Download Investigations of cytoskeletal elements in cultured bovine

Investigations of Cytoskeletol Elements in Cultured
Bovine Meshwork Cells
Ion Grierson, Lynn Millar, Jiang De Yong,* Joanne Day, Nicol M. McKechnie,f
Cafhryn Hitchins, and Michael Boulronf
An ultrastructural, immunohistochemical, and functional study was conducted on cultured bovine meshwork cells. Particular emphasis was placed on the organization of the cytoskeleton, and the cells were
viewed either as whole cells or following detergent extraction. For ultrastructural examination, several
modes of viewing were adopted, including a) a detector situated above the specimens collecting secondary
electrons (SE), b) a detector situated beneath the specimen collecting transmitted electrons (STEM),
and c) conventional transmission electron microscopy at 100 KV (TEM). In whole cell mounts, information
was obtained about the organization of the cytoskeleton and its relationship to other cytoplasmic organelles. Extraction procedures removed much of the plasma membrane and most organdies. The nucleus
and cytoskeleton remained and stress fibers were prominent. Immunohistochemistry showed that the
actin content of the cytoskeleton could be preserved after detergent extraction. Detergent-extracted cells
decreased their surface area when exposed to MgATP in a dose-dependent manner. The decrease in
surface area was associated with disassembly of cytoskeletal stress fibers and was optimal with 1 mM
MgATP. Whether or not the change in surface area could be considered a "contractile event" was
discussed. Invest Ophthalmol Vis Sci 27:1318-1330, 1986
The cytoskeletal and contractile filament system
within the cells of the trabecular meshwork has been
of considerable interest to various investigators. The
cells have been shown by conventional transmission
electron microscopy to contain microtubules, abundant 8-10 nra diameter intermediate filaments, and
many smaller 4-6 nm diameter microfilaments within
their cytoplasm. 12 The 4-6 nm diameter microfilaments have been subject to particular attention and,
on the basis of their selective binding of heavy meromyosin subfragment 1, they can be considered to be
actin microfilaments.3'4
The actin microfilament distribution has been studied in human trabecular meshwork cells established in
primary culture by indirect immunofluorescence.2'5
Also, when human meshwork cells, which have been
established in long term culture,6 are examined by
electron microscopy, a well-developed cytoskeleton is
evident.7 Indeed, there is some evidence to indicate
that the actin microfilament system is depleted in the
cytoplasm of cells grown from the chamber angle of
glaucomatous patients in primary culture 5 but, it
should be mentioned, that the successful establishment
of glaucomatous trabecular meshwork cells is not generally accepted.
The significance of a possible lesion in the cytoplasmic microfilaments associated with glaucoma is
obscure. However, it has been proposed that the contractile microfilaments play an important role in the
maintenance of the cell cover on the trabeculae in the
normal outflow tissues.4 In addition, the possibility that
meshwork cell contractility could influence outflow resistance by the active alteration of the size of the intertrabecular spaces has been expressed.3 For that matter, drugs like cytochalasin B which influence actin
polymers and colchicine which destabilizes microtubules, both reduce the resistance to aqueous outflow
when infused into the eye. 89 Tripathi and Tripathi 10
have recently observed that epinephrine induced the
retraction of cultured meshwork cells. Available evidence indicates that the mechanism of actin is mediated
through adrenoceptors, and that it ultimately involves
the cellular cytoskeletal system.10
Clearly, detailed study of the organization and function of the cytoskeletal elements and the actin filaments
of meshwork cells would be of value. Considerable insights into subcellular organization have been obtained
by culturing cells on a suitable substrate and viewing
From the Department of Pathology, Institute of Ophthalmology,
London, U.K. ""Visiting Fellow in association with Hunan Medical
College, China. f'Fight for Sight" Fellow.
Supported by Medical Research Council Grant No. G83O1O5OW,
and in part by Wellcome Research Foundation Grant No. 10998/
1.5.
Submitted for publication: September 5, 1985.
Reprint requests: Ian Grierson, PhD, Department of Pathology,
Institute of Ophthalmology, 17/25 Cayton Street, London, EC IV
9AT, U.K.
1318
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DOVINE MESH WORK CELLS CYTOSKELETON / Grierson er ol.
