* Department of Physics, Northwestern School of Law, and § Department of Mathematics, Lewis & Clark College, Portland,
Oregon 97219; and
Biophysics Research Division and Department of Physics, University of Michigan, Ann Arbor, Michigan
48109
Translational dynamics of chromatin in interphase nuclei of living Swiss 3T3 and HeLa cells was
studied using fluorescence microscopy and fluorescence recovery after photobleaching. Chromatin was
fluorescently labeled using dihydroethidium, a membrane-permeant derivative of ethidium bromide. After
labeling, a laser was used to bleach small (~0.4 µm radius) spots in the heterochromatin and euchromatin of
cells of both types. These spots were observed to persist
for >1 h, implying that interphase chromatin is immobile over distance scales 0.4 µm. Over very short
times (<1 s), a partial fluorescence recovery within the
spots was observed. This partial recovery is attributed
to independent dye motion, based on comparison with
results obtained using ethidium homodimer-1, which
binds essentially irreversibly to nucleic acids. The immobility observed here is consistent with chromosome
confinement to domains in interphase nuclei. This immobility may reflect motion-impeding steric interactions that arise in the highly concentrated nuclear milieu or outright attachment of the chromatin to
underlying nuclear substructures, such as nucleoli, the
nuclear lamina, or the nuclear matrix.
In recent years, models of the structure and organization of the interphase nucleus have changed dramatically. The interphase nucleus was once believed to be
a largely homogeneous organelle, containing little structural specialization beyond the nucleoli (Manuelidis, 1990 Considerably less is known about the mobility of chromatin and other constituents of interphase nuclei than is
known about their organization. Despite being poorly understood, mobility has been the subject of interest for at
least two reasons. First, the mobility of nuclear constituents should reflect and influence important aspects of their
organization. Indeed, in the case of interphase chromatin,
mobility has been most commonly inferred indirectly from
observed changes in organization and distribution (Manuelidis, 1985 To date, data bearing on the mobility of chromatin in interphase nuclei are not only limited, they are also somewhat contradictory. For example, some studies suggest that
chromatin is relatively immobile during interphase. Notable examples include observations of nonrandom organization of chromosomal substructures and of chromosome
confinement to domains in fixed interphase nuclei (Moroi et al., 1981 Data bearing on the mobility of nonchromosomal molecules and other objects in interphase nuclei are also somewhat limited and contradictory. In situ hybridization studies
have shown that RNA molecules may move nonrandomly
along "tracks" in the nucleus, perhaps because their motion is constrained by an underlying nuclear matrix (Lawrence et al., 1989 In this study, we have directly monitored the translational motion of fluorescently labeled chromatin in the nuclei of living Swiss 3T3 and HeLa cells using FRAP. These
cell lines were chosen because microscopy studies of nuclear organization have frequently employed cultured cells,
including Swiss 3T3 and HeLa, generating a considerable
body of germane literature (e.g., Fey et al., 1986 Cell Culture and Chromatin Labeling
Swiss 3T3 fibroblasts and HeLa cells were grown on 25-mm-diam round
glass No. 1 coverslips (Fisher Scientific Co., Pittsburgh, PA) in Dulbecco's
modified Eagle medium (low glucose) (GIBCO BRL, Gaithersburg, MD)
supplemented with 10% fetal calf serum. Cells were maintained at 37°C in
a 10% CO2/90% air incubator and used before cells had reached confluence. Chromatin in living cells was labeled with ethidium by a 10-45 min
incubation at 37°C with 2.5-10 µg/ml dihydroethidium (Molecular Probes,
Inc., Eugene, OR). (The longest labeling times and highest dihydroethidium concentrations were used to prepare cells for photography.) Dihydroethidium is a membrane permeant, chemically reduced derivative of
ethidium bromide that is dehydrogenated to ethidium bromide. After labeling, cells were washed three times and then maintained in a physiological buffer (Hanks' or Dulbecco's phosphate buffered saline; both from GIBCO BRL). In some cases, cells were fixed in buffer containing 4%
paraformaldehyde for 10 min before labeling and then labeled with 0.5 µg/ml ethidium homodimer-1 (Molecular Probes, Inc.) or ethidium bromide. For FRAP experiments and imaging, cells were mounted in buffer
in Sykes-Moore Chambers (Bellco Glass, Inc., Vineland, NJ). Most experiments were conducted at 23°C, but some were also conducted at 37°C to
verify that the results remain valid at physiological temperatures.
Cell viability during and after photobleaching experiments was assayed
in two ways. First, short-term viability during photobleaching experiments
was demonstrated by simultaneously labeling cells for 10 min at 37°C with
dihydroethidium and with 10 µM calcein acetoxymethyl (AM) ester (Molecular Probes, Inc.), a "premier indicator of cell viability" (Haugland,
1996 Second, long-term viability and absence of DNA damage were demonstrated by verifying that cells were able to divide and proliferate after they
were photobleached. To facilitate identification of cells over long periods
of time, cells were grown on coverslips that had been scratched with a diamond pen. Cells were labeled with dihydroethidium and calcein AM well
before they had reached confluence and were then transferred to a sterile
Sykes-Moore chamber for photobleaching experiments. Cells in an easily
identified region of the scratch (such as the end) were bleached and returned to the incubator after ~1 h. Cells were then examined frequently under a dissecting microscope; individual bleached cells could thus be
tracked at low cell density even if they moved slowly. Division and proliferation were monitored for ~72 h after bleaching.
