[CANCER RESEARCH 40, 4552-4564,
0008-5472/80/004
0-OOOOS02.00
December 1980]
Accumulation of Actin Microfilaments in Adult Rat Hepatocytes Cultured
on Collagen Gel/Nylon Mesh1
W. Wai-Nam Mak, Carol A. Sattler, and Henry C. Pilot2
McArd/e Laboratory for Cancer Research. University of Wisconsin, Madison, Wisconsin 53706
ABSTRACT
Adult rat hepatocytes cultured on a collagen gel/nylon
mesh support were shown previously to exhibit fetal properties
as a function of time in culture and to accumulate numerous
microfilament structures as seen with electron microscopy. The
present study characterizes the accumulation and properties
of such microfilaments. Electron microscopy revealed that microfilaments, 40 to 60 A in diameter, accumulated as a compact
network almost exclusively beneath the plasma membrane at
the interface with the culture medium. Treatment with heavy
meromyosin resulted in the formation of the characteristic
arrowhead complexes on these microfilaments, indicating that
they contain actin. Extraction of glycerinated hepatocytes with
low-ionic-strength
solutions removed most of the microfilaments, which could also be disrupted by treatment of the cells
with cytochalasin B. The disruption was reversible, and reap
pearance of the network of microfilaments, though less com
pact, was observed as early as 30 min after removal of cyto
chalasin B. At later times in culture, the microfilaments devel
oped into a more compact and extensive network, and their
reappearance was not dependent on protein synthesis. Sodium
dodecyl sulfate-polyacrylamide
gel electrophoresis of proteins
extracted from hepatocytes labeled with L-[35S]methionine or
from acetone powders of the labeled hepatocytes showed a
major band of a molecular weight of 42,000 that comigrated
with skeletal muscle actin on the sodium dodecyl sulfate-poly
acrylamide gels. The amount of radioactivity in this band in
creased with the age of the hepatocyte culture; this indicated
an increase in the synthesis of the actin-like protein as well as
a greater degree of polymerization into microfilaments. This
finding was further confirmed by the demonstration of an
increase in the proportion of the filamentous form of actin in
the late cultures by means of a DNase I assay. The studies
reported in this paper indicate that (a) the microfilaments in
cultured hepatocytes contain actin, with properties similar to
those found in other nonmuscle cells, and (b) the large accu
mulation of microfilaments is due in part to the synthesis of
actin and the formation of new microfilaments.
INTRODUCTION
In many eukaryotic cells, a cytoskeletal
system of microfila-
ments and microtubules provides the mechanochemical basis
for diverse cellular activities, including the maintenance of cell
shape, cytoplasmic streaming and saltatory movements, endocytosis and secretory processes, cell division, regulation of
the topographical distribution of membrane proteins, and pos' Supported in part by grants from the National Cancer Institute (CA-07175
and CA-22484).
2 To whom requests for reprints should be addressed.
Received April 10, 1980; accepted September 15, 1980.
4552
sibly the functional interconnection of the cell membrane with
the nucleus (for reviews, see Refs. 10, 12, 24, 31, 42, 46, 51,
58, 63, and 67), as well as the establishment of antiviral states
(7). The components of this cytoskeleton in nonmuscle cells
have been extensively studied, and the microfilaments have
been demonstrated to be similar in structure and in function to
those in muscle cells (1, 38) and the microtubules similar to
those in cilia and flagella (1, 55). To date, most studies of the
cytoskeletal system have been carried out in cultured cells of
mesenchymal origin, especially fibroblasts. Investigations of
the cytoskeletal system of hepatocytes in vivo and in vitro have
been reported (14, 41, 53). In addition, other secretory cells
including the /8-cells of the islets of Langerhans (18, 47) and
adrenocortical cells (17) contain a network of microfilaments
at the periphery of their cytoplasm. In hepatocytes, such a
network is particulary evident around bile canaliculi (4, 43, 69);
this network contains actin (15, 20, 43, 65).
The development in this laboratory of a floating collagen gel
substratum (39) and a collagen gel/nylon mesh substratum
(59) for hepatocyte cultures has provided a means to maintain
adult rat hepatocytes in stationary culture for up to 2 weeks
(33); this allows functional studies of viable differentiated hep
atocytes in vitro for relatively extended periods. Microfilament
structures accumulate beneath the apical cell surface of adult
rat hepatocytes cultured either on floating collagen gels (56)
or on collagen gel/nylon meshes (59). To understand the
underlying mechanism of the accumulation of these microfilament structures in hepatocytes cultured on collagen gel/nylon
mesh, we have attempted to establish and characterize the
identity and accumulation of these structures.
MATERIALS
AND METHODS
Cell Culture Conditions. Hepatocytes were isolated from
normal adult male albino rats (Holtzman; 180 to 220 g) by a
collagenase perfusion method, as described previously (39,
59). The cells were plated at a concentration of 10 to 12 x 106
cells in 10 ml into 100-mm Falcon tissue culture dishes con
taining collagen gel/nylon meshes (mesh pore diameter, 0.25
mm) (59). Cultures were maintained at 37° in a humidified
incubator with temperature-controlled
air flow. Medium used
for the cultures was Leibovitz (L-15) medium, pH 7.4, supple
mented with 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic
acid (18 ITIM), albumin (2 mg/ml), penicillin (100 units/ml),
streptomycin (100 jug/ml), insulin (0.5 /¿g/ml), and glucose
(1.5 mg/ml) (hereafter called L-15 culture medium). For the
first 4-hr period of attachment of the hepatocytes to the gel/
mesh substratum, the L-15 culture medium was supplemented
with 5% fetal bovine serum (KC Biological, Inc.). After the 4-hr
period, the meshes were transferred to culture dishes contain
ing 10 ml of fresh L-15 culture medium at 37°,with or without
other reagents. After a further incubation at 37°for 20 hr, the
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
Microfilament
"Day 1" cultures
had a medium change. The medium was
changed every 24 hr thereafter.
The hepatocyte cultures were harvested by the use of collagenase (59). The gel/meshes were first washed 3 times with
PBS,3 pH 7.4, and incubated at 37°for 10 min with 5 ml of a
solution of collagenase (1 mg/ml; Worthington Biochemical
Corp.)-soybean trypsin inhibitor (0.1 mg/ml, Sigma Chemical
Co.) in PBS. The released cells were collected by centrifugation
at 200 x g for 5 min at 4°,washed twice with cold PBS, and
stored frozen at —70°for subsequent analyses.