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the cells (without subsequent embedding and sectioning) in the high voltage electron microscope
(HVEM).11>12 HVEM systems are extremely expensive
and scarce, so that examination of whole cells by this
technique is not available to most research morphologists. Nonetheless, adequate images of whole cells can
be obtained with conventional transmission electron
microscopes (TEM).13
If cells are exposed to a non-ionic detergent in a
suitable buffer then, depending on the time of exposure,
the plasma membrane can be perforated, partially removed, or totally removed. The cytosol and many of
the major organelle systems are extracted, but the nucleus, cytoskeletal filaments, and actin-containing microfilaments are preserved.1415 The extracted cells can
be examined readily by electron microscopy from
which the three-dimensional organization of the cytoskeleton can be appreciated.16'17 The cytoskeletons
can be stained to demonstrate various types of microfilamentous protein. 1518 Finally, the intrinsic contractility of the cell can be evaluated by measuring changes
in configuration of the cytoskeleton in response to
MgATP.19-20
It seemed to us that a combination of some of the
techniques outlined would represent a particularly
powerful means of investigating the contractility of the
microfilament network, its organization, and its relationship to other cytoskeletal elements within cultured
meshwork cells. The cells used for this study were third
passage cells grown from the bovine drainage angle.
The growth characteristics21 and the synthetic activities22 of third passage bovine meshwork (BMW) cells
have been the subjects of earlier communications. Intact and detergent-extracted cultured bovine meshwork
cells were examined by a variety of electron microscopic techniques. Immunofluorescence was used to
investigate the actin distribution within the meshwork
cells. Finally, the decrease in surface area of detergentextracted cytoskeletons was measured following exposure to various concentrations of MgATP.
Materials and Methods
Culture Procedures
The isolation of BMW cells and the conditions of
culture have been the subject of an earlier report.21
Second passage cells were grown to confluence in standard 25 cm2 flasks (Sterilin, England) and removed by
trypsinization. The trypsinized cells were spun down,
resuspended, and plated out at 5 X 104 cells per cm2
onto either gold grids or glass coverslips placed at the
bottom of 25 cm 2 flasks or 8 well slides (Lab Tech
Products, Naperville, IL). Thereafter, the cells were
grown to pre-confluence prior to further investigation.
1319
Gold Grid Preparation
The gold grids (200 mesh, EM scope) were acid
cleaned, washed in phosphate buffered saline, rewashed
in distilled water, placed on clean coverslips, and overlaid with a formvar film.1323 Thereafter, the gold grids
with their formvar film and glass support were carbon coated and subsequently sterilized by U.V. light
before use.
Cell Extractions
Cell extractions to produce cytoskeletons were performed in the non-ionic detergent Triton X-100 (modified from Masuda et al20) or glycerol (from Kreis and
Birchmeier19).
For Triton X-100 extraction, the cells were washed
with phosphate buffered saline and exposed to 0.2%
Triton X-100 in a stabilization buffer at room temperature. The stabilization buffer consisted of 0.01 M
Tris-HCl (pH 7.6), 0.14 M NaCl, 0.005 M Mg Cl 2 , and
4% polyethylene glycol 6000. The time of exposure
varied between 1 and 5 min. Glycerination involved
the washing of cells in phosphate buffered saline.
Thereafter, the cells were treated with varying concentrations of glycerol (up to 50%) in 0.05 M KC1, 0.005
M ethylenediamine-NjNjN^N1 tetraacetic acid, 0.01
M Tris-HCl (pH 7.0) at 4°C for time periods between
1 and 24 hr.
The optimum extraction period was determined by
the ultrastructural and topographical appearance of the
extracted cells, the intensity of immunostaining for actin, and the decrease in the protein content of the cells.
Protein determinations were conducted according to
Lowry et al.24
Morphology
The BMW cells seeded down onto either glass slides
or gold grids were monitored throughout their growth
period with an inverted microscope (Olympus, Japan)
using positive phase optics. For ultrastructural examination, the intact whole cells and extracted cells on
gold grids were fixed with 3% glutaraldehyde in cacodylate buffer for at least 1 hr. The specimens were postfixed in 1% buffered osmium tetroxide and dehydrated
through graded alcohols. To date, a comprehensive investigation of optimum fixation conditions has not
been conducted. Thereafter, the specimens were critical-point dried (Polaron Equipment Ltd., England),
coated with carbon, and viewed with minimal delay
in an Hitachi 600. The Hitachi 600 provided facilities
for fine structural examination in three modes of viewing. As well as conventional transmission imaging
(TEM), a scanning detector was situated above the
specimen to collect secondary electrons (SE), and a
second scanning detector was located beneath the
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1986
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Fig. 1. A light micrograph
showing the growing cells by
phase contrast (X220).
specimen collecting transmitted electrons (STEM). All
observations were made at 100 KV in dark field emission. Dark field emission improved the contrast of the
image in TEM mode and facilitated rapid interchange
between TEM, SE, and STEM. Following examination
in the Hitachi 600, some specimens were coated with
gold in a sputter coater (Polaron) and observed in a
scanning electron microscope (Hitachi S520). The
scanning electron microscope (SEM) provided low
magnification plan views of the entire grid which was
not possible with the SE detector.