Antibody Samples
As a FRAP control, samples containing a fluorescently labeled macromolecule undergoing Brownian diffusion were prepared by diluting a rhodamine-labeled goat anti-rabbit antibody into Hanks' buffer.
FRAP Experiments
Chromatin dynamics was monitored using FRAP (Axelrod et al., 1976 The FRAP apparatus used in these experiments has been described
previously (Yuan and Axelrod, 1995 Light was focused onto the sample using a 63× (NA = 1.4) Zeiss objective (Carl Zeiss Inc., Thornwood, NY), producing an in-focus spot of radius ~0.4 µm in the sample plane. The 514-nm (green) line of the argon-ion laser was used to excite fluorescence; excitation of ethidium with visible light, as contrasted with ultraviolet radiation, reduces the probability
of radiation-induced damage to the cells. The bleaching power at the sample was typically in the range ~4-60 mW. The ratio of the intensities of
the bleach and probe beams was ~10,000:1, ensuring insignificant bleaching during the probe phase of the experiment.
Photography
Photographs of cells were taken at defined times before and after photobleaching using Kodak TMX 400 film. Cells were illuminated for photography by inserting a defocusing lens into the optical path of the photobleaching apparatus, thereby expanding the laser beam to cover the
entire field of view.
Data Analysis
Diffusion coefficients and mobile fractions were obtained by fitting photobleaching data to an equation describing photobleaching recovery in a
system containing one immobile fraction and one mobile fraction diffusing in two dimensions (Axelrod et al., 1976 Fig. 1 shows a series of photographs of two dihydroethidium-labeled Swiss 3T3 cells taken (a) before; (b) a few min
after; and (c) ~60 min after the nucleus in the upper cell
was exposed to a brief pulse of intense laser light. A several hundred millisecond bleaching pulse was sufficient to
bleach the ethidium molecules in the illuminated region
and create a dark spot in the euchromatin (Fig. 1, b and c).
The radius of the spot was calculated to be ~0.6 µm, using
known magnification parameters and the size of the photograph. This spot persisted for at least 1 h (Fig. 1 c), indicating that a significant fraction of the interphase euchromatin in Swiss 3T3 cells is translationally immobile on the
0.6 µm distance scale. (This distance scale can be lowered
to ~0.4 µm; see below.) Moreover, the spot did not move
within the nucleus, providing further evidence for chromatin immobilization during interphase. Similar results were
observed after photobleaching HeLa cells and cells maintained at 37°C (data not shown).
More quantitative translational mobility data were obtained by monitoring the temporal dependence of the
post-bleach fluorescence using FRAP curves. Fig. 2, a and
b, shows representative curves for Swiss 3T3 and HeLa
cells, respectively. Both euchromatic and heterochromatic
regions of the nucleus were studied. When the light used in
the FRAP experiments was focused down to a small ~0.4
µm radius spot, the recovery curves were biphasic, reflecting the presence of two fractions. One fraction was "mobile," leading to partial recovery on a time-scale of several
hundred milliseconds; the other fraction was immobile,
leading to an incomplete recovery in the FRAP curves.
FRAP curves were fitted to the theoretical expression
for photobleaching recovery in two dimensions, assuming
one mobile and one immobile fraction. The results of the
fits for dihydroethidium-labeled Swiss 3T3 cells are 27 ± 4% immobile, 73 ± 4% mobile, and D = 1.1 (± 0.5) × 10 There are two important differences between the results
shown in the photographs and in the photobleaching recovery curves. First, fluorescence from out-of-focus planes
inevitably appears in the photographs (Scalettar et al.,
1996 Second, the FRAP curves show an initial partial recovery of the fluorescence in the spots (mobile fractions ~64-
73%) that is not evident from the photographs. The recovery time associated with this component was studied as a
function of the size of the illuminated spot to identify its
origin. If molecular motion is the source of the initial partial recovery, the recovery time should increase systematically as the size of the illuminated spot is increased. For
example, for a recovery driven by Brownian diffusion, the
recovery half-time is expected to increase as the square of
the spot size (Axelrod et al., 1976
A motion-derived origin for the partial recovery is also
suggested by results from two additional experiments; see
Fig. 4, a and b. First, when samples are dried down and immobilized, the recovery largely disappears. Second, when
hydrated samples are deoxygenated, the recovery time is
essentially unaltered, which indicates that the initial recovery does not arise from reversible photobleaching (see
Discussion). It is important to eliminate this latter possibility because the effects of reversible photobleaching have
previously been observed in FRAP experiments on DNA
and chromatin (Scalettar et al., 1990
The mobile fraction could represent chromatin, RNA,
or a subpopulation of the ethidium bromide that transiently comes off the chromatin. To distinguish these possibilities, FRAP control experiments were performed on
cells fixed and labeled with either ethidium bromide or
ethidium homodimer-1, which binds essentially irreversibly to DNA and RNA (Gaugain et al., 1978
A potential artifact in interpretation could arise if the
lack of full fluorescence recovery is due to depletion of the
finite reservoir of fluorescence in the nucleus, and not to
an immobile chromatin fraction. However, the photographs show that the volume of the bleached region is
small, indicating that the bleach is unlikely to significantly
deplete the total nuclear fluorescence. Moreover, if the
"immobile fraction" simply reflected depletion of that fraction of the total nuclear fluorescence, a few bleaches would
render the nucleus essentially nonfluorescent. This was not found to be the case. Finally, the long-lived spots shown in the photographs demonstrate that the nucleus does not
become uniformly less fluorescent with time after the
bleach.
