ICANCER RESEARCH 46, 4738-4745, September 1986]
Effect of Growth State and Heat Shock on Nucleolar Localization of the
110,000-Da Heat Shock Protein in Mouse Embryo Fibroblasta1
Thung-Tai Shyy, John R. Subjeck,2 Roy Heinaman, and Garth Anderson
Division of Radiation Biology and Department of Cell and Tumor Biology, Roswell Park Memorial Institute, Buffalo, New York 14263
ABSTRACT
We have shown previously that the mammalian 110,000-Da heat shock
protein (hspllO) associates with nucleoli in several cell types and that in
2-day postconfluent mouse KM'1; cells, a segregation of the antigen from
the nucleolar phase-dense body is seen (J. R. Subjeck, T. Shyy, J. Shen,
and R. J. Johnson. J. Cell Biol. 97:1389-1395, 1983). Here we further
characterize the nucleolar segmentation of hspllO in mouse lOT'/z and
3T3 cells with respect to the formation of this structure in dense cultures
and investigate the behavior of this protein following conditions (serum
deprivation, actinomycin D, and heat shock) known to affect the func
tional and morphological integrity of the nucleolus. It is shown that in
addition to its nucleolar locale, an affinity of hspl 10 for the nonnucleolar,
nuclear compartment in actively proliferating cells is also observed. When
proliferating cells are treated with actinomycin D (l Mg/ml) for 8 h,
hspl 10 separates from the nucleolar phase-dense body to form a fluores
cent nucleolar cap which resembles that seen in confluent cultures. This
drug also results in a disappearance of hspllO from the nucleoplasm.
Incubation of cells for 24 h in media without serum also results in the
nucleolar segmentation of hspllO and a reduction in nucleoplasmic
staining. A moderate nonlethal heat treatment does not lead to segmen
tation of hspl 10 in proliferating cells but conversely results in a transient
reversal of segmentation in confluent cultures. Examination of segmented
nucleoli of postconfluent cells by immunoelectron microscopy reveals that
hspllO is associated with the fibrillar component of these nucleoli, the
site of ribosomal DNA.
INTRODUCTION
Although many studies have focused on the molecular mech
anisms responsible for the induction of heat shock proteins as
well as their function and intracellular localization, the role of
these proteins and of the heat shock response is still unclear
(reviewed in Refs. 1-3). Earlier studies using cell fractionation,
autoradiography, and microdissection have shown that some of
the heat shock proteins exhibit a nuclear localization (4-7),
suggesting a nuclear role for these proteins. We have previously
described the preparation of a polyclonal antiserum against the
major mammalian heat shock protein at 110,000 Da and have
demonstrated that this antigen is associated with nucleoli in
several cell types (8). In addition, antisera against another
mammalian heat shock protein approximately 70 kDa (9, 10)
indicates that this heat shock protein exhibits an affinity for
nucleoli. The nucleolar localization of two different mammalian
heat shock proteins suggests that aspects of nucleolar function
may be associated with the role(s) which these proteins play in
the cell.
It is also recognized that the nucleolus is highly sensitive to
changes in environmental conditions, i.e., temperature ( 11-14),
growth state (15-17), and certain drugs (18), all of which can
modify its functional and morphological integrity. Therefore it
is of interest to determine if such conditions can affect the
staining pattern of anti-hspllO3 as a nucleolar marker. This
Received 12/23/85; revised 5/6/86; accepted 6/10/86.
The costs »Ipublication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by USPHS Grant 40330.
1To whom correspondence and requests for reprints should be addressed.
1The abbreviations used are: hsp, heat shock protein; PBS, phosphate-buffered
saline without calcium and magnesium; TBS, Tris-buffered saline.
study presents an analysis of the effects of heat shock, growth
state, serum deprivation, and actinomycin D on the nucleolar
localization of the hspl 10 antigen in mouse embryo fibroblasts.