Contraction Studies
All the contraction studies were conducted on extracted cells produced by the exposure of bovine meshwork cells to 0.2% Triton X-100 in stabilization buffer
at room temperature for 2 min. Following extraction,
the cells were washed in phosphate buffered saline and
placed on the stage of an inverted microscope (Olympus). The inverted microscope was encased by an incubation chamber maintained at 37°C. The test cytoskeletons were located under the X20 phase objective
lens and photographed on 35 mm film for reference.
Then the phosphate buffered saline was replaced by
either contraction buffer alone or contraction buffer
containing various concentrations of MgATP (0.1-10
mM). Photographs were taken of the reference field
every minute for the first 10 min and subsequently
every 10 min for 1 hr. The contraction buffer19 consisted of 0.01 M TRIS-HC1, 0.03 M KC1, 10 »M CaCl2,
and 0.005 M MgCl2. The 35 mm film negatives from
each "contraction run" were passed through a film
reader (Zeiss, Jena) and the cell outline was drawn to
give a magnification of X17.5 to the negative. The area
of the drawing was measured with a semi-automated
image analyser (Mop Videoplan Zeiss) and the appropriate software.
Bovine aortic smooth muscle cells, extracted in a
similar manner to the BMW cells and exposed to 0.1
and 1.0 mM MgATP, served as reference controls.
Some protracted experiments were conducted in which
the extracted cells were exposed to contraction buffer
containing MgATP and photographed under X20 objective phase optics. At 1 hr, the buffer with MgATP
was replaced with either EAGLES buffer alone or EAGLES buffer containing the smooth muscle relaxant
papaverine (Sigma). The extracted cells were photo-
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BOVINE MESHWORK CELLS CYTOSKELETON / Grierson er ol.
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1021
Fig. 2. A, SE of surface topography of BMW cell, microvilli are present (arrow
heads) on a cytoplasmic fan
with surface ruffles (arrow)
(XI ,800). B, STEM view of
A in which the nucleus (n)
and cytoplasmic organdies
can be seen (XI,800). C,
STEM showing a mitochondrion (m) with cristae and a
stress fiber(s) deep in the cell
cytoplasm (X27.00O).
IV*
r.
graphed every 10 min for 2 hr and the films were analysed as described previously.
Immunohistochemistry
Indirect immunofluorescence and immunogold
procedures were employed to investigate the distribution of actin within cultured BMW cells. The anti-actin
antibody was raised in rabbits against chick gizzard
actin (Miles Laboratories, Elkhart, IN). Indirect immunofluorescence was conducted in accordance with
procedures outlined in a previous study.25 The cells
were grown on coverslips and the detergent-extracted
or non-extracted specimens were air dried prior to
staining. Actin patterns demonstrated by immunofluorescence were examined in a Zeiss epifluorescent microscope equipped with an FITC interference filter.
Controls were conducted in the following manner: 1)
omission of the primary antiserum, 2) incubation with
an irrelevant antiserum, and 3) incubation with antiactin serum absorbed with actin.
Results
Anatomy of Whole BMW Cells
The cells seeded down onto gold grids were fixed
when they were in the log phase of growth (Fig. 1). By
this time, they were well-spread, flattened, and had an
epithelioid morphology (Fig. 2A). Examination of the
surface of the cells with the SE detector showed focal
patches of microvilli, and when there was an anterior
fan, surface ruffles were prominent (Fig. 2A). However,
many areas on the surface of the BMW cells were featureless. If detectors were switched from SE to STEM,
then the nucleus and the distribution pattern of the
organelles beneath the surface of the cell could be appreciated (Fig. 2B). The arrangement of heterochromatin was seen in the nucleus and, in some cells, the
nucleus was distinguished from the adjacent cytoplasm
by a lucent halo which was essentially organelle free
(Fig. 2B). The highest density of organelles was in the
perikaryon (Fig. 2B) and mitochondria, stress fibers
and electron-dense inclusion bodies were evident (Fig.
2C). Although many organelles were difficult to recognize, mitochondria were distinctive. They were
identified as long, ribbon-like structures measuring up
to 7 fxm in length (Fig. 2C). The STEM system at our
disposal was adequate to resolve the cristae of mitochondria situated deep within the cell cytoplasm (Fig.