Glycerination of the Hepatocytes. The hepatocytes were
glycerinated according to the method of Ishikawa ef al. (30).
The hepatocyte culture was washed twice with 5 ml of SSS
and glycerinated in situ at 4°for 24 hr with 50% glycerol/SSS,
then for 12 hr with 25% glycerol/SSS,
and finally for 12 hr
with 5% glycerol/SSS. The glycerinated culture was stored in
5% glycerol/SSS containing 0.1% sodium azide before use.
Decoration of the Microfilaments with HMM. The glycerin
ated hepatocytes were incubated for 12 hr at 4°with HMM
solution diluted immediately before use with an equal volume
of SSS. HMM was kindly supplied by Dr. L. F. Lemanski,
Department of Anatomy, University of Wisconsin-Madison. The
HMM solution was prepared as described (36) and contained
10 to 12 mg protein per ml in 25% glycerol/SSS.
Control
samples (from the same hepatocyte culture) were incubated
for the same period of time at 4°in a solution of SSS either
without HMM or with HMM and 0.2 HIM ATP. After incubation,
the samples were rinsed for 1 hr in 0.1 M KCI before being
processed for electron microscopy (30).
Negative Staining with HMM. A 100-rnrn dish of hepatocyte
culture on a collagen gel/nylon mesh (containing 5 to 8 x 106
cells) was harvested with collagenase treatment and washed
twice with cold PBS. The pelleted hepatocytes were homoge
nized at 0°in 1 ml of SSS containing 1 mw EDTA by rapidly
drawing the mixture in and out of a 1-ml syringe without a
needle (30, 36). A drop of the homogenate or a drop of a lowspeed supernatant of the homogenate was placed on a Formvar-coated carbon-stabilized grid and treated for 1 min with a
drop of HMM solution (diluted immediately before use with 0.1
M KCI to approximately 1 mg protein per ml). The grid was
rinsed with several drops of 0.05 M KCI, stained 1 min with 1%
uranyl acetate, and examined at high magnification with a
Hitachi H-500 electron microscope. Controls were prepared
from the homogenates without the HMM treatment.
Extraction of Microfilaments. The glycerinated hepatocytes
were treated with a series of "actin depolymerization" solutions
according to Spooner era/. (60). The glycerinated hepatocytes
were incubated at 4°for 12 hr successively with the following
solutions: (a) distilled water; (b) distilled water containing ATP
(0.2 mw); (e) Tris buffer [2 mw Tris-HCI, 0.2 mw CaCI2 (pH 8.0)]
containing ATP; and (d) SSS without KCI [6 mw potassium
phosphate buffer, 5 mM MgCI2 (pH 7.0)]. The control was
prepared by incubating the glycerinated hepatocytes with SSS
at 4°for 48 hr. After incubation, all samples were processed
for electron microscopy.
Cytochalasin B Treatment and Recovery. A Day 8 culture
3 The abbreviations used are: PBS, phosphate-buffered saline [per liter: NaCI.
8g; KCI. 0.2 g; Na2HPO4, 1.15g; KH2PO<, 0.2 g; CaCI2-2H20, 0.132 g; MgCI26 H2O, 0.1 g (pH 7.4)]; SSS, standard salt solution [100 mM KCI, 5 mM MgCI2,
6 mM potassium phosphate buffer (pH 7.0)]; HMM, heavy meromyosin; SDS,
sodium dodecyl sulfate.
of hepatocytes
Accumulation
in Cultured Hepatocytes
was treated for 1 hr at 37° with 10 ml L-15
culture medium containing 20 jtiM cytochalasin B (Aldrich
Chemical Co.). After treatment, the cultures were washed twice
with warm L-15 culture medium and then incubated at 37°in
L-15 culture medium with or without additional reagents. This
period of incubation was arbitrarily termed "recovery."
At
different time points of the recovery, a sample was removed
for electron microscopy by cutting a section out of the collagen
gel/nylon mesh culture.
Electron Microscopy. The hepatocyte culture after experi
mental treatment was fixed in 3% glutaraldehyde, postfixed
with 2% osmium tetroxide, dehydrated, and embedded in
Epon-Araldite. Thin sections of hepatocytes cultured on colla
gen gel/nylon meshes (mesh pore diameter, 0.25 mm) were
cut at an oblique angle closely parallel to the substratum
surface. The sections were stained and examined with a Hitachi
H-500 electron microscope. In some experiments in which the
hepatocytes were cultured on collagen gel/nylon meshes with
larger mesh pore diameter (1 mm), it was possible to orient the
cells during embedding so that nearly vertical sections through
the cell and substratum were obtained.
Radioactivity Labeling of Hepatocytes with L-[35S]Methionine. Hepatocyte cultures to be labeled were first washed 3
times with warm L-15 culture medium lacking methionine and
then incubated for 4 hr at 37°in 10 ml of L-15 culture medium
lacking nonradioactive methionine but supplemented with L[35S]methionine (New England Nuclear; 670 Ci/mmol; 1 juCi/
ml). Labeling for longer periods, up to 24 hr, did not alter the
results. After the labeling period, the medium was removed,
and the cultures were washed 3 times with cold PBS before
being harvested with collagenase as described above.
Protein samples of the total extracts of labeled hepatocytes
were prepared by boiling the cells at 100°for 2 min (32) with
O'Farrell's sample buffer (44) [2.3% SDS, 5% /î-mercaptoethanol, 10% glycerol, 0.0625 M Tris-HCI (pH 6.8)] and subse
quent clarification by centrifugation at 30,000 x g for 30 min.
Acetone powders of labeled hepatocytes were prepared by
successive extraction at 4° with water, 90% acetone, and
100% acetone. The dried acetone powders were extracted
with Buffer A [2 mM Tris-HCI, 0.2 mM ATP, 0.2 mw CaCI2, 0.5
mw /8-mercaptoethanol (pH 8.0)] as described by Spudich and
Watt (62). These extracts were prepared for gel analyses by
being adjusted to contain 1% SDS, 5% /8-mercaptoethanol,
and 10% glycerol.
SDS-Polyacrylamide Gel Electrophoresis and Fluorography. Protein samples were analyzed on 10% slab gels contain
ing SDS by the method of Laemmli (32). After electrophoresis,
the gels were stained with Coomassie brilliant blue and destained successively with 30% ethanol/7% acetic acid, 5%
ethanol/10% acetic acid, and 10% acetic acid. The destained
gels were prepared for fluorography according to Bonner and
Laskey (6). Radioactivity contained in the dried gel was de
tected with Kodak X-Omat R film at -70°. Exposure on the
film was quantitated with a microdensitometer (Joyce, Loebel
& Co., Inc.), the area under a peak on the densitometric tracing
being measured either by the weighing method or with a
planimeter.