It is demonstrated that these conditions and treatments signif
icantly affect both the abundance of the antigen in the nucleus
and its nuclear/nucleolar distribution.
MATERIALS
AND METHODS
Cell Culture. Mouse C3H 10T'/2 fibroblasts (19) originally obtained
from Dr. John Bertram (University of Hawaii) were cultured as monolayers on glass coverslips (18 x 18 mm) in plastic Petri dishes (Corn
ing) and incubated at 37°Cin a humidified 5% CO2 atmosphere. The
medium used was Eagle's basal medium with Earle's salts [Gibco
Laboratories, Grand Island, NY] supplemented with 10% heat-inacti
vated fetal bovine serum [Gibco] and antibiotics [penicillin G (50 ^g/
ml), streptomycin (50 fig/ml), and neomycin (100 fig/ml) (Gibco)].
Cultures were seeded at 2 x IO4 cells/100-mm plate. Swiss mouse
embryo 3T3 cells (20) were plated at 2 x 10s cells on glass coverslips
in a 100-mm Petri dish with Dulbecco's modified Eagle's medium
(Gibco) supplemented with 10% calf serum (Gibco). Cultures were
maintained at 37°Cin a 10% CO; environment.
For heat shock experiments, Petri dishes containing coverslips were
heated by placing the dishes 7-8 mm deep in a water bath (Haake III
2) on the surface of an aluminum frame which allowed contact between
the dish bottom and the bath. In this way dishes were exposed to a
temperature of 45"C ±0.1'C (SD) for 15 min and then postincubated
at 37"C as required. For serum deprivation experiments, cells were
grown with complete medium for 3 days followed by incubation in
serum-depleted medium for 24 h. Actinomycin D (Sigma) was used at
a concentration of 1.0 fig/ml. Following serum deprivation cells re
sumed normal growth after addition of serum and exhibited normal
plating efficiency. Actinomycin D exposure did not significantly alter
cell morphology during the exposure but led to a significant reduction
in attached cells by 72 h posttreatment.
Antibody. Rabbit antiserum raised against hspl 10 was prepared and
characterized as described previously (8).
Indirect Immunofluorescence. Coverslips containing the cells were
washed in Hanks' balanced salt solution (Gibco) and fixed in 2%
formaldehyde in PBS for 20 min at room temperature. They were then
rinsed in PBS followed by extraction in PBS containing 0.5% Triton
\ KM)for 30 min at room temperature. Ani i hspl 10 antiserum at a
1:40 dilution was then added to the coverslips and they were incubated
in a moist chamber for 60 min at room temperature. The coverslips
were then washed again in PBS, following which a 1:150 dilution of
fluorescein-conjugated goat anti-rabbit IgG (Miles-Veda, Rehovot, Is
rael) was added and incubated again at room temperature for 45 min.
Coverslips were then rinsed well and mounted in Elvanol (21). Cells
were examined using a Zeiss photomicroscope II (Carl Zeiss, Inc.,
Thornwood, NJ) and photographed using Kodak Tri-X Pan film (East
man Kodak Co., Rochester, NY) with the film speed automatic control
set at ASA 400.
Immunoelectron Microscopy. Postconfluent cells were used in this
study. Cells were fixed for 20 min in l % glutaraldehyde-Puck's solution
(0.11 mM CaCI2-5.4 mM KCI-1.1 mM KH2PO4-0.6 mM MgSO4-0.14 M
NaCl-1.1 mM Na2HPO4-6.1 mM D-glucose). The cells were then washed
in Puck's solution and dehydrated in a graded ethanol series. After
dehydration the cells were transferred to 100% 2-propanol and em
bedded in Epon. Silver thin sections were cut and mounted on nickel
grids and processed for colloidal gold labeling. Etching was not per
formed on these sections. For labeling the nickel grids containing thin
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sections of cells were incubated for 30 min on a drop of 0.02 M TBS
containing 0.5% bovine serum albumin. The girds were then transferred
to 1 drop of 1:40 diluted primary antiserum and incubated for 2 h at
room temperature. After a washing in TBS the girds were labeled with
1:10 diluted S-nm goat anti-rabbit colloidal gold (Janssen Pharmaceutica. Life Sciences Products Division) for 60 min at room temperature.