2C). However, details of the smallest components of
the cytoplasm were not resolved, e.g., we could identify
stress fibers, but not the individual micronlaments
which make up the stress fiber (Fig. 2C, see also Fig.
4A for comparison).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1986
Vol. 27
Fig. 3. TEM of whole
BMW cells. A, Part of a cell
in which it can be seen that
the cell center is too thick for
electron penetration and cytoplasmic detail is only possible in the periphery
(X2.300). B, Toward the
periphery, stress fiber(s) (arrows) and long branching
mitochondria (arrow heads)
are evident (X3,2OO). C,
Stereo-pair showing thick
stress fibers) and aggregates
of mitochondria (arrowheads) (X6,000). D, Stereopair of thin stress fiber(s);
note the long mitochondria
(arrow heads) and cytoplasmic vesicles (arrows)
(X8,000).
With TEM at 100 KV5 little ultrastructural detail
was observed in the thicker perikaryon (Fig. 3A). On
the other hand, if relatively thin portions of the BMW
cells were examined, remarkable detail was possible
(Figs. 3, 4). Large vesicles, micropinosomes, coated
vesicles, cisternae, mitochondria, dark inclusions
(lipid), and ribosomes were identified within the cytoplasm. Individual mitochondria frequently had
branches, and several had a complex contorted shape
(Figs. 3B, C). The mitochondrial matrix was dense and
homogenous, but short cristae were easily identified at
high power (Fig. 4B). Stereo-pairs showed the mitochondria to be cylindrical rather than the ribbon-like
structures as seen in single images (Fig. 4B).
A network of filamentous elements were recognized
throughout the cytoplasm forming the cell cytoskeleton
(Figs. 3C, D, 4). Intermediate filaments (approximately
10 nm in diameter), micronlaments (4-6 nm in diameter) (Fig. 4A), and microtubules (around 20 nm in
diameter) were distinguished. Many organelles were
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BOVINE MESHWORK CELL5 CYTOSKELETON / Grierson er QI.
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Fig. 4. TEM of whole
BMW cells. A, Stereo-pair of
stress fiber(s) show it to consist of aggregated microfilaments. Four to six nm microfilaments from the surrounding cytoskeleton link
with the stress fiber. Reticulum (r), coated vesicles (arrowheads) and 10-12 nm intermediate filaments (arrows)
are indicated (X40,000). B, In
a stereo-pair, the mitochondria appear cylindrical. Cristae are evident in a dense
matrix (X52,OOO).
intimately associated with the component filamentous
elements of the cytoskeleton, and these associations
were best appreciated by examining stereo-pairs (Fig.
4). Associations of organelles with intermediate filaments and microtubules were identified, but microfilament associations with organelles such as mitochondria (Fig. 4B), cisternae of reticulum, and coated vesicles (Fig. 4A) were particularly common.
The stress fibers could be seen more clearly (away
from the cell center) by TEM than had been possible
with STEM. These structures could be as thick as 8
jum (Fig. 3C) or as thin as 0.1 fxm (Fig. 3D), and consisted of aggregates of micronlaments (Fig. 4A), although larger diameter filament/microtubules could
also be seen joining with the stress fibers. Clearly, some
detail of the cytoskeleton could be determined, but,
because of the presence of many organelles and cyto-
plasmic material, it was difficult to get a meaningful
appreciation of the full extent and the three-dimensional organization of the cytoskeletal matrix as a
whole. However, it could be appreciated from immunofluorescence that a substantial component of the
general cytoskeleton (as seen by diffuse fluorescence)
and the stress fibers (as recognized by streaks of fluorescence) was rich in actin (Fig. 5A).
Production of Cytoskeletons
Glycerination was found to be an unsatisfactory
technique because it was protracted, taking from 1-24
hr to produce a suitable extraction. Also, a large proportion of the cells detached from the substrate. As
many as 60% of the extracted cells were lost in some
experiments. On the other hand, Triton X-100 pro-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1986
posed, at least, to 0.2% buffered detergent for 2 min,
by which time around 50% of the cellular protein was
removed (Fig. 6). Fluorescent staining intensity of the
stress fibers remained comparable to that of the intact
cells, and there was only a minor reduction in diffuse
cytoplasmic fluorescence when extracts (Fig. 5B) were
compared to whole BMW cells (Fig. 5A). Longer exposures to 0.2% buffered detergent (up to 5 min) were
not associated with substantially greater loss of protein
(Fig. 6), but there was an unacceptable reduction in
actin fluorescence.