DNase I Assay for the Monomeric and Filamentous Forms
of Actin. Preparation of the substrate, calf thymus DNA (type
I; Worthington), and the enzyme, bovine pancreas DNase I
(Worthington), and the assay for the monomeric and filamen-
DECEMBER 1980
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
4553
W. W-N. Mak et al.
tous forms of actin in the hepatocyte pellets harvested from
the cultures of different ages were carried out essentially as
described (5).
RESULTS
Adult rat hepatocytes maintained in culture on floating col
lagen gels (39) or collagen gel/nylon mesh supports (59)
exhibit many ultrastructural characteristics of hepatocytes in
vivo; in addition, they accumulate microfilaments in their apical
cytoplasm as a function of time in culture (56, 59). Fig. 1, a
and b, show typical patterns of well-developed networks of
microfilaments that have accumulated beneath the plasma
membranes of hepatocytes cultured as a monolayer for 8 and
10 days on collagen gel/nylon mesh supports. The accumu
lation of microfilaments begins in Day 3 cultures of hepato
cytes and progresses to complex networks with further time in
culture. Moreover, microfilament accumulation is localized
mainly beneath the plasma membranes interfacing with the
culture medium. Microfilaments are virtually absent beneath
the plasma membranes interfacing with the collagen gel sub
stratum. The complexity of this accumulation is manifested by
the densely stained microfilament clusters (Fig. 1a), their ex
tension into and interweaving with intermediate filaments in the
cytoplasm, as well as tonofilaments of the desmosomal com
plexes on the lateral plasma membranes of adjacent cells (Fig.
10). The characterization of these microfilaments, other than
by their size (40 to 60 A) as measured with the electron
microscope, has been further established by the following
studies.
Decoration of Microfilaments with HMM. The complex net
work of microfilaments accumulated in late cultures was found
to contain actin, as evidenced by HMM decoration (23, 30).
Late cultures, i.e.. Day 6, Day 8, or Day 10 cultures, were
selected for electron microscopic studies because of the pres
ence of a well-developed network of microfilaments in the
cultured hepatocytes. Fig. 2a shows a control sample of he
patocytes cultured for 6 days which still retain the network of
microfilaments after glycerination and incubation with SSS.
After treatment with HMM, individual microfilaments from either
Day 6 (data not shown) or Day 8 (Fig. 20) cultures of hepato
cytes display the characteristic
arrowhead complexes. Al
though the arrowheads on each microfilament are unidirec
tional, the overall polarity of the microfilaments appears to be
randomly oriented, mainly because of the complexity of the
interwoven network of microfilaments. Such complexity fur
ther aggravates the difficulty in estimating the periodicities of
the arrowheads. The HMM-decorated structures were reason
ably stable and did not dissociate after repeated rinsing in 0.1
M KCI solution. In the presence of ATP, however, the HMM
decoration was not observed (data not shown), since this
compound or pyrophosphate is capable of dissociating the
HMM-actin complexes (30). These cytochemical reactions es
tablished the microfilaments to be actin-containing.
Further
more, the negatively stained homogenates of a Day 6 culture
of hepatocytes (Fig. 3) corroborated the observations of the
sectioned samples seen in Fig. 2. Without HMM treatment, the
microfilaments were represented as filaments of approximately
40 A in diameter in the negatively stained homogenates (Fig.
3a), a size similar to those (40 to 60 A) measured in other cell
types. With HMM treatment, the arrowhead complexes were
4554
visible on the microfilaments. The periodicities of these arrow
heads were estimated to be approximately 400 A (Fig. 3b), an
interval similar to those (340 to 380 A) reported previously
(30).
Extraction of Microfilaments. To demonstrate the effects of
extraction of the network of microfilaments, a Day 10 culture
of hepatocytes with extensive networks of microfilaments was
chosen. Fig. 4a shows a control sample of a Day 10 culture of
hepatocytes glycerinated and incubated with SSS. The exten
sive network of microfilaments appeared to remain intact after
the incubation, similar to a Day 6 culture (Fig. 2a). Incubation
of glycerinated hepatocytes of the same Day 10 culture in lowionic-strength solutions (the series of "actin depolymerization"
solutions described in "Materials and Methods") resulted in
the disappearance of identifiable microfilaments (Fig. 4b).
Areas normally occupied by networks of microfilaments were
devoid of such networks, although other cellular organelles
were not removed by this treatment. In some hepatocytes,
however, the extraction was not sufficient to remove the entire
network completely, especially where it was very complex
before the extraction. In these areas, a light network remained
which was much less in quantity than in the control (Fig. 4a).
In addition, collagen fibrils of the substratum (not shown) and
many intermediate filaments (Fig. 4b) were not extracted; this
indicates that not all filamentous protein polymers could be
extracted by these treatments.
Cytochalasin B Treatment and Recovery. Hepatocyte cul
tures were examined for their sensitivity towards cytochalasin
B and their subsequent recovery from its effects. The treatment
with 20 juM cytochalasin B for 1 hr of a Day 8 culture resulted
in the disappearance of the network of microfilaments (Fig. 5a)
that normally accumulated beneath the plasma membrane in
terfacing with the culture medium (cf. Fig. 1a). Neither microfilaments nor compact masses derived from actin filaments (61 )
could be found in other areas in the cytoplasm in this or other
sections studied. Other cellular organelles appeared to be
normal after the cytochalasin B treatment, and intermediate
filaments were found in the cytoplasm of the treated hepato
cytes (Fig. 5a). The recovery from the cytochalasin B effect
was assessed by the reappearance of the microfilaments be
neath the plasma membrane interfacing with the culture me
dium. After cytochalasin B treatment, the hepatocyte cultures
were washed 3 times with fresh L-15 culture medium to remove
any residual cytochalasin B and were then allowed to recover
in fresh L-15 culture medium. Vertical sections were obtained
from samples at different periods of recovery. At 30 min of
recovery, a small but noticeable accumulation of microfilaments
was observed (Fig. 50), with the microfilaments parallel to the
plasma membrane. In addition, such an accumulation was not
inhibited by the presence of cycloheximide (0.1 (TIM)during the
recovery phase (Fig. 5c), nor was it enhanced by the presence
of polyamines (putrescine, spermidine, and spermine; data not
shown), which have been reported to induce the polymerization
of the monomeric form of actin to its filamentous form in an in
vitro system (48). The presence of cyanide (20 mM), however,
resulted in total disintegration of the hepatocytes after 30 min
of recovery (data not shown), although Holtzer ef al. (29)
reported that cyanide blocked the recovery, which was as
sessed in terms of morphological transformation. At 4 hr of
recovery, the network of microfilaments began to emerge and
progressively accumulated to a greater extent and complexity
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
Microfilament
Accumulation
in Cultured Hepatocytes
(Fig. 5cO. Microfilament clusters were seen at the closely
packed areas of the microfilaments. At 12 hr of recovery (Fig.