OF hspllO
They were then thoroughly washed in TBS with 0.5% bovine serum
albumin, rinsed in distilled water, and dried. Staining with 5% aqueous
uranyl acetate and Reynold's lead citrate was performed before exami
nation under a Siemens Elmiskop 1A electron microscope. In order to
demonstrate the specificity of the labeling, control studies using preim
mune serum were performed.
Fig. 1. Rearrangement of h-.pl 10 as Idi ' .•
cells reach a confluent state. Sequential changes of phase contrast (a, c, <•)
and corresponding hspl 10 fluorescence (b,
d, /) in growing and confluent lO'l ' •
cells. In cells in exponential growth state (a, b), hspl 10 fluorescence is uniformly distributed in the phase-dense body. As the
cells become initially confluent (c, </). hspl 10 localizes with a phase-dense ring structure. After 48 h of confluency (<•,/).hspl 10 migrates into a phase-lucent cap
attached to a phase-dense structure to form the segmented nucleolus. Note general nucleoplasmic staining. Arrows, some sites where anti-hspllO appears to react.
Bar, 7.5 urn.
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OF hspllO
Fig. 2. Effect of heat shock on the localization of
hspllO in actively proliferating in I'.- cells, a, c, phase
contrast; b, d, hsp 110 fluorescence; a, b, control; r, </.heatshocked cells. Nucleoli are round and of reduced size after
heat. Bar, 7.5 urn.
RESULTS
Sequential changes in phase density and nuclear patterns of
hspl 10 fluorescence in lOT'/z cells as they reach confluency are
shown in Fig. 1. During exponential growth the nucleoli appear
large and irregular in shape, containing small light vacuoles
(Fig. la). At this stage fluorescent localization of the hspllO
antigen coincides exactly with the phase-dense body (Fig. Ib).
However, as cells reach a confluent, contact-inhibited state, the
nucleoli concurrently undergo major structural alterations and
a rearrangement of hspl 10 is seen to occur (Fig. 1, c and d).
During this phase, nucleoli become more spherical and smaller.
Occasionally phase-dense patches appear to migrate to the
periphery of the nucleolus and seem to exclude the fluorescent
stain and in some instances an affinity of hspllO for the
periphery of the nucleolus is observed (a ring-like structure). In
many cells a total nucleolar segregation of hspl 10 fluorescence
from the phase-dense body is evident, even at these early stages
of confluency. As a period of time of postconfluency increases
(24 to 48 h) the segregation of the hspllO fluorescent caps
becomes more obvious and in most instances the phase-dense
bodies generally exclude this antigen (Fig. 1, e and/). However,
not all nuclear phase-dense structures interact with anti-lisp 110.
In addition to the studies on lOT'/z cells, Swiss mouse embryo
3T3 cells also exhibit an identical pattern of nucleolar segmen
tation (data not shown) of hsp 110 fluorescence which occurs
concomitantly with the onset of confluency in this contactinhibited cell line.
In addition to the specific fluorescent staining of the nucleo
lus, the extranucleolar nucleoplasm of both cell lines also clearly
binds the antisera, although to a reduced degree. The nucleoplasmic staining observed in growing 10T'/2 and 3T3 cells is
brighter than that found in confluent cells, indicating an en
hanced accumulation of hspllO in this compartment during
growth.
The nucleolus is thought to be a particularly sensitive nuclear
site to supranormal temperatures (13, 14, 22). Cells in expo
nential phase of growth or in a postconfluent state were heated
and the nucleolar/nuclear localization of hsp 110 was examined.