Anatomy of Cytoskeletons
Examination by SE of cytoskeletons prepared in
0.2% detergent for 2 min showed that the plasma
membrane was not removed completely. The membranous remnant was perforated by many holes of
varying sizes (Fig. 7B). The internal contents of the
detergent treated cells could be observed with the SE
detector, including the nuclear remnant (Fig. 7B) and
a three-dimensional network of filaments in the cytoplasm.
TEM examination at low magnification revealed
that, after detergent treatment, there was sufficient
electron penetration to observe structural detail
throughout the cell, including the central region, which
was obscured in whole cell mounts (Figs. 7A, 3A). The
extraction process often left the nuclei with a dark
granular appearance (Figs. 7A, C). However, stress fibers were most conspicuous, there being extremely
good contrast between these structures and the extracted cytoplasm (Fig. 7). The stress fibers frequently
extended across the whole cell (Figs. 7A, C), and they
were cylindrical in the central region (Figs. 7C, D),
Fig. 5. Immunofluorescence to demonstrate the actin distribution
pattern in BMW cells. A, A BMW cell in which stress fibers are
evident. Diffuse fluorescence is prominent around the nucleus (n)
(X600). B, A BMW cell exposed to 0.2% Triton X-100 for 2 min
(X600). C, Similar to B but having been in 0.1 mM MgATP for 1
hr. The insert shows a rounded up, extracted cell which was in 1.0
mM MgATP for 1 hr <X600),
duced extracts within a few minutes (Fig. 6), and detachment was negligible. Therefore, glycerination was
abandoned in favour of detergent extractions for the
remainder of this study.
The optimum time period for the exposure of buffered Triton X-100 to the BMW cells was determined
on the basis of several criteria. Adequate resolution of
cytoskeletal elements required that the cells were ex-
time(mins)
Fig. 6. Protein content of BMW cells exposed to 0.2% Triton X100 for various time periods. Each point is the mean ± sem of five
flasks of cells.
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BOVINE MESH WORK CELLS CYTOSKELETON / Grierson er QI.
1025
Fig. 7. BMW cells extracted with 0.2% Triton X100 for 2 min. A, Low power
TEM, the nuclei (n) and
stress fibers (arrowheads) of
several cells on a grid square
can be seen clearly (X700). B,
SE showing the nucleus (N)
and perinuclear region
(X4,500). C, A stereo-pair by
TEM showing the nucleus in
relation to stress fibers. Organelle remnants are indicated '(arrow) (X 2,800). D,
Stereo-pair of stress fiber(s)
and the cytoskeletal honeycomb (X25,OOO).
flattening off towards the cellular periphery. Unfortunately, filament detail within the fibers was not clear,
as the fibers tended to have an amorphous appearance
at high power (Fig. 7D).
The bulk of the extracted cell consisted of a "honeycomb" of cytoskeletal material where the individual
filaments were obscured by adherent material (Fig. 7D).
The amorphous material may either have been residual
ground substance, or the remnants of partially extracted
organelles. The only recognizable organelles which remained (apart from the nucleus) were a few scattered,
electron-dense inclusions (Fig. 7C). Towards the periphery of the cell, the detergent extraction left a
"cleaner" preparation than close to the cell center (Fig.
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / September 1986
Vol. 27
8). However, there was substantial variation between
cells, so that, in the 1 mM MgATP group, one cell
decreased in area by as little as 10%, whereas some
were in the region of 60% or more (Fig. 9). A total of
16 photographs were taken of each cell during the hour,
one each minute for the first 10 min, then one every
10 min over the remaining 50 min. The single cell
demonstrated in Figure 10 was not typical in that it
displayed a greater than 60% change in area. On the
other hand, it did demonstrate a feature which was
common to our extracted cells, i.e., that most of the
area change took place in the first 10 min. The decrease
in area of the example in Figure 10 was 3.9%:per minute
over the first 10 min, but only 0.7% per minute over
the remaining 50 min.
Extracted cells on gold grids were examined by TEM
after exposure to 1 mM ATP for 1 hr. At low magnification, the contracted cytoskeletons appeared to have
produced folds in the support film (Fig. 11 A). It was
noted that both cells with a pronounced response to
MgATP (Fig. 11A) and those with a more modest response (Fig. 1 IB) had reduced numbers of stress fibers.
60
Fig. 8. The percentage of the'initial area of extracted cells exposed
to contraction buffer alone (star), 0.1 mM MgATP (circle), 1.0 mM
MgATP (triangle), and 10 mM MgATP (square) for 1 hr (mean
± sem). The change in area is significant for each dose level of MgATP
(Paired T-test P < 0.01 to P < 0.001), but there is no significant
change if extracts are exposed to buffer alone. The most pronounced
alteration is associated with 1.0 mM MgATP (20 cells in each group).
