September 1986
Vol. 27/9
Investigative Ophthalmology
& Visual Science
A Journal of Dosic and Clinical Research
Articles
The Cytoskeleton of the Cynomolgus Monkey
Trabecular Cell
/. General Considerations
Mark I. Ryder* and Robert N. Weinrebf
Many cellular functions involve the complex network of actin filaments, microtubules, and intermediate
filaments collectively known as the cytoskeleton. Stereo transmission electron microscopic observations
of whole cynomolgus monkey trabecular cells, which were extracted, S-l labeled, and critical-point
dried, were employed to simultaneously identify these three major cytoskeletal systems and visualize
their three-dimensional nature. A double fluorescence technique for actin and microtubules was used to
provide a broad view of cytoskeletal relationships within the cell. Actin microfilaments were the most
prominent elements of the cytoskeleton. They appeared as bundles in stress fibers. Between stress fiber
bundles, a continuous meshwork of microfilaments and intermediate filaments could be seen. Numerous
microtubules radiated from the centriole region to the cell periphery. This comprehensive overview of
the cytoskeleton of the cynomologus monkey trabecular cell can be used to understand structure-function
relationships of the trabecular cell cytoskeleton and its influence on outflow facility. Invest Ophthalmol
Vis Sci 27:1305-1311, 1986
Trabecular cells perform many of the activities which
have been hypothesized to be necessary for maintainence of the structural integrity and normal function of
the trabecular meshwork.1"3 As a lining of the aqueous
channels in the outflow pathway, they must remain
attached to the trabecular beams, possess the ability to
spread to cover beams that become denuded, and
maintain a flat appearance to prevent aqueous flow
turbulence. Trabecular cells also have phagocytic capabilities which allow them to remove materials, such
as pigment or inflammatory debris, which could obstruct the outflow of aqueous humor.4"6 Further, these
cells regulate the deposition and degradation of the extracellular matrix in the trabecular meshwork, 27 "" as
well as functioning as secretory12 and target cells for
drugs' and hormones. 13 As in other cells,14"16 these cel-
lular functions most likely involve the complex, threedimensional network of actin filaments, microtubules,
and intermediate filaments collectively known as the
cytoskeleton. To understand these functions of the trabecular cells and their influence on outflow facility,
therefore, it is important to characterize the structurefunction relationships of the trabecular cell cytoskeleton.
Two morphologic techniques, transmission electron
microscopic (TEM) observation of thin-sectioned material and immunofluorescence, previously have been
employed to study the cytoskeleton of trabecular cells.
Both Ringvold17 and Gipson 18 used S-l labeling in glycerinated cells to localize filamentous actin in TEM
sections. In unlabeled thin sections, Grierson19 observed both 6 nm filaments (probably actin) and 10
nm (intermediate filaments), while Alvarado 2021 noted
microfilaments and microtubules. However, thin sections can only provide a limited view of the cytoskeleton, and glycerination distorts the cell shape. Grierson19 and Tripathi 22 used antibody techniques to localize actin in trabecular cells. These immunofluorescent methods can be used to localize other cytoskeletal elements as well.
In the current investigation, we employed stereo
pairs of whole cells which were extracted, S-1 labeled,
From the *Department of Oral Biology, University of California,
San Francisco, California, and the fDepartment of Ophthalmology,
University of California, San Diego, California.
Supported in part by NIH Grants DE06681 (MIR) and EY05990
(RNW).
Submitted for publication: November 7, 1985.
Reprint requests: Robert N. Weinreb, MD, University of California,
San Diego, Department of Ophthalmology (T-014), La Jolla, CA
92093.
1305
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1986
and critical-point dried to simultaneously identify all
three major cytoskeletal systems (actin filaments, microtubules, and intermediate filaments) and visualize
the three-dimensional nature of the cytoskeleton. Further, we used a double fluorescence technique for actin
and microtubules to provide a broad view of cytoskeletal relationships within the same cell. With these techniques, we have obtained a comprehensive overview
of the cytoskeleton of the cynomolgus monkey trabecular cell. These data can be used as a basis for comparison with pharmacologic and physiologic investigations of outflow facility in the cynomolgus
monkey eye.
