analyzed in muscle fibers of OPMD patients and in primary cultures of human myoblasts are the relationships between nuclear speckles and INIs, and the contribution of the former to the biogenesis of the latter. While nuclear speckles concentrate snRNP splicing factors and PABPN1 in control muscle fibers, they are depleted of PABPN1 and appear closely associated with INIs in muscle fibers of OPMD patients. The induction of INI formation in human myoblasts expressing either wild type GFP-PABPN1 or expanded GFP-PABPN1-17ala demonstrates that the initial aggregation of PABPN1 proteins and their subsequent growth in INIs occurs at the edges of the nuclear speckles. Moreover, the growing of INIs gradually depletes PABPN1 proteins and poly(A) RNA from nuclear speckles, although the existence of these nuclear compartments is preserved. Time-lapse experiments in cultured myoblasts confirm nuclear speckles as biogenesis sites of PABPN1 inclusions. Given the functional importance of nuclear speckles in the post-transcriptional processing of pre-mRNAs, the INI-dependent molecular reorganization of these nuclear compartments in muscle fibers may cause a severe dysfunction in nuclear trafficking and processing of polyadenylated mRNAs, thereby contributing to the molecular pathophysiology of OPMD. Our results emphasize the potential importance of nuclear speckles as nuclear targets of neuromuscular disorders.
    Full-text · Article · Jan 2012 · Neurobiology of Disease
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