7C). In these "cleaner" regions, we were able to identify
individual filamentous components of the cytoskeleton.
The size of the filaments was surprisingly variable; small
"branch" filaments no more than 2 nm in diameter
were observed, as were very thick filamentous structures in excess of 25 nm. Nonetheless, the majority
of the filaments had diameters in the range of 8-14
nm. Filaments of all sizes joined with the stress
fibers (Fig. 7D).
Contraction Studies
Our contraction assay was based on the alteration
in area of individual extracted cells which had been
exposed to 0.2% buffered Triton X-100 for 2 min. Over
1 hr, the shape of the extracted cells did not alter significantly in contraction buffer alone, but did exhibit
a significant reduction in surface area in response to
the addition of MgATP. The response was dose-dependent. The greatest change in area was associated
with the addition of 1 mM MgATP and was a reduction
of approximately 40%; 10 mM was supermaximal (Fig.
30
60
time (mins)
Fig. 9. When 20 cells are exposed to 1.0 mM MgATP in contraction
buffer they all decrease in surface area over 1 hr, but the variation
between cells is pronounced.
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BOVINE MESHWORK CELLS CYTOSKELETON / Grierson er ol.
No. 9
80
60
10
TIME IN MINS
30
Fig. 10. The percentage of the initial area of one cell at various
time periods up to 1 hr following exposure to 1.0 mM MgATP. A
copy of the initial and final drawings made with the image analyser
are shown.
Those fibers which remained were frequently insubstantial (Figs. 7C, 1 IB). Apart from the apparent loss
of stress fibers, there were no other obvious alterations
in the organization of the cytoskeleton in response to
MgATP that were evident from qualitative examination.
Bovine aortic smooth muscle cells, by way of comparison, decreased their surface area by almost 50% in
1 hr when exposed to 0.1 mM MgATP in contraction
buffer, whereas 1 mM MgATP, which produced the
optimal response from BMW cells, was supermaximal
for the smooth muscle cells (Table 1). If BMW cells,
which had reduced their surface area by 25% or more
when exposed to 0.1 mM MgATP in contraction buffer,
were then exposed to 0.26 mM papaverine (a smooth
muscle relaxant) in EAGLE's buffer, there was a relaxation by 2 hr of 44.1 ± 10.2%; (n = 10; mean
± SEM) compared to 9.0 ± 4.7 (n = 13; mean ± SEM)
Fig. I I . Extracted cells exposed to I mM MgATP for I hr as seen
by TEM. A, Two cells rounded to the edge of the grid bars. Distinct
folds are evident in the support film (arrows) (X625). B, A cell which
has undergone only minor change in shape, but stress fibers are rare
and those that are present are small (arrows) (X2,000).
in buffer alone. The difference was significant at the
level of P < 0.02 using the Student T-test. However,
cells with less than a 25% reduction in surface area did
Table 1, MgATP-induced alteration in cell area: a comparison of the response
of bovine aortic smooth muscle and BMW cells
Cell type
Bovine aortic smooth
muscle
Bovine mesh work
%of
Time
period
(min)
Area of cells (in
nm) (mean ±
SEM)
original
area
Paired
t-test
0
10
60
1290.1 ± 105.2
889.9 ± 101.7
692.5 ± 82.1
100
69.0
53.7
P<0.00l
P< 0.001
20
20
20
0.1 mM
0.1 mM
0.1 mM
1 mM
1 mM
1 mM
0
10
60
1357.0 ± 99.8
1144.3 ± 83.9
948.2 ± 77.3
100
84.3
69.9
P< 0.001
.P<0.00l
20
20
20
20
20
20
0.1 mM
0.1 mM
0.1 mM
1 mM
1 mM
1 mM
0
10
60
1372.2 ±
1258.3 ±
1169.8+
1553.2+
1344.0 ±
954.9 ±
100
91.7
85.2
100
86.5
61.5
No. of
cells
20
20
20
MgATP in
contraction
buffer
0
10
60
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140.3
159.4
143.9
93.3
90.3
84.5
P < 0.05
P < 0.01
P< 0.001
P< 0.001
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / September 1986
not relax significantly beyond that which occurred in
buffer alone.
Discussion
The main objectives of the present study were a) to
investigate the fine structure of whole BMW cells grown
on gold grids, b) to study the organization of the cytoskeletal components, c) to prepare cytoskeletons by
detergent extraction of BMW cells with minimal loss
of contractile elements, and d) to determine whether
the detergent-extracted cells contract when exposed to
MgATP.