In the case of actively proliferating cells the coincident pattern
of hspllO fluorescence with the phase-dense body is unaltered
(Fig. 2). However, the nucleoli appear to be round and of a
reduced size after heat. When postconfluent cells are subjected
to a heat treatment, the segmented nucleoli observed in this
situation are seen to undergo a marked change (Fig. 3). The
fluorescent cap exhibited by confluent cells (Fig. 3, a and h) is
eliminated by heat shock and the phase and fluorescent bodies
are again observed to coincide. This disappearance of segrega
tion is observed as early as 1 h after heat shock and continues
up to 8 h postshock (Fig. 3, c and d). However, at later times
(8 h+, postshock) nucleoli once again begin to segregate and
the fluorescent cap reappears (Fig. 3, e and/). This disappear
ance and reappearance of segregation of hspl 10 in response to
heat shock correlates with the induction and repression phases
of the heat shock response as determined using a pulse label
analysis of protein synthesis (data not shown). In addition,
shortly after heat treatment, a distinct increase in nuclear
fluorescent staining, indicative of a rapid accumulation of
hspl 10 in the nucleus, is observed (Fig. 3d). However, at later
times when the response has subsided, the nucleoplasm is again
observed to exhibit a diminished fluorescence (Fig. 3/), sug
gesting a reduction of hspl 10 levels in the nucleus. Thus the
presence of coincident nucleoli appears to predict enhanced
nucleoplasmic hspl 10 levels.
Both confluent and serum-depleted cells are considered to be
arrested in the Gìphase of the cell cycle (16, 23), in a state of
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OF hspllO
Fig. 3. Effect of heat shock on the localization of hspllO in postconfluent IO! '•.•
cells, a, c, e, phase contrast; b, d,f, hspllO fluorescence. Nucleolar segmentation
(a, b) disappears at 1 to 8 h after heat shock (c, d) and then reappears at times exceeding 8 h postshock (e, /). Note general nucleoplasmic staining. Arrows, some
sites where anti-hspl 10 reacts. Bar, 1.5 urn.
reduced rRNA synthesis (16,17). The effect of serum depriva
tion on nucleolar localization of hspl 10 in exponential growing
lOT'/z cells is shown in Fig. 4. Cells are grown with complete
medium for 3 days and then switched into a serum-depleted
medium for 24 h. Under normal growth conditions the hspl 10
fluorescence is observed to precisely coincide with the phase-
dense structure (Fig. 4, a and b) as described above. However,
in serum-starved cells which are subconfluent, a segregation of
fluorescence from the phase-dense body is again seen (Fig. 4, c
and d), consistent with the above study of postconfluent cells
(Fig. 1, e and /). Therefore, arrest of the cell cycle by serum
deprivation leads to a similar morphology with respect to
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OF hspllO
Fig. 4. Effect of serum deprivation on the nucleolar localization of hspl 10 in growing lOT'/i cells, a, c, phase contrast; b, d, hspl 10 fluorescence. Exposing growing
cells to serum deprivation Tor 24 h. the coincident hspl 10 fluorescent pattern with the phase-dense body (a, b) undergoes a segregation phenomenon (c. ,I\ similar to
that observed in postconfluent cells. Arrows, some sites where anti-hspl 10 reacts. Bar, 7.5 ¡an.
hspl 10 localization as is seen in postconfluent, contact-inhib
ited cells. Low doses of actinomycin D have been shown to
inhibit rRNA synthesis (18). When actinomycin D at a concen
tration of 1 Mg/ml is added to a subconfluent, proliferating
population of cells for 8 h, a segregation of hspl 10 fluorescence
and the phase-dense body is once again observed (data not
shown), identical to the data obtained for serum deprivation
and postconfluency. In addition, intermediate degrees of seg
mentation, analogous to that seen in Fig. 1, occur at shorter
time intervals.