Materials and Methods
Monkey Trabecular Cell Culture
Trabecular tissue was obtained for cell culture from
one eye of a 3.2 kg cynomolgus monkey immediately
postmortem and propagated as we have previously described.23 We used third passage cells.
Prior to cell culture, a 1 cm2 coverslip was placed
at the bottom of each culture well. Cells were then
grown on the coverslip for 7-10 days. For light microscopic observations (Nomarski and fluorescence), the
cells were grown directly on the coverslip surface. For
transmission electron microscopic (TEM) observations
of whole cells, 150 or 200 mesh gold grids were attached
to the coverslip with an overlying formvar film layer.
The trabecular cells were then grown on the film surface. All coverslips were sterilized under ultraviolet light
prior to placement in the wells.
For TEM observations of sectioned cells, they were
grown directly onto the plastic surface of small petri
dishes (3.5 cm diameter).
Light Microscopy
For Nomarski observations, coverslips with attached
cells were rinsed briefly in 0.1 M phosphate buffered
saline (PBS) with 10 mM sodium azide, and then fixed
for 20 min in a mixture of 1.0% gluteraldehyde and
0.5% Osmium tetroxide in the same buffer. The coverslips were then stored in the buffer. Fixed cells were
photographed with a Nomarski optics system on an
inverted microscope.
For fluorescent labeling of actin and microtubules,
the cells were double-labeled. First, they were labeled
for tubulin with an indirect rhodamine conjugated antibody technique, and then for actin with a direct NBDphallacidin technique. With these techniques, cells were
washed once in the PBS-sodium azide buffer, and then
fixed and extracted simultaneously for 20 min in 2.0%
formaldehyde, 0.2% Triton X-100 and PHEM buffer.24
After two rinses in PBS-sodium azide, the cells were
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Vol. 27
incubated with mouse monoclonal antibody to alphatubulin for 20 min. The mouse monoclonal antibody
was previously diluted 1:100 in PBS-sodium azide with
0.1% Triton X-100 and 1.0% bovine serum albumin.
All incubations were performed at room temperature.
The cells were then rinsed twice in PBS-sodium azide
and incubated with rhodamine-conjugated goat antimouse antibody for 30 min. This secondary antibody
was diluted 1:50 in the same buffer as the mouse
monoclonal. After another rinse in PBS-sodium azide,
the cells were incubated with NBD-phallacidin (Molecular Probes, Inc., Junction City, OR) diluted 1:20
in PBS-sodium azide for 20 min. The coverslips were
then rinsed and mounted in 50% glycerol-50% PBS
onto a glass slide. Fluorescent micrographs were obtained with the appropriate exitation and barrier filters
to screen only NBD-fluorescence for actin (green) or
rhodamine fluorescence for microtubules (red). Color
slides were converted to black and white prints with
an intermediate negative using Tech Pan film (Kodak,
Rochester, NY).
Electron Microscopy
Cells grown directly onto small petri dishes for TEM
sectioning were fixed in 0.5% paraformaldehyde and
1.0% gluteraldehyde in 0.1 M sodium cacodylate with
0.2 mM CaCl 3, pH 7.4.21 Cells were then post-fixed in
either a reduced (with 1.5% potassium ferrocyanide)
or non-reduced (no potassium ferrocyanide) 1.0% osmium tetroxide in 0.1 M sodium cacodylate buffer.
The cells were then stained with 1.0% uranyl acetate
for 2 hr, dehydrated in ethanol, and embedded in the
dish with Epon-Araldite. The embedded cells were then
separated from the petri dish, re-embedded in EponAraldite, sectioned, and stained with uranyl acetate and
lead citrate.