Artifacts in interpretation could also arise if the cells
were not viable or were damaged during the FRAP experiments. For this reason, cell viability during and after photobleaching experiments was tested using the two methods
described under Materials and Methods. No leakage of the
viability dye calcein from bleached or unbleached cells
was observed during the ~1-h period that the cells remained on the microscope stage, demonstrating viability during the experiments. In contrast, substantial leakage of
calcein occurred <2 min after cells were placed in buffer
containing 0.1% Triton X-100. Moreover, bleached cells in
an easily identified (scratched) region of the coverslip
were observed to divide and proliferate after being returned to the incubator. Both of these results strongly suggest that the FRAP data reflect interphase chromatin dynamics in living and unperturbed Swiss 3T3 and HeLa cells.
Several other results, observations, and aspects of the
experimental protocol argue against light-induced artifacts
in the FRAP experiments. First, the mobility results were
independent of light intensity and bleaching time. Second,
only a very small fraction of the nuclear volume was typically exposed to light. Finally, ethidium was excited through
its visible absorption band, using green light; this is likely
to result in significantly less nuclear damage than would
excitation of ethidium through its UV absorption band.
The experiments described here were directed at (a) measuring the translational mobility of chromatin, especially
euchromatin, in living interphase cells using FRAP; and
(b) relating the results to current ideas about chromatin
function and organization during interphase.
Visually Constrained Motion
Even in the absence of photobleaching data, it is evident
that there are some constraints on chromatin motion in
living interphase Swiss 3T3 cells. For example, the photographs in Fig. 1 show that heterochromatin is largely immobile, since the bright heterochromatic spots remain in
approximately the same place in the nucleus during long
periods of observation.
The mobility of euchromatin in living Swiss 3T3 cells,
and of chromatin in general in small HeLa cells, is more
difficult to assay visually. Hence, we turned to photobleaching techniques to give us more complete information on chromatin mobility in interphase cells. When we
bleached the fluorescence in euchromatic regions of Swiss
3T3 nuclei, the subsequent temporal dependence of the
fluorescence could be monitored both photographically
(Fig. 1) and through photobleaching recovery curves (Fig.
2). Both types of data show that it is possible to bleach a
long-lived spot in Swiss 3T3 euchromatin, indicating that a
substantial fraction of Swiss 3T3 chromatin is immobile
over distance scales comparable to, or larger than, the size
(~0.25 µm) of a chromatin looped domain (Nelson et al.,
1986 Origin of Initial Recovery
Although long-lived, the bleached spots quickly refill partially, as manifest in the partial recoveries in Fig. 2. We
have studied the properties of this initial recovery in detail
to verify that it is attributable to molecular motion. This is
essential because there are mechanisms that can lead to
recovery in FRAP experiments, such as reversible photobleaching, which are not mobility derived (Velez and
Axelrod, 1988 In our case, several lines of evidence suggest that the initial recovery arises from motion and not effects such as reversible bleaching. First, the relaxation time associated with
mobility-derived recoveries is expected to increase with
increasing size of the illuminated spot, an effect which can
be seen experimentally in the data in Fig. 2. Also, the relaxation time associated with reversible recoveries is expected to be independent of spot size but dependent on
oxygen concentration, effects that we do not observe experimentally (Figs. 2 and 4). (The dependence on oxygen concentration is expected because reversible recovery reflects a long-lived residence in a triplet state, and triplet
lifetimes are very sensitive to oxygen concentration; Scalettar et al., 1990 Effects of High and Low Affinity Binding of
Ethidium Bromide
To identify the molecular species giving rise to the partial
recovery, control experiments were performed on fixed
samples that were labeled with either ethidium bromide or
ethidium homodimer-1, which binds to naked DNA about
1,000 times more tightly than does ethidium bromide (Gaugain et al., 1978 In contrast, the mobile fraction was absent from fixed,
ethidium homodimer-1-labeled samples. Because fixing
and labeling with ethidium bromide leaves the mobile
fraction unaltered, whereas fixing and labeling with the
more tightly binding ethidium homodimer-1 eliminates
the mobile fraction, we have attributed the initial recovery
to independent motion of ethidium bromide arising from its weaker binding to DNA. In addition, the effects of on/
off binding of ethidium bromide have been previously observed in FRAP data (Icenogle and Elson, 1983a Over the time-scale we have probed, only a subpopulation of the ethidium molecules is transiently moving independently of the chromatin. If all ethidium molecules were