Segmentation has been shown previously to separate nucleoli
into two ultrastructural elements, the fibrillar and granular
segments, as presented by others (18). To ultrastructurally
examine the localization of hspl 10 in segmented nucleoi, post
confluency was chosen as the method of inducing nucleolar
segmentation. Fig. 5a presents a typical electron micrograph of
a segregated nucleolus from 48-h postconfluent lOT'/z cells,
which has been reacted with the antibody and labeled with
colloidal gold. The gold staining is found exclusively on the
densely uranyl acetate-lead citrate-stained nucleolar segment
which strongly resembles the fibrillar component recognized by
others (18), while no binding is observed in the less electrondense granular segment. Control specimens utilizing preim
mune serum showed no specific or significant labeling of either
nuclear or cytoplasmic structures (Fig. 5¿>).
Therefore the seg
regated fluorescent hsp 110 cap structure described above ap
pears to represent the nucleolar fibrillar element. In addition
to this association, gold may also associate with other nuclear
electron-dense structures.
DISCUSSION
Several laboratories have recently used an immunological
approach to study the localization and behavior of specific heat
shock proteins. Valazquez and Lindquist (24) prepared a mono
clonal antibody against the Drosophila 70-kDa hsp and have
demonstrated that it moves into the nucleus during periods of
stress. Welch and Feramisco (10) have shown that the mam
malian nonconstitutive hsp near 70 kDa is found transiently in
the nucleolus after heat shock and Pelham (9) has recently
demonstrated a similar effect at the mammalian level using a
Drosophila monoclonal antibody. In addition to these studies
we have reported (8) that mammalian hspl 10 occupies a nu
cleolar site in proliferating cells in the absence of heat shock
and that this hsp separates from the phase-dense component of
the nucleolus in 2-day postconfluent lOT'/z cells. We demon
strate in this study that the initiation of this segregation occurs
as cells begin to reach confluency and proceed through certain
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OF hspllO
•500
500
n m
nm
Fig. S. Immunoelectron micrography of a segregated nucleolus from a 48-h postconnuent 10l1: cell. In a, staining with colloidal gold is found exclusively on the
fibrillar component (/I and no staining is observed in the granular component (G). Gold is also bound to other nuclear structures (arrows), b, corresponding
preimmune control.
transition forms which precede total segregation. It is also
shown that other conditions inhibitory to RNA synthesis also
result in a similar change in nucleolar morphology. Interest
ingly, this segmented nucleolus is not a terminal structure but
readily reverts to a coincident state by a mild (nonlethal) heat
shock, following which the segmented form reappears. These
rapid changes, which appear to result from changes in nucleolar
activity, suggest a highly labile or reversible association between
the hspl 10 and an active nucleolar structure. Immunoelectron
microscopy additionally indicates that during the quiescent
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NUCLEAR
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phase hspl 10 is associated with the component of the nucleolus
which is the locale of ribosomal DNA.
We have shown here that both serum deprivation and actinomycin D, two conditions known to affect the morphological
and functional integrity of the nucleolus, also induce significant
changes in the localization of anti-hspl 10. In addition to these
conditions, heat shock itself interferes with nucleolar function
and would therefore be expected to result in a similar inhibitory
effect (18, 22) in proliferating cells as described above using
actinomycin D and serum deprivation. We show here that not
only does heat have little effect on the nucleolar localization of
hspl 10 in proliferating cells, but during the recovery period it
actively reverts the inactive nucleolar morphology observed in
confluent cultures to a coincident form. In contrast to this,
Welch and Suhan (25) have recently reported that heat shock
results in the redistribution of hspl 10 (utilizing the same antisera as described here) to form a "ring-like" structure in which
antigen is concentrated at the nucleolar periphery. A similar
ring pattern is also described in this report as a transition form
leading to complete nucleolar segmentation in confluent cells.
Our study utilizes a "slow transition" 15-min, 45°Cshock
(which is nonlethal) and examines the cells following various
recovery times at 37°C,while the study of Welch and Suhan
examines cells immediately following a 42°C,3-h exposure.