5e), the network extended into and intensified at the lateral
plasma membranes of adjacent cells, interweaving with some
intermediate filaments and possibly with some tonofilaments of
the desmosomal complexes. At this time, the network had
accumulated to a great extent, and microfilament clusters were
numerous. Other cellular organelles were excluded from this
extensive network. Further recovery led to an extent and com
plexity of the microfilaments comparable to those of a Day 9
culture.
Analysis of Actin by SDS-Polyacrylamide Gel Electrophoresis. The total protein content of hepatocytes was analyzed
on SDS-polyacrylamide gels to detect the major components
of the hepatocyte proteins. Actin was identified on SDS-poly
acrylamide gels in this study as a band of m.w. 42,000, which
comigrates with skeletal muscle actin. The hepatocytes in
culture were first labeled with a pulse of L-{35S]methionine and
then solubilized in an SDS-containing sample buffer, as de
scribed in "Materials and Methods." The protein components
were detected by fluorography and subsequent densitometric
tracings. In Chart 1,4, one of the major bands detected has a
B
40
20
Doys in culture
Chart 2. A, densitometric tracings of proteins extracted from acetone powders
of hepatocytes. Adult rat hepatocytes in culture for 2, 4, or 6 days were labeled
with L-[35S]methionine, and acetone powders of the labeled hepatocytes were
prepared. Extracts of the acetone powders were prepared and analyzed on 10%
SDS-polyacrylamide gels. Each sample contained equivalent amount of proteins.
Arrows, positions of "C-labeled molecular weight markers analyzed in a different
slot on the same gel, as detailed in Chart 1; open arrow, position of a m.w.
42,000 protein, which comigrates with skeletal muscle actin. B, increase in the
area of the actin peak indicated by the open arrow in A, with time in culture.
80
molecular weight of 42,000, as determined by the '"C-labeled
40
Days in culture
Chart 1. A, densitometric tracings of total hepatocyte proteins. Adult rat
hepatocytes in culture tor 2. 4. or 6 days were labeled with L-{35S]methionine.
and total protein extracts of the labeled hepatocytes were prepared and analyzed
on 10% SDS-polyacrylamide gels. Each sample contained equivalent amount of
proteins. Arrows, positions of "C-labeled molecular weight markers analyzed in
a different slot on the same gel (phosphorylase a, 92.500; albumin, 68,000;
ovalbumin, 46.000; and carbonic anhydrase, 30,000; purchased from New
England Nuclear); open arrow, position of a m.w. 42,000 protein, which comi
grates with skeletal muscle actin. ß.increase in the area of the actin peak
indicated by the open arrow in A, with time in culture.
molecular weight markers coelectrophoresed on the same gel.
In addition, this band exhibited the same mobility on the gel as
skeletal muscle actin after Coomassie brilliant blue staining.
The area under this actin peak increased with time in culture
(Chart 16), indicating an increase in the synthesis of actin. By
comparison of the area of the actin peak with the total area on
the densitometric tracing of a sample, such as shown in Chart
'\A, the percentage of actin in terms of the total protein in the
hepatocytes cultured for various days was found to increase
between Day 2 (6 ±1%) and Day 4 (10 ±2%) in culture but
did not increase further after Day 6 (11 ±2%) in culture.
By the same criterion, actin was the major band on analysis
of proteins extracted from the acetone powders of hepatocytes
(Chart 2A). Acetone powders of hepatocytes presumably con
tain the insoluble cytoskeleton and thus the filamentous form
DECEMBER 1980
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
4555
IV. W-N. Mak et al.
Table 1
Distribution of monomeric and filamentous forms of actin in the hepatocytes
The contents of monomeric and filamentous forms of actin in the hepatocytes
cultured from 2 to 10 days were measured by the DNase I assay as described in
the "Materials and Methods." The percentage of monomeric actin in the hepa
tocytes is expressed as the number of inhibitor units relative to the total number
of inhibitor units. The remaining portion is considered to be the proportion of the
filamentous form of actin in the hepatocytes.
Days in
culture246810Monomeric (%)1
form
form
00
2a56
±
543±
239±
536±
±3Filamentous
<%)0
244±
557±
261±
564 ±
±3
Mean ±S.E. from 3 separate experiments.
of actin. With Buffer A, which was used to extract actin from
acetone powders of muscle (62), other tissues (60), and he
patocytes (Fig. 4), the filamentous form of actin was solubilized
and could be quantitated by the SDS-gel analysis. The area of
the actin peak increased from Day 2 to Day 6 in culture (Chart
2B); this indicates an increasing proportion of the filamentous
form of actin. The increasing actin content as well as this
increasing proportion of the filamentous form of actin could
account for the accumulation of HMM-binding microfilaments
in late cultures of hepatocytes (Fig. 20).
DNase I Assay for the Monomeric and Filamentous Forms
of Actin. An assay of monomeric and filamentous forms of
actin in extracts of human platelets, lymphocytes, and HeLa
cells, reported by Blikstad ef al. (5), was based on the obser
vation that actin was a naturally occurring inhibitor of DNase I
(35). With application of the DNase I assay to hepatocytes
cultured for 2 to 10 days, the proportion of monomeric and
filamentous forms of actin could be estimated. In a preliminary
study, the proportion of monomeric and filamentous forms of
actin in a normal human fibroblast culture was found to be 55
and 45%, respectively. The values obtained are in agreement
with those reported in the literature (5, 8). The proportion of
the monomeric form of actin in cultured hepatocytes de
creased, whereas that of the filamentous form of actin in
creased with time in culture (Table 1). The increase of the
filamentous form agrees with the increase of actin content in
the acetone powders of labeled hepatocytes (Chart 2A), as
well as with the accumulation of microfilaments observed by
electron microscopy (Fig. 1a).