Vol. 27
shrinkage.28 Indeed, the "halo" observed around some
nuclei may have been produced by differential shrinkage with respect to the rest of the cell.
Clearly, extensive research is required into all aspects
of preparation including optimum fixation, processing,
drying, and staining before the whole mounts can be
interpreted with the same confidence as ultrathin sections. However, whole cell mounts represent a valuable
and accurate means of conducting volumetric, stereological quantitation of organelle distribution, shape,
and numbers.
Cytoskeletons
Whole Cells
The three-dimensional fine structure of intact whole
cells grown on gold grids has been investigated in some
detail using HVEM by Porter et al.11-12-26 The HVEM
provides images of exceptional quality, so that organelles throughout the cell can be identified and, by means
of stereo pairs, the interrelationships of the various organelles can be investigated. However, we have shown,
as have others,13'27 that useful information can be obtained with the conventional TEM. At 100 KV, satisfactory images of organelles, such as mitochondria,
endoplasmic reticulum, micropinocytotic vesicles,
coated vesicles, microtubules, intermediate filaments,
microfilaments, and stress fibers, were photographed,
but only in the thinner parts of the cell. In the thicker
portion of the cell cytoplasm, which incorporated the
nucleus and abundant cytoplasmic organelles, electron
penetration was poor, therefore, useful images were
not obtained. To some extent, we were able to overcome this problem by using the STEM mode of detection. Indeed, we were able to resolve the cristae of mitochondria situated deep within the cytoplasm of the
perikaryon. On the other hand, we were not able to
obtain as good a quality image with the STEM as the
conventional TEM where restrictions of cell thickness
did not apply, because the optimum resolution with
the STEM system available to us was only about 3 nm.
It would seem to us that conventional TEM alone or
TEM in combination with STEM provided a valuable
means of examining organelle distribution and their
three-dimensional organization. For example, we were
impressed with the elongated, branching mitochondria
because their complex form had not been predicted
from previous ultrastructural studies of ultrathin plastic
sections of BMW cells in culture.22
Because the images provided by the TEM and STEM
in particular were complex and "unusual", preparation
artifacts were difficult to identify. We have not, as yet,
investigated optimium conditions for tissue drying and,
undoubtedly, the critical-point drying procedure we
used throughout the investigation produced organelle
For the purpose of the present discussion, cytoskeleton refers to the contractile elements as well as the
usual structural components of the cell cytoplasm. In
whole cells, the cytoskeleton was seen to consist of a
complex network of microfilaments, intermediate filaments, and microtubules. The organization of the 4 6 nm microfilaments were of considerable interest, and
formed an extensive web of criss-crossing elements
making up the microtrabecular lattice described by
Porter.26 Aggregates of microfilaments gave rise to stress
fibers which stretch in various directions across the cell
and link with the microtrabecular lattice throughout
the cytoplasm. Stress fibers can be seen in living cells
in vitro by phase contrast.29 Also, the fibers are present
in cells in vivo, having been identified in such diverse
cells as vascular endothelium 30 and the myofibroblasts
of granulation tissue.31 The myofibroblasts of experimental vitreal scars recently have been shown to contain structures which satisfy the morphological criteria
ncessary to classify them as stress fibers32 and, of significance to the present communication, stress fibers
may exist in situ in normal monkey meshwork cells.4
Therefore, although stress fibers are seen readily in cultured cells, it is likely that they are an intrinsic component of many cells in vivo. The role of stress fibers
in cells is controversial,33 but they would appear to
have a role in cellular adhesion, locomotion, and contraction.
Detergent extraction has been used by a variety of
authors as a means of making the cytoskeleton readily
visible for anatomical studies while preserving its threedimensional organization.14"18'34 By removing the
plasma membrane, most organelles, and much of the
labile ground substances, there is easy access for immunohistochemical and histochemical stains to reach
the cytoskeleton. In this way, actin, myosin, tropomyosin, filamin, a-actinin, intermediate filament keratin, and the tubulin of microtubules have been located
and identified in the cytoskeletons of some cultured
cells.1518'34"36 Detergent extraction in this investigation
preserved the stress fibers, which could be seen clearly
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No. 9
BOVINE MESHWORK CELLS CYTOSKELETON / Grierson er ol.
even at relatively low magnification. The bulk of the
extracted cell consisted of a "honeycomb" of cytoskeletal material. Thus, in several areas, the individual filaments were obscured by what may have been either
residual ground substance or the remnants of partially
extracted organelles.