moving independently, the FRAP curves could be described by a single recovery time reflecting the fraction of
ethidiums bound and the diffusion coefficient of ethidium
alone (Icenogle and Elson, 1983a Previous studies (Lawrence and Daune, 1976 This interpretation is further strengthened by the quantitative agreement between mobile fractions in living dihydroethidium-labeled samples and fixed ethidium bromide-labeled
samples. The mobile fractions in the living dihydroethidium labeled Swiss 3T3 samples were 73 ± 4% and in the
fixed ethidium bromide-labeled Swiss 3T3 samples were
71 ± 6%. Thus, the entire mobile fraction can be attributed to transiently unbound ethidium bromide, and the
FRAP data show that interphase chromatin is immobile
on the Comparison with Previous Studies of Chromosome
Organization and Dynamics
Results obtained in a number of studies suggest that chromosome substructures can be nonrandomly organized and
that chromosomes are confined to domains within interphase nuclei. From a dynamic perspective, these results
suggest that there are restrictions on the translational mobility of interphase chromatin, as observed here.
Examples of nonrandom organization and confinement
to domains are numerous. For example, a nuclear membrane-apposed/peripheral distribution has been observed
for inactivated X chromosomes in fibroblasts, centromeres
in some types of cells, and centromeres and telomeres of
polytene chromosomes in Drosophila salivary gland nuclei
(Moroi et al., 1981 More direct evidence for constraints on chromosome
lateral mobility during interphase comes from particularly
pertinent recent studies of the mobility of centromeres in
living HeLa cells (Shelby et al., 1996 In contrast, some studies have indicated that chromatin
can be mobile during interphase. For example, in living interphase neurons the position of heterochromatin has
been shown to change in tandem with changes in nucleoli
position (DeBoni and Mintz, 1986). Similarly, centromeres
have been observed to relocate in a variety of interphase
cells (Manuelidis, 1985 Our work is in basic agreement with the body of work
suggesting the existence of constraints on interphase chromatin mobility. A unique feature here is that the results
reflect the mobility of interphase chromatin in general,
rather than the mobility of a particular chromosome or
chromosome substructure. The results thus give a global
view of chromatin mobility while also defining a distance
scale for chromatin immobilization. In addition, the results
were obtained from living cells, unlike many of those discussed above.
Chromatin in Transformed and Cultured Cells
Swiss 3T3 and HeLa cells are commonly used in studies of
chromatin organization and nuclear structure. Nevertheless, there are potential differences between nuclear structure in these cell lines and nuclear structure in untransformed, uncultured cells. For example, transformed cells
may have less heterochromatin and a somewhat altered
nuclear matrix protein composition when compared with
untransformed cells (for review see Manuelidis, 1990 Origin of Chromatin Immobilization on the There are two fundamentally different types of interactions that could act alone or in combination to produce the
chromatin immobilization observed here. These are (a)
outright attachment of chromatin to nuclear substructures,
such as nucleoli, the nuclear lamina, or the nuclear matrix;
and (b) motion-impeding steric interactions inherent in a
highly concentrated milieu such as the nucleus. We consider each of these possibilities, in turn.
Interphase chromatin is known to be attached to nucleoli and the nuclear envelope (Gerace and Burke, 1988 Interphase chromatin may also be attached to the nuclear matrix (Berezney et al., 1995 Finally, chromatin will interact sterically with the broad
spectrum of macromolecules found in the congested nuclear milieu (Livolant and Maestre, 1988 Similarly, large DNAs in concentrated ("semi-dilute")
solution are significantly more mobile than interphase
chromatin. For DNA molecules with radii of gyration on
the order of 0.5 µm, diffusion is somewhat slower in semi-dilute solution than in dilute solution and diffusion coefficients range from 10 Steric interactions alone can produce such major inhibition of mobility in highly entangled polymer solutions
(Luby-Phelps et al., 1987 We have shown that interphase euchromatin and heterochromatin in living Swiss 3T3 fibroblasts and HeLa cells
are translationally immobile over distance scales ;
Berezney et al., 1995
). In contrast, the interphase nucleus
is now widely believed to be inhomogeneous, with many nuclear substructures and functions localized to specific
nuclear subdomains (for review see van Driel et al., 1995).
In particular, a considerable body of evidence suggests
that the most prominent nuclear constituent, chromatin, is
organized so that each chromosome occupies its own discrete domain (Cremer et al., 1982
; Lichter et al., 1988
;
Leitch et al., 1990
; for review see van Driel et al., 1995; Eils et
al., 1996
).