DISCUSSION
Microfilaments have been described in several organizational
forms in a variety of nonmuscle cells, as determined by electron
microscopy. These forms include bundles, meshworks, and
networks (25, 67). Interconversions between the bundle and
meshwork forms and between the bundle and network forms
have been observed in normal and adenovirus-transformed
fibroblasts (26) and in the petal coelomocytes of sea urchins
(11, 25), respectively. In the present study, the accumulation
of microfilaments in adult rat hepatocytes cultured on the
collagen gel/nylon mesh supports was seen in the form of
networks, localized mainly beneath the plasma membrane inracing with the culture medium. Meshwork and bundle forms
r microfilaments were largely lacking in the cytoplasm, al
though bundles of intermediate filaments were frequently found
below the network of microfilaments and deep in the cytoplasm
4556
of these cultured hepatocytes. Intermediate filaments were
tentatively identified in this study by their size (100 A) and their
resistance towards cytochalasin B treatment (Fig. 5a). Microfilament networks have been described in vivo at the periphery
of normal and regenerating liver cells as pericanalicular webs
(19, 43) and at the cell margins of yß-cellsof the islets of
Langerhans as cell webs (18, 47). The apical but not peripheral
organization of microfilament networks found in the hepato
cytes in this stationary culture system appears to be different
from that found in liver in vivo, although a few instances of
similar organization can be cited in other epithelial cells. In
normal corneal epithelium in vivo, actin microfilaments were
found as an apical network under the microplicae of superficial
cell layers (21). In embryonic chick pigmented retina epithelial
cells (40) or rat hepatocytes (41 ) cultured on plastic dishes for
24 to 48 hr, a similar organization, but much less in quantity
and complexity, was reported. However, the extensive accu
mulation of microfilaments with time in culture appears to be
characteristic of this culture system.
The HMM decoration technique revealed that the microfilaments contain actin. The fact that the microfilaments could be
extracted from glycerinated cells by solutions that also extract
actin-like protein from acetone powders further links the HMMbinding microfilaments to the actin-like protein resolved on
SDS-polyacrylamide gels. Cytochalasin B disruption and sub
sequent recovery indicated that the microfilaments were sen
sitive to the drug, as are actin-containing microfilaments in
other nonmuscle cells (22, 64, 68). This finding provided a tool
to manipulate the dissolution and formation of the network.
Radioactivity labeling of hepatocyte proteins showed that actin
is one of the major proteins in cultured hepatocytes, as has
also been reported for lymphocytes (3).
The accumulation of microfilaments appears to be due in
part to the biosynthesis of actin and to the formation of new
microfilaments. Increased biosynthesis of actin has been re
ported during chick embryogenesis (54). The amount of actin
in rat liver was estimated to be about 1 to 2% of the total
protein (27), whereas that in Day 2 hepatocyte cultures, esti
mated by radioactivity labeling, was about 6%. The relative
rate of actin synthesis increased further with time in culture,
thus providing a valuable system for possible studies on the
control of gene expression and of microfilament formation.
Since the microfilament accumulation is observed in hepato
cytes cultured on collagen substratum, the role of collagen, as
well as any contaminating components in the collagen prepa
ration, in bringing about such an accumulation remains open.
Collagen may play an important role in the control of cell
proliferation and differentiation (28, 57). It will be interesting to
investigate the possibility of a stimulatory or regulatory role of
collagen in this system other than providing a substratum for
the maintenance of hepatocytes in a functional and viable state.
The formation of new microfilaments could be achieved by the
increased actin level in the cell since an elevated concentration
of monomeric G-actin would favor polymerization into the
polymeric form (F-actin) according to the G (monomer) ^±F
(polymer) equilibrium (31 ). On the other hand, possible removal
or degradation of inhibitors of polymerization, such as DNase
I (35) and profilin (9), may lead to an increased level of
polymerization of actin. Furthermore, enrichment in the hepa
tocytes of a motility-related high-affinity cytochalasin-binding
complex (37), which may control the polymerization and memCANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
Microfilament
brane attachment of actin-containing microfilaments, may be
causal to the extensive accumulation of microfilaments beneath
the plasma membrane.
The microfilament clusters described in this study are seen
as densely stained areas on the microfilament network. Their
appearance seems to be more pronounced in the later day
cultures as the microfilaments accumulate. It has been reported
in nonmuscle cells (52, 61) that the regularly spaced densities
appearing on microfilament bundles are reminiscent of Z-bands
in striated muscle cells. Furthermore, these densities may
coincide with the distribution of a-actinin, a protein that may be
involved in the organization of microfilaments (34). However,
the microfilament clusters seen in the cultured hepatocytes
appear to be less regularly spaced and more pronounced in
density on the microfilament networks than the "regularlyspaced densities" reported in fibroblasts and glial cells (52,
61). Their composition and function await further characteri
zation.
In light of the findings that an increase in microfilaments
occurrs in vivo in carcinogen-induced
preneoplastic hepato
cytes (45), regenerating hepatocytes (19), and fetal hepato
cytes (54), the accumulation of microfilaments in this hepatocyte culture system could indicate a possible link to abnormal
cellular processes, such as transformation (2, 16, 49), transi
tion from adult to fetal-like states (59), and aging (66). More
over, since the network of microfilaments in the hepatocytes of
this culture system is being synthesized and polymerized at the
expense of cellular energy, the accumulation of these microfil
aments would imply some kind of functional significance or a
degenerative process. The state of polymerization of actin may
be inversely correlated with the susceptibility of the cells to the
toxic effects of the drug chlorpromazine and its metabolites
(13). That the components of the cytoskeleton could be mod
ulated by this clinically useful drug would imply a role of the
cytoskeleton in mediating the drug effect. Networks of microfilaments can reversibly appear under certain physiological
conditions in some cells, such as granulation tissue fibroblasts,
regenerating hepatocytes, and epidermal cells growing over a
skin wound (20). It has also been suggested that microfilament
dysfunction may be a cause of intrahepatic cholestasis (50).
However, the function of the network of microfilaments in the
hepatocytes of this culture system has not been elucidated. It
could be a contractile role, resulting in tension in the epithelial
cell sheets (39, 41 ) or in cell migration (1, 21 ), or a cytoskeletal
role, involving various cellular activities (see "Introduction").
With the network of microfilaments characterized in this report,
further investigations could be initiated. For instance, investi
gation of cellular activities such as endocytosis, secretion, and
transport processes in the hepatocytes with or without the
accumulation of microfilaments would shed light on the function
of these microfilaments.