Details of individual filaments could be appreciated
in most parts of the extracted cell. The filaments were
variable in thickness, but those in the size range 8-12
nm predominated. However, it should not be considered that these were all or even predominantly filaments of the intermediate type. It has to be remembered
that accurate sizing of filaments is hazardous in detergent-produced cytoskeletons, because adherent ground
substance and carbon coating tends to increase their
thickness beyond the size normally seen in whole
mounts or tissue sections.15>35>36
MgATP Studies
In the preparation of detergent-extracted cells, a balance is required between excessive loss of cytoplasmic
constituents (including cytoskeletal elements) and obtaining a "clean" preparation for morphological examination. Routinely, we extracted BMW cells with
0.2% buffered Triton X-100 for 2 min which was associated with a 50% loss of cellular protein. This provided an adequate extraction for fine structural study
(see previous section) and, on the basis of immunohistochemistry, did not seem to produce an excessive
loss of actin. However, careful biochemical analysis of
the actin and contractile protein of cytoskeletons is
required for future investigations.
When these detergent-extracted cells were exposed
to MgATP, a dose-dependent decrease in surface area
was measured over a period of 1 hr. A variety of nonocular cells have been shown to contract in the presence
of MgATP following either glycerination37"39 or treatment with Triton X-100.20 The question arose whether
the change in shape, which was relatively slow, was a
true contraction. Isolated smooth muscle cells respond
in vitro to electrical stimulation with a shortening of
over 40% in 30 sec,40 whereas the BMW extracts took
60 min to produce a 40% decrease in surface area when
exposed to 1 mM MgATP. The slow alteration in BMW
cell shape in comparison to the electrically stimulated
contraction of single smooth muscle cells may, in part,
be explained by intrinsic differences between the cell
types, but differences in assay design should also be
considered.
It is thought that intact cultured cells on rigid substrates do not respond to MgATP because the cell must
be opened for easy access of MgATP and the cells are
too strongly adherent to the substrate.33 Thus, detergent
is thought not only to open the cell, but also to loosen
1329
surface attachment. 33 Depending on how loosely adherent the cell is to its rigid surface, contraction forces
will be expressed in either an isotonic or an isometric
manner. Clearly, adhesion to anything but a totally
elastic scaffold will modify and dampen the isotonic
contraction. Therefore, it was of value to have found
that (in identical conditions to BMW cells) bovine aortic smooth muscle cells produced only a moderately
greater decrease in surface area over a similar time period, albeit the optimal response was to a lower concentration of MgATP.
It has been shown that isolated stress fibers are contractile41 and shortening of individual subunits within
stress fibers have been noted in response to MgATP.19
However, our immunofluorescent and electron microscopic investigations of contracted BMW cells did not
demonstrate a shortening of stress fibers. Indeed, it was
evident that, in cells with a decrease in area of over
40%, few, if any, stress fibers remained after 1 hr. Cells
with a more limited decrease in surface area contraction
had extremely thin stress fibers. Therefore, our preliminary observations indicated that shortening of the
stress fibers did not occur in this system. It would seem
that there was either a disassembly of stress fibers and
reorganization of the contractile filaments as part of a
"contractile event," or that we were not looking at a
"contractile process" but merely the consequences of
its extrusion of contractile protein from the extracted
cells during the exposure to MgATP plus buffer.
Clearly, monitoring the actin content of extracted cells
is an essential step in future studies. However, that
exposure to buffer alone did not produce a decrease in
the surface area of the extracted cells and that papavarine could reverse part of the MgATP effect indicated that a contraction-like event was the more likely
of the two.
In Vivo Significance
The significance of the possible contractility of
meshwork cells remains obscure. Nonetheless, it provides further support for the proposal of Ringvold3 that
feedback mechanisms which control the shape and size
of the intertrabecular spaces may be present in the
drainage angle. Tripathi and Tripathi, 10 who showed
that epinephrine produced a retraction of cultured human cells, have emphasized the importance of the cytoplasmic contractile protein system within meshwork
cells in such diverse processes as phagocytosis, synthetic
activities, maintenance of cell shape, and reactivity to
neuronal and hormonal agents. The techniques used
in the present study represent valuable tools by which
such processes can be studied in meshwork cells in
vitro.
Key words: cytoskeleton, bovine meshwork cells, ATP, actin
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1330
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1986
Acknowledgments
We would like to thank Mr. R. C. Howes for his technical
support and Miss S. M. Pavitt for her secretarial assistance.
21.
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