; Bartholdi, 1991
; Ferguson and Ward, 1992
;
Janevski et al., 1995
). Second, the mobility of nuclear constituents should reflect their function. In the case of interphase chromatin, a functional significance for mobility is
strongly suggested by recent observations of changes in
centromere and chromosome distribution in response to
cell differentiation, transcription signals, and stage of the
cell cycle (Manuelidis, 1984
; Bartholdi, 1991
; Ferguson and
Ward, 1992
; Funabiki et al., 1993
; Janevski et al., 1995
; Pluta et al., 1995
; LaSalle and Lalande, 1996
).
; Lichter et al., 1988
; Manuelidis and Borden,
1988
; Dyer et al., 1989
; Leitch et al., 1990
; Popp et al., 1990
;
for review see van Driel et al., 1995). Moreover, in living
HeLa cells, centromeres are generally motionless during
interphase (Shelby et al., 1996
), and in living Drosophila
embryos, chromosomes undergo late decondensation and
subsequent condensation at the same sites on the nuclear
envelope (Hiraoka et al., 1989
). Finally, photobleaching studies of chromatin in isolated interphase nuclei indicate
that chromatin reorientational mobility is highly restricted
(Selvin et al., 1990
). In contrast, some studies have shown
that chromatin can reposition during interphase; notable
examples include the three-dimensional movement of heterochromatin in nuclei of living interphase neurons (De
Boni and Mintz, 1986), the coalescing and dispersing of
centromeres in cultured cells in late G2 and early G1, respectively (Manuelidis, 1985
), and the occasional slow movement of a centromere in living HeLa cells (Shelby et al., 1996
).
). Studies of the trajectories of large, naturally occurring cytoplasmic inclusions have shown that the mobility of inclusion-sized objects in the nuclei of newt pneumocytes is several hundredfold slower than in dilute
solution (Alexander and Rieder, 1991
). In contrast, translational FRAP experiments on fluorescently labeled dextrans (3-150 kD) have shown that translation of dextrans
in the nuclei of Hepatoma cells is only about sevenfold
slower than in dilute solution (Lang et al., 1986
). These
rather disparate results leave considerable uncertainty surrounding the rates and determinants of motion in the nucleus.
; He et al.,
1990
; Berezney et al., 1995
; Shelby et al., 1996
). Our results
show that interphase euchromatin and heterochromatin are substantially immobile in these cell lines. This immobility is consistent with chromatin attachment to nuclear
substructures and with chromosome confinement to discrete domains during interphase. Moreover, this immobility provides a dynamics-based foundation for the many
observations of nonrandom organization of chromosomal substructures in fixed interphase cells.
Materials and Methods
). The latter molecule is nonfluorescent and membrane permeant but
is converted into the green fluorescent molecule calcein by intracellular
esterases in viable cells. After conversion, calcein is retained by cells only
so long as their plasma membranes are not compromised. Thus, green cells
are viable cells, and cell death can be assayed by monitoring for a loss of cytoplasmic green fluorescence. This was done visually and electronically to
demonstrate short-term viability during photobleaching experiments. In
addition, to demonstrate that calcein is not retained after cells are compromised, calcein-labeled cells were placed in buffer containing 0.1% Triton X-100 (Bio Rad Laboratories, Hercules, CA) and loss of cytoplasmic
green fluorescence was then monitored.
).
In a FRAP experiment, a brief, intense pulse of laser light is used to
bleach (render nonfluorescent) many of the fluorescent molecules in a
small subregion of a fluorescent sample. The return of fluorescence to this
subregion is then monitored by shining much attenuated laser light onto
the bleached spot and monitoring the temporal dependence of the post-bleach (probe) fluorescence. The time-scale over which the fluorescence
recovers is determined by the rate at which the fluorescently labeled molecules diffuse and can be used in simple systems to determine a diffusion
coefficient. Failure of the fluorescence to recover indicates that the fluorescently labeled molecules are immobile.
). Briefly, its major components are
(a) an argon-ion laser, which is used as a high-intensity source of light; (b)
an acousto-optic modulator, which is used to modulate the intensity of the
light incident on the sample; (c) an inverted microscope, which is used to
focus the light onto the sample and to collect the fluorescence emitted by
the sample; and (d) an avalanche photodiode ("single photon counting module" [SPCM-100]; EG&G Optoelectronics, Vaudrevil, Quebec, Canada), which is used to detect the fluorescence. An aperture diaphragm placed in
the image plane of the microscope minimizes detection of background and
out-of-focus light. Data were collected on a 486-based PC using custom
FORTRAN software and a custom-programmed counter/timer board
(CTM-05; Keithley Metrabyte Instruments Inc., Cleveland, OH).