ACKNOWLEDGMENTS
We are greatly indebted to K. Babcock and L Romano for their excellent
technical assistance and to G. Sattler for photographic work. We thank Dr. L.
Lemanski for his generous gift of HMM and Drs. A. Sirica, D. F. Mosher, and L.
Lemanski for helpful discussions.
REFERENCES
1. Adelstein, R. S., Scordilis, S. P., and Trotter, J. A. The cytoskeleton and cell
movement: general considerations. Methods Achiev. Exp. Pathol., 8:1-41,
1979.
Accumulation
in Cultured Hepatocytes
2. Asch, B. B., Medina, D., and Brinkley. B. R. Microtubules and actin-contain
ing filaments of normal, preneoplastic, and neoplastic mouse mammary
epithelial cells. Cancer Res.. 39: 893-907, 1979.
3. Barber, B. H., and Delovitch, T. L. The identification of actin as a major
lymphocyte component. J. Immunol., 122: 320-325, 1978.
4. Biara, C. G. Studies on cholestasis. A re-evaluation of the fine structure of
normal human bile canaliculi. Lab. Invest., 73. 840-864, 1964.
5. Blikstad, I., Markey, F., Carlsson. L., Persson, T.. and Lindberg, U. Selective
assay of monomeric and filamentous actin in cell extracts, using inhibition of
deoxyribonuclease I. Cell, 15: 935-943, 1978.
6. Bonner, W. M., and Laskey, R. A. A film detection method for tritium-labeled
proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem., 46: 8388, 1974.
7. Bourgeade, M. F., and Chany, C. Effect of sodium butyrate on the antiviral
and anticellular action of interferon in normal and MSV-transformed cells.
Int. J. Cancer, 24: 314-318, 1979.
8. Bray, D., and Thomas, C. The actin content of fibroblasts. Biochem. J., 147:
221-228, 1975.
9. Carlsson, L., Nystrom, L.-E., Sundkvist, I., Markey, F., and Lindberg, U.
Profilin, a low-molecular weight protein controlling actin polymerisability. In:
S. V. Perry, A. Margreth, and R. S. Adelstein (eds.). Contractile Systems in
Non-Muscle Tissues: Proceedings, pp. 39-49. New York: Elsevier-North
Holland Publishing Co., 1977.
10. Clark, M., and Spudich, J. A. Nonmuscle contractile proteins: the role of
actin and myosin in cell motility and shape determination
Annu. Rev.
Biochem., 46. 797-822. 1977.
11. Edds, K. T. Microfilament bundles. I. Formation with uniform polarity. Exp.
Cell Res., 108: 452-456. 1977.
12. Edelman. G. M. Surface modulation in cell recognition and cell growth.
Science (Wash. D. C.), 792. 218-226, 1976.
13. Elias, E., and Boyer. J. L Chlorpromazine and its metabolites alter polym
erization and gelation of actin. Science (Wash. D. C.). 206: 1404-1406,
1979.
14. Fisher, M. M., and Phillips, M. J. Cytoskeleton of the hepatocyte. In: H.
Popper and F. Schaffner (eds.). Progress in Liver Diseases, Vol. 6, pp. 105121. New York: Gruñe& Stratton, Inc., 1979.
15. French, S. W., and Davies, P. L. Ultrastructural localization of actin-like
filaments in rat hepatocytes. Gastroenterology, 68: 765-774, 1974.
16. Gabbiani, G. The cytoskeleton in cancer cells in animals and humans.
Methods Achiev. Exp. Pathol.. 9: 231-243, 1979.
17. Gabbiani, G., Chaponnier, C., and Lüscher,E. F. Actin in the cytoplasm of
adrenocortical cells. Proc. Soc. Exp. Biol. Med., 749: 618-621, 1975.
18. Gabbiani, G., Malaisse-Lagae, F., Blondel, B., and Orci, L. Actin in pan
creatic islet cells. Endocrinology, 95: 1630-1635.
1974.
19. Gabbiani, G., and Ryan, G. B. Development of a contractile apparatus in
epithelial cells during epidermal and liver regeneration. J. Submicrosc.
Cytol., 6: 143-157, 1974.
20. Gabbiani, G.. Ryan, G. B.. Lamelin, J.-P.. Vassalli, P., Majno, G., Bouvier,
C. A., Cruchaud. A., and Lüscher,E. F. Human smooth muscle autoantibody:
its identification as antiactin antibody and a study of its binding to "nonmuscular" cells. Am. J. Pathol., 72: 473-488, 1973.
21. Gipson, I. K., and Anderson, R. A. Actin filaments in normal and migrating
corneal epithelial cells. Invest. Ophthalmol. Visual Sci., 76: 161-166, 1977.
22. Godman, G. C., and Miranda. A. F. Cellular contractility and the visible
effects of cytochalasin. In: S. W. Tannenbaum (ed.), Cytochalasins. Bio
chemical and Cell Biological Aspects, pp. 279-429. New York: ElsevierNorth Holland Publishing Co.. 1978.
23. Goldman, R. D. The use of heavy meromyosin binding as an ultrastructural
cytochemical method for localizing and determining the possible functions
of actin-like microfilaments in nonmuscle cells. J. Histochem. Cytochem.,
23: 529-542, 1975.
24. Goldman, R. D.. Milsted, A., Schloss, J. A., Starger, J., and Yerna. M.-J.
Cytoplasmic fibers in mammalian cells: cytoskeletal and contractile ele
ments. Annu. Rev. Physiol., 41: 703-722, 1979.
25. Goldman, R. D., Schloss, J. A., and Starger. J. M. Organizational changes
of actin-like microfilaments during animal cell movement. Cold Spring Harbor
Conf. Cell Proliferation, 3: 217-245, 1976.
26. Goldman, R. D., Yerna, M.-J., and Schloss, J. A. Localization and organi
zation of microfilaments and related proteins in normal and virus-transformed
cells. J. Supramol. Struct., 5: 155-183, 1976.
27. Gordon, D. J.. Boyer. J. L.. and Korn. E. D. Comparative biochemistry of
non-muscle actin. J. Biol. Chem.. 252, 8300-8309,
1977.
28. Hauschka, S. D.. and Königsberg, I. R. Influence of collagen on muscle
clones. Proc. Nati. Acad. Sei. U. S. A., 55: 119-126, 1966.