Thus our study describes morphology associated with recovery
and that of Welch and Suhan describes morphology during the
heat exposure. It is likely that longer heat exposures than those
used in this report would also result in one or more forms of
segmentation morphology, when examined immediately follow
ing the exposure.
While hspl 10 expresses a strong affinity for nucleoli, a
significant affinity of the antisera for the nucleus as a whole
was also observed. The level of this antigen in the nucleoplasm
reflects the state of segmentation of the nucleolus. As cells
reach confluency (or are subject to serum deprivation or acti
nomycin D), general nucleoplasmic staining is significantly
diminished. The level of hspl 10 in the nucleus is also briefly
increased by a mild heat exposure of confluent cells. Welch and
Feramisco (10) report that the nonconstitutive hsp at approxi
mately 70 kDa is observed in the nucleolus after heat shock,
from which it appears to migrate to a secondary site in the
nucleus. A similar phenomenon was also observed in a study
by Pelham (9). All of these studies are reminiscent of the earlier
work of Roti Roti and Winward (26) who showed a significant
increase in protein:DNA ratio after heat shock. Although
hspl 10 is observed in the nucleolus and nucleoplasm in prolif
erating cells in the absence of heat, perhaps a continuous
migration of hspl 10 through the nucleolus into the nucleo
plasm is occurring.
In addition to the nucleolar locale of hspl 10, a major nucleo
lar protein designated C23 by Orrick et al. (27) has a molecular
size of 100,000 to 110,000 Da and contains several phosphorylated and highly acidic regions (28). Protein C23 is a major
nucleolar silver-staining protein (29), is localized in the fibrillar
structure of segregated nucleoli (30), and possesses properties
which suggest that it may have an organizational role (31, 32).
In comparison, hspl 10 is released from the nucleus following
digestion with RNase (8). We have recently compared C23 with
hspl IO4and have shown by immunochemical and peptide map
OF hspllO
second component of a hspl 10 gene family, analogous to the
situation already known to occur in the case of hsp70 (reviewed
in Ref. 2). Kistler et al. (33) have recently described a monoclo
nal antibody which cross-reacts with a 110-kDa nucleolar pro
tein in HeLa cells. We have shown that this antibody reacts
with hspl 10 and stains nucleoli of mouse lOT'/z cells in a
manner which parallels that described for the polyclonal antisera studied here.5
It is well known that the major function of the nucleolus is
the biogenesis of rRNA. Numerous proteins seem to participate
in this synthesis and processing of pre-rRNA (34) or are in
volved in maintaining the structural integrity of nucleolus (35).
Segregation of nucleoli into separate granular and fibrillar
components following application of drugs, such as D-galactosamine (36, 37) or actinomycin D (18), has been well studied. In
addition a similar phenomenon appears to occur during the
differentiation of frog erythroblasts (38) and during maturation
of human epithelial cells (39). This segregation phenomenon
appears to reflect a state of relative nucleolar inactivity in rRNA
accumulation (16, 40). Thus, the specific and reversible rear
rangement of hspl 10 during this segregation phenomenon sug
gests that an association between hspl 10 and rRNA synthesis
or processing may occur. Since the fibrillar component of the
nucleolus is believed to be a transcription unit, hspl 10 may be
associated with actively transcribed rRNA genes.
ping criteria that while C23 and hspl 10 are clearly distinct
proteins, they share a limited degree of homology. While it
remains to be determined, it is possible that C23 represents a
4 T-T. Shyy, M. O. J. Olson, and J. R. Subjeck, manuscript in preparation.
ACKNOWLEDGMENTS
We appreciate the expert advice of Dr. Jennifer Black.
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4745
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1986 American Association for Cancer Research.
Effect of Growth State and Heat Shock on Nucleolar
Localization of the 110,000-Da Heat Shock Protein in Mouse
Embryo Fibroblasts
Thung-Tai Shyy, John R. Subjeck, Roy Heinaman, et al.
Cancer Res 1986;46:4738-4745.
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