). Complex generalizations of
this equation describing diffusion in three dimensions have recently been
derived (Blonk et al., 1993
). These generalized equations show that the recovery time along each dimension is proportional to the square of the
characteristic distance along that dimension. This implies that the fastest
recovery time will be dominated by diffusion along the smallest dimension. For a focused spot, the smallest dimension is the characteristic e
2
bleached radius of the spot (0.4 µm here), rather than the corresponding e
2 axial distance over which substantial bleaching occurs (2.5 µm here).
Thus, for the focused spot used in these experiments, the simpler photobleaching recovery equations describing diffusion in two dimensions can
be used. In addition, our conclusion that a large fraction of interphase
chromatin is immobile (diffusion coefficient [D]1 <10
13 cm2/s) is a
model-independent result, which is also demonstrated visually (see Results).
Results
Fig. 1.
Pre- (a) and post-bleach (b and c) photographs of two
dihydroethidium-labeled Swiss 3T3 cells in interphase. The ethidium stains nucleic acid in both the nucleus and cytoplasm, and reveals the distribution of euchromatin and heterochromatin within
the nucleus. a shows the cells immediately before bleaching; b
and c show the cells a few minutes and 1 h after bleaching the upper cell, respectively. The long-lived dark spot in the upper cell in
b and c was created in an initially fluorescent region of euchromatin by illuminating the region with a 300 ms pulse of 514 nm
(green) light from an argon-ion laser (Power 50 mW at the
sample). The failure of this spot to fill back up with fluorescently
labeled chromatin demonstrates that a large fraction of the chromatin is immobile. The focus may have shifted slightly or the cell
may have moved slightly in c after the sample spent 60 min on the microscope stage. Some nonuniformity in fluorescence in the
photographs may arise from interference in the illuminating light.
Bar, 10 µm.
[View Larger Versions of these Images (96 + 89 + 88K GIF file)]
Fig. 2.
Typical FRAP curves and associated theoretical recovery curves obtained from dihydroethidium-labeled Swiss 3T3
cells (a) and HeLa cells (b). The FRAP curves were smoothed using an eleven-point fit to a fourth-order polynomial (Savitzky and
Golay, 1964) to facilitate distinguishing the different recovery
curves. The bleach pulse was 10 ms in duration, and data were
collected in 10-ms increments. The upper (diamonds) and lower
(squares) curves in each graph were obtained from relatively
smaller (focused) and larger (defocused) spots, respectively. The
defocused spot was obtained by translating a lens along the optical path so that the light was not focused exactly on the sample
plane. Appropriate neutral density filters were inserted into the
path of the bleach beam so that the bleach depths for the two
curves in each graph were comparable; results did not depend on
bleach depth (data not shown), because relatively shallow bleach
depths were employed. Two important features are qualitatively
apparent from the FRAP curves. First, a large fraction of the fluorescence fails to recover, indicating that a large fraction of the
chromatin is immobile. Second, the recovery time associated with
the fraction of the fluorescence that does recover increases with
increasing spot size, indicating that molecular motion is the
source of the initial recovery.
[View Larger Version of this Image (21K GIF file)]
9 cm2/s, where the quoted errors are standard deviations and typically data from
20 different nuclei were averaged. The analogous results for dihydroethidium-
labeled HeLa cells are 36 ± 6% immobile, 64 ± 6% mobile, and D = 2.9 (± 0.2) × 10
9 cm2/s. These results were
independent of the depth of bleach. The mobile fractions
obtained from the fits are fairly large but will be shown to
represent a population of less tightly bound ethidium molecules, and not mobile chromatin. No more than a few
percent of interphase chromatin is mobile on the
0.4 µm
distance scale (see Discussion).
) but is preferentially excluded from the photobleaching recovery curves by the image-plane diaphragm. Because laser beam divergence causes the spot radius to increase in out-of-focus planes, out-of-focus pick-up may
cause the spots shown in the photographs to appear larger
than their in-focus size. Nevertheless, the spot radius obtained by direct measurement from the photographs
closely agrees with the in-focus spot size produced by the
63× Zeiss objective.
). Fig. 3 illustrates this effect in data obtained from a fluorescently labeled antibody solution. Fig. 2, a and b, shows FRAP curves and associated fitted theoretical recovery curves obtained from two
different nuclei samples for two different spot sizes. As
with the antibody samples, the initial recovery time for the
nuclei samples increases as spot size is increased, implying
that molecular motion is the source of the initial recovery.
Fig. 3.
Typical FRAP curves and associated theoretical recovery curves obtained from a rhodamine-labeled antibody undergoing Brownian diffusion in dilute solution (~0.5 mg/ml antibody)
for smaller (diamonds) and larger (squares) spots. The bleach
pulse was 200 µs in duration, and data were collected in 100-µs
increments. The upper curve represents an average of 1,000 experiments and the lower an average of 3,000 experiments, and
each was smoothed using an 11-point fit to a fourth-order polynomial. The fluorescence recovers almost completely, indicating
that all molecules are mobile. In addition, the recovery time increases with increasing spot size.
[View Larger Version of this Image (14K GIF file)]
; Selvin et al., 1990
).
Fig. 4.