29. Holtzer, H., Sanger, J. W., Ishikawa, H., and Strahs, K. Selected topics in
skeletal myogenesis. Cold Spring Harbor Symp. Quant. Biol., 37: 549-566,
1972.
30. Ishikawa, H., Bischoff, R., and Holtzer, H. Formation of arrowhead com
plexes with heavy meromyosin in a variety of cell types. J. Cell Biol., 43:
312-328. 1969.
31. Korn, E. D. Biochemistry of actinomyosin-dependent
cell motility. Proc. Nati.
Acad. Sei. U. S. A., 75: 588-599, 1978.
32. Laemmli, U. K. Cleavage of structural proteins during the assembly of the
DECEMBER 1980
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
4557
W. W-N. Mak et al.
head of bacteriophage T4. Nature (Lond.). 227. 680-685, 1970.
33. Laishes, B., and Williams, G. M. Conditions affecting primary cell cultures of
functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and
maintenance of morphology. In Vitro (Rockville), 12: 821-832, 1976.
34. Lazarides, E., and Burridge, K. a-Actinin: immunofluorescent localization of
a muscle structural protein in nonmuscle cells. Cell, 6. 289-298, 1975.
35. Lazarides, E., and Lindberg, U. Actin is the naturally occurring inhibitor of
deoxyribonuclease I. Proc. Nati. Acad. Sei. U. S. A., 71: 4742-4746,
1974.
36. Lemanski, L. F., Mooseker, M. S., Peachey, L. D., and lyengar, M. R. Studies
of muscle proteins in embryonic myocardial cells of cardiac lethal mutant
Mexican axolotls (Ambystoma mex/canum) by use of heavy meromyosin
binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis. J.
Cell Biol., 68. 375-388, 1976.
37. Lin, D. C., and Lin, S. Actin polymerization induced by a motility-related
high-affinity cytochalasin binding complex from human erythrocyte mem
brane. Proc. Nati. Acad. Sei. U. S. A., 76. 2345-2349.
1979.
38. Lindberg, U., Carlsson, L., Markey. F., and Nyström,L. E. The unpolymerized
form of actin in non-muscle cells. Methods Achiev. Exp. Pathol., 8. 143170, 1979.
39. Michalopoulos, G., and Pitot, H. C. Primary culture of parenchymal liver cells
on collagen membranes. Morphological and biochemical observations. Exp.
Cell Res., 94: 70-78, 1975.
40. Middleton, C. A., and Pegrum, S. M. Contacts between pigmented retina
epithelial cells in culture. J. Cell Sci., 22. 371-383, 1976.
41. Miettinen, A., Virtanen, I., and Linder, E. Cellular actin and junction formation
during reaggregation of adult rat hepatocytes into epithelial cell sheets. J.
Cell Sci.. 31: 341-353, 1978.
42. Nicolson, G. L. Transmembrane control of the receptors on normal and
tumor cells. Biochim. Biophys. Acta, 457. 57-108, 1976.
43. Oda. M.. Price. V. M.. Fisher. M. M., and Phillips, M. J. Ultrastructure of bile
canaliculi, with special reference to the surface coat and the pericanalicular
web. Lab Invest.. 31: 314-323, 1974.
44. O'Farrell. P. H. High resolution two-dimensional electrophoresis of proteins.
J. Biol. Chem., 250. 4007-4021.
1975.
45. Ogawa, K.. and Mediine. A. Architecture and ultrastructure of carcinogen
induced putative preneoplastic hepatocytes during liver carcinogenesis.
Proc. Am. Asso. Cancer Res., 19: 63, 1978.
46. Olmsted. J. B., and Borisy, G. G. Microtubules. Annu. Rev. Biochem., 42:
507-540. 1973.
47. Orci, L.. Gabbay, K. H., and Malaisse, W. J. Pancreatic beta-cell web: its
possible role in insulin secretion. Science (Wash. D. C.), ) 75. 1128-1130,
1972.
48. Oriol-Audit, C. Polyamine-induced actin polymerization. Eur. J. Biochem.,
87 371-376. 1978.
49. Ozzello, L. Electron microscopic study of functional and dysfunctional human
mammary glands. J. Invest. Dermatol., 63. 19-26. 1974.
50. Phillips. M. J., Oda. M., Mak, E., Fisher, M. M., and Jeejeebhoy, K. N.
Microfilament dysfunction as a possible cause of intrahepatic cholestasis.
Gastroenterology. 69. 48-58, 1975.
4558
51. Pollard. T. D., and Weihing, R. R. Actin and myosin and cell movement. CRC
Crit. Rev. Biochem., 2. 1-65, 1974.
52. Porter, K. R. Introduction: motility in cells. Cold Spring Harbor Conf. Cell
Proliferation, 3: 1-28, 1976.
53. Rouiller, C., and Jezequel. A. M. Electron microscopy of the liver. In: C.
Rouiller (éd.).The Liver, pp. 195-264. New York: Academic Press, Inc.,
1963.
54. Santerre, R. F., and Rich, A. Actin accumulation in developing chick brain
and other tissues. Dev. Biol., 54: 1-12, 1976.
55. Salir, P. Introductory remarks: cilia, eukaryotic flagella and an introduction
to microtubules. Cold Spring Harbor Conf. Cell Proliferation, 3: 841-846.
1976.
56. Sattler. C. A.. Michalopoulos. G.. and Pitot. H. C. Ultrastructure of adult rat
hepatocytes cultured on floating collagen membranes. Cancer Res.. 38.
1539-1549.
1978.
57. Schor, A. M., Schor, S. L., and Kumar, S. Importance of a collagen substra
tum for stimulation of capillary endothelial cell proliferation by tumor angio
genesis factor. Int. J. Cancer, 24: 225-234, 1979.
58. Silverstein, S. C., Steinman, R. M., and Conn, Z. A. Endocytosis. Annu. Rev.
Biochem., 46: 669-672, 1977.
59. Sirica. A. E., Richards, W., Tsukada, Y.. Sattler, C. A., and Pitot, H. C. Fetal
phenotypic expression by adult rat hepatocytes on collagen gel/nylon
meshes. Proc. Nati. Acad. Sei. U. S. A., 76. 283-287. 1979.
60. Spooner, B. S., Ash, J. F.. and Wessels. N. K. Actin in embryonic organ
epithelia. Exp. Cell Res., 114: 381-387, 1978.
61. Spooner, B. S., Yamada, K. M., and Wessels, N. K. Microfilaments and cell
locomotion. J. Cell Biol., 49: 595-613, 1971.
62. Spudich, J. A., and Watt, S. The regulation of rabbit skeletal muscle
contraction. I. Biochemical studies of the interaction of the tropomyosintroponin complex with actin and the proteolytic fragments of myosin. J. Biol.
Chem., 246. 4866-4871,
1971.
63. Stossel, T. P. Contractile proteins in cell structure and function. Annu. Rev.
Med., 29. 427-457, 1978.
64. Tannenbaum, S. W. (ed.). Cytochalasins. Biochemical and Cell Biological
Aspects. New York: Elsevier-North Holland Publishing Co., 1978.
65. Trenchev, P., Sneyd. P., and Holborow, E. J. Immunofluorescent tracing of
smooth muscle contractile protein antigens in tissue other than smooth
muscle. Clin. Exp Immunol., )6 125-136. 1974.
66. van Gänsen, P., Devos. L., Ozoran. Y.. and Roxburgh. C. Phenotypes des
Fibroblastes d'Embryons de Souris Vieillissant in Vitro (SEM et TEM). Biol.
Cell., 34: 255-270, 1979
67. Weihing, R. R. The cytoskeleton
Exp. Pathol. 8: 42-109, 1979.
and plasma membrane. Methods Achiev.
68. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley. M. O.. Luduena, M. A.,
Taylor, E. L., Wrenn, J. T., and Yamada, K. M. Microfilaments in cellular and
developmental processes. Science (Wash. D. C.), 171: 135-143, 1971.
69. Wood. R. L. Some structural features of the bile canaliculus in calf liver.
Anat. Ree., 140: 207-215. 1961.
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
Microfilament
Accumulation
in Cultured Hepatocytes
XMF
JF'
''
1.0pm
MF
••«•••l
••
•••••••
I
Fig. 1. Morphology of the network of microfilaments, a. Day 8 culture of hepatocytes containing well-developed network of microfilaments (MF) beneath the
plasma membrane (PMmi interfacing with the culture medium. Individual microfilaments and densely stained microfilament clusters (MC) are seen in the network of
microfilaments. Intermediate filaments (IF), which are assembled in long bundles, are observed in the cytoplasm beneath the network of microfilaments. x 11,400.
b, Day 10 culture of hepatocytes showing the complex network of microfilaments (MF) beneath the plasma membrane interfacing with the culture medium. The
network of microfilaments extends into and interweaves with intermediate filaments (IF) in the cytoplasm and apparently with tonofilaments (JF) of the desmosomal
complexes on the lateral plasma membranes of adjacent cells, x 21.600.
DECEMBER
1980
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
4559
W. W-N. Mak et al.
l.Opm
.
-
MF
*
r
0.1 pm
Fig. 2. Decoration of microfilaments with HMM. a, Day 6 culture of hepatocytes
(MF) appears to remain intact after the glycerination and extraction in this control
perfusion as the Day 6 culture shown in a These hepatocytes were glycerinated
complexes are observed on the microfilaments in the network. The polarity of these
4560
glycerinated and incubated with SSS for 12 hr. The network of microfilaments
sample, x 35,700. b. Day 8 culture of hepatocytes obtained from the same
and incubated with an HMM solution for 12 hr. The characteristic arrowhead
arrowheads appears to be randomly oriented. X 73,500.
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
Microfilament
Accumulation
in Cultured Hepatocytes
Fig. 3. Negative staining with 1% uranyl acetate of a homogenate of a Day 6 culture of hepatocytes. In a, numerous filaments measuring approximately 40 A in
diameter are found in the homogenate. x 145.750. In b, in the homogenate treated with an HMM solution, several filaments (arrow) are observed to display the HMMdecorated arrowheads, x 100,800.
DECEMBER
1980
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
4561
W. W-N. Mak et al.
Fig. 4. Day 10 culture of hepatocytes
glycerinated
and extracted
with SSS (a) or a series of
actin depolymerization"
solutions (o). In a, the closely packed
network of microfilaments (MF) appears to remain intact after the extraction. Some bundles of intermediate filaments OF) are seen to extend into the network of
microfilaments, x 30.500. In b, the extraction renders an almost complete removal of the microfilaments that are normally found beneath the plasma membrane
(PMm) interfacing with the culture medium and at the lateral plasma membranes (LPM) of adjacent cells, x 24,500.
4562
CANCER
RESEARCH
VOL.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
40
Microfilament
Accumulation
in Cultured Hepatocytes
l.Oum
H-H
Fig. 5 a-c.
Cytochalasin B treatment and recovery of a Day 8 culture of hepatocytes.
In a, the hepatocytes were treated with 20 JIMcytochalasin
B for 1 hr at 37°.
The complex network of microfilaments seen in untreated Day 8 cultures of hepatocytes (Fig. 1a) is absent throughout the cytoplasm of the treated hepatocytes.
Plasma membrane interfacing with the culture medium (PMm) and intermediate filaments (IF) are shown, x 12,500. In b to e, after the treatment and subsequent
removal of cytochalasin B, the hepatocytes were incubated in fresh L-15 culture medium for a period of recovery, b. recovery for 30 min. A band of microfilaments
(MF) that accumulates beneath and orients parallel to the plasma membrane (P/vfm) interfacing with the culture medium is apparent, x 25,200. c, recovery for 30 min
in the presence of 0.1 HIM cycloheximide. Similar accumulation of microfilaments (MF) as seen in b is also observed, x 31.500
DECEMBER
1980
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
4563
W. W-N. Mak et al.
Fig. 5 d-e. d, recovery for 4 hr. The network of microfilaments (MF) has increased significantly in size beneath the plasma membrane. Densely stained
microfilament clusters (MO are noticeable near the lateral plasma membranes of 2 adjacent cells. Bundles of intermediate filaments (IF) interweave into the network
of microfilaments, x 19,800. e, recovery for 12 hr. A complex network of microfilaments (MF) and numerous microfilament clusters (MC) have formed. The network
excludes all of the other cellular organelles and intensifies at the lateral plasma membranes (LPM) of adjacent cells Intermediate filaments (IF) are found below the
network of microfilaments in the cytoplasm, x 10,300.
4564
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.
Accumulation of Actin Microfilaments in Adult Rat Hepatocytes
Cultured on Collagen Gel/Nylon Mesh
W. Wai-Nam Mak, Carol A. Sattler and Henry C. Pitot
Cancer Res 1980;40:4552-4564.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/40/12/4552
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research.