Typical FRAP curves obtained from Swiss 3T3 cells
that were labeled with dihydroethidium and then (a) dried down
or (b) placed in a deoxygenated buffer (diamonds) or an atmosphere-equilibrated buffer (squares). Bleach and data collection
times were 10 ms. The virtual absence of the initial recovery in a
sample immobilized by drying (a) supports the idea that the recovery is motion derived. The failure of the initial relaxation time
to vary appreciably with oxygen concentration in the hydrated
samples (b) indicates that the initial relaxation does not represent
reversible photobleaching. Six times more power was required to
achieve bleaching in the deoxygenated sample comparable to
that achieved in the nondeoxygenated sample. This reflects the
greater difficulty of doing irreversible bleaching in the absence of
oxygen, and shows that the deoxygenation was successful.
[View Larger Version of this Image (19K GIF file)]
; Markovits et al.,
1985
; Rye and Glazer, 1995
). Fig. 5 shows that the mobile fraction is present in the fixed ethidium bromide-labeled
samples but is absent from the fixed ethidium homodimer-1-labeled samples. Specifically, the results of curve fits for
the fixed ethidium bromide-labeled samples are 29 ± 6%
immobile, 71 ± 6% mobile, and D = 7.1 (± 0.1) × 10
10
cm2/s. The results for the fixed ethidium homodimer-1-
labeled samples are essentially 0% mobile, 100% immobile, and D
0 cm2/s. Fixation should largely inhibit motion of protein-containing macromolecules on the FRAP
distance scale. This, together with the fact that only the
nucleic acid label differs between the two control samples,
strongly suggests that the mobile fraction is a subpopulation of the ethidium bromide that transiently comes off the
chromatin and diffuses freely (see Discussion). The mobile chromatin fraction is thus small (or zero).
Fig. 5.
Typical FRAP curves obtained from fixed ethidium
bromide-labeled (diamonds) and ethidium homodimer-1-labeled
(squares) Swiss 3T3 cells. Note that the initial component is absent when the homodimer is used as a label.
[View Larger Version of this Image (31K GIF file)]
Discussion
). Similar results were obtained after photobleaching
a spectrum of regions in HeLa nuclei, indicating that chromatin is similarly immobile in this human cell line. This
immobility of interphase chromatin is in marked contrast to the known mobility of ethidium bromide and ethidium
bromide-stained naked DNA in isolated samples (Icenogle and Elson, 1983a
,b
; Scalettar et al., 1989
).
; Scalettar et al., 1990
; Selvin et al., 1990
).
; Selvin et al., 1990
.) Second, the relaxation
time associated with reversible photobleaching of ethidium bromide when bound to DNA or chromatin is <2 ms
(Scalettar et al., 1990
), about two orders of magnitude
shorter than that of the initial recovery observed here. Finally, the initial recovery disappears when the molecules
are dried down; drying leads to molecular immobilization but does not eliminate reversible bleaching (Scalettar et al., 1988b
).
). Fixed samples were used because neither
dye is membrane-permeant; however, fixation in formaldehyde was not expected to immobilize the dye, because
formaldehyde cross-links amino groups (Baker, 1958
) and
because the dye was added after fixation. This expectation
was borne out by the fact that the mobile fraction was still
present in fixed, ethidium bromide-labeled samples and in
dihydroethidium-labeled samples that were fixed after labeling.
,b
).
,b
; Kao et al., 1993
). Our
data show two recovery times (one approximately several
hundred milliseconds and one around infinity), indicating
that not all ethidiums are moving independently.
) of the interaction of ethidium bromide with chromatin suggest a
mechanism that could give rise to two ethidium populations in our samples. These studies identified high and low
affinity ethidium binding sites, which differ by close to
three orders of magnitude in binding constant (Lawrence
and Daune, 1976
). Moreover, the low affinity sites constitute the dominant fraction (~82%) of the ethidium sites in
native chromatin and are present in formaldehyde-fixed chromatin. It thus seems likely that the mobile species observed here is ethidium that diffuses after release from low
affinity sites. This interpretation is strengthened by the
fact that our mobile fractions (~64-73%) are in reasonable agreement with the fraction of sites in native chromatin that have a low affinity for ethidium bromide.
0.4 µm distance scale.
; Mathog et al., 1984
; Manuelidis and Borden, 1988
; Dyer et al., 1989
). Similarly, chromosomal
confinement to domains has been observed or deduced
from in situ hybridization studies and DNA irradiation
studies (Cremer et al., 1982
; Hens et al., 1983
; Schardin et al.,
1985
; Cremer et al., 1988
; Lichter et al., 1988
; Manuelidis
and Borden, 1988
; Pinkel et al., 1988
; Leitch et al., 1990
;
Popp et al., 1990
). Finally, active genes can exhibit nonrandom organization, concentrating in the nuclear periphery
of mouse L and P19 embryonal carcinoma cells and near the borders of condensed chromatin in nucleated newt
erythrocytes (Hutchison and Weintraub, 1985
; de Graaf
et al., 1990). These examples all suggest constraints on
chromosome lateral mobility during interphase.
). Motion of centromeres was tracked by labeling centromeres with a fusion protein consisting of green fluorescent protein and
CENP-B, a centromeric satellite DNA-binding protein.
Most centromeres were found to remain motionless for a
time period of up to 2 h.
; Bartholdi, 1991
; Ferguson and Ward,
1992
; Janevski et al., 1995
; Shelby et al., 1996
), as have
X chromosome centromeres in certain pathological states
(Borden and Manuelidis, 1988
). Finally, chromosome arms in polytene nuclei (Hochstrasser and Sedat, 1987
) and
neuronal cells (Manuelidis and Borden, 1988
) can exhibit
some randomness in configuration and position.
; Nickerson et al., 1995
). Similarly, chromatin in cultured cells
may partially expand to a more euchromatic state (Manuelidis, 1990
). However, because these potential changes
in chromatin structure would probably tend to "loosen
up" chromatin and increase its mobility, our results should
represent a lower bound on the immobility of interphase chromatin in cells in general.
0.4 µm
Distance Scale
;
Manuelidis, 1990
; Taniura et al., 1995
). Such attachment
will certainly hinder chromatin motion; moreover, it might
completely immobilize chromatin if the attachment is extensive enough. Alternatively, less extensive attachment
might also lead to immobilization if interphase chromatin is relatively rigid, and segmental motion between attachment sites thus cannot occur on the
0.4 µm distance
scale. Indeed, some data suggest that interphase chromatin is quite rigid (Selvin et al., 1990
), making the latter
mechanism more plausible.
). In the matrix model,
interphase chromatin is organized into looped domains on
the order of 0.25 µm in size and containing 5-200 kb of DNA
(Nelson et al., 1986
; Davie, 1995
). Because these tethered
loops are smaller than the FRAP spot, segmental motion
of the loops would not be expected to produce a FRAP recovery. Thus, the immobility observed in the FRAP experiments is also consistent with the tethering postulated in
the matrix model.
; Selvin et al., 1990
),
including other chromatin. These steric interactions will
also impede mobility (Lang et al., 1986
; Scalettar et al.,
1989
); however, it has been shown experimentally (Lang
et al., 1986
; Scalettar et al., 1989
; Hou et al., 1990
) and theoretically (Scalettar et al., 1988a
; Abney et al., 1989
) in a
variety of systems that steric interactions usually do not
lead to complete immobilization of an untethered species.
For example, dextrans in nuclei (Lang et al., 1986
), tracer
particles in the cytoplasm of Swiss 3T3 cells (Luby-Phelps et al., 1987
; Kao et al., 1993
), and proteins in biological
membranes that are not attached to cytoskeletal components (for review see Jacobson et al., 1987
) all diffuse
translationally, albeit more slowly than in dilute solution.
These examples all suggest that steric interactions, in the
absence of some attachment, are unlikely to produce complete molecular immobilization.
9 cm2/s to 10
8 cm2/s (Thomas et al.,
1980
; Scalettar et al., 1989
). Associated FRAP recovery
times are on the order of 100 ms, assuming the spot radius is 0.4 µm. In contrast, the FRAP recovery times reported
here for interphase chromatin are
1 h. This and the overall immobility of the FRAP spot imply that interphase
chromosomes, which occupy discrete micron-sized domains,
diffuse at least four orders of magnitude more slowly than
comparably sized DNA molecules.
, 1988). Significantly, individual
chromosomes appear not to be entwined (for reviews see
Manuelidis, 1990
; Jackson, 1991
; van Driel et al., 1995),
suggesting that entanglement does not have a major effect
on chromatin motion. Steric interactions can also produce
such major inhibition of mobility if the system contains a
very high concentration of immobile obstacles that block
all potential paths for motion (Luby-Phelps et al., 1987
, 1988; Saxton, 1993
). However, if such a situation exists in
interphase nuclei, it would once again suggest that immobile nuclear substructures extensively bind nuclear constituents, thereby producing a high concentration of immobile
obstacles.
Conclusions
0.4 µm.
This immobilization may reflect motion-impeding steric
interactions that arise in the highly concentrated nuclear
milieu or outright attachment of the chromatin to underlying nuclear substructures, such as nucleoli, the nuclear
lamina, or the nuclear matrix. However, independent of its
origin, this immobilization ensures that spatial organization of chromosomes within interphase nuclei is maintained, as postulated in current models of a highly ordered
interphase nucleus.
Received for publication 27 December 1996 and in revised form 25 March 1997.
1. Abbreviation used in this paper: D, diffusion coefficient.We thank Dr. John Langmore of the University of Michigan for a critical reading of the manuscript.
This work was supported by grants CC3819 from Research Corporation (to B.A. Scalettar), BIR-9510226 from the National Science Foundation (to B.A. Scalettar), and MCB-9405298 from the National Science Foundation (to D. Axelrod).
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