For TEM observations on the cytoskeletons of whole
unsectioned cells, the cells were extracted, labeled with
the S-l myosin subfragment to localize filamentous
actin, fixed, and critical-point dried. These methods
have been previously described.25 The only modifications we employed in this study were: 1) 0.1% formaldehyde was added to the Triton X-100 extraction
solution; and 2) all procedures, up to the critical point
drying of the cells, were done with the TEM grids attached to the coverslip.
Grids with either thin sections of cells or whole critical-point dried cells were scanned and photographed
on a Philips 300 TEM operated at 60 KV (for sections)
or 100 KV (for whole cells). Stereo pairs were obtained
by maneuvering the tilting stage on this microscope.
Results
Cultured monkey trabecular cells grown either directly on glass coverslips or formvar film appeared at
No. 9
TRABECULAR CELL CYTO5KELETON: I. GENERAL CONSIDERATIONS / Ryder and Weinreb
1307
Fig. 1. Nomarski micrograph of
confluent monkey trabecular cells.
In general, the cells appear broad,
flat, and polygonal in shape. Each
cell has a "granular" cytoplasm
and a round nucleus with two
prominent nucleoli (arrows)
(X3,400). Fig. 2. Low power TEM
of a cross section of a trabecular
cell from a confluent culture processed with non-reduced osmium.
The cells appear in flat monolayers.
In cross section, the nucleus appears flat and ovoid, with a pominent nuclcolus (NE). Several electron dense inclusions (G) are seen
within the cytoplasm. At the free
surface, microvillar-like processes
are noted (arrows) (X5,400). Fig.
3. A, High power TEM of the nuclear region processed with reduced osmium. There is a fine euchromatic staining pattern and
prominent nuclear envelope (arrows) (X49,000). B, High power
TEM of the nuclear region processed with non-reduced osmium.
There are spike-like condensations
of chromatin at the nuclear periphery (arrows). A prominent nuclear envelope is absent (X49,000).
Fig. 4. A, Low power TEM of the
junction between two trabecular
cells (GJ-gap junction, CP-coated
pit, F-filamentous
material
(X25.000). B, High power insert of
the gap junction between two cells.
The apposing cell membranes (arrows) are thickened, with an electron dense line between these
membranes (X83,000). C, Highpower insert of a filamentous region. The filaments are oriented
parallel to the cell membrane (arrows) (X83,000).
confluence as a continuous sheet of broad, flat polygonal cells when viewed under Nomarski optics (Fig.
1). Each cell had a "granular" cytoplasm and a round
or oval nucleus with two prominent nucleoli. When
viewed in TEM cross sections, the cells appeared in
flat monolayers (Fig. 2). The most prominent features
of the cytoplasm were the numerous electron dense
inclusions and microvillar-like projections at the upper
surface of the cell (Fig. 2). With reduced osmium, the
nucleus of the cells appeared to contain a fine euchromatin pattern with a prominent nuclear envelope (Fig.
3A). On the other hand, with non-reduced osmium,
the chromatin condensed into "spike-like" conden-
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sations at the nuclear periphery (Fig. 3B). In addition,
the nuclear envelope was not prominent. Intercellular
junctions between overlapping cell processes were not
a prominent feature. However, gap junctions, coated
pits, and coated vesicles were observed (Figs. 4A, B).
With conventional TEM, the most prominent cytoskeletal features were short lengths of filaments which
were oriented parallel to the length of the cell (Figs.
4A, C). These filaments were especially prominent at
the upper and lower surfaces of the cell.
A more complete view of the cytoskeleton could be
seen with the fluorescence micrographs and with the
whole critical-point dried cells. Under fluorescence, the
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / September 1986
Vol. 27
Fig. 5. Trabecular cells fluorescently stained for filamentous actin
using NBD-phallacidin. Stress fiber
organization (arrows) ts prominent, both within the cell and at
the cell periphery {X3,800). Fig. 6.
The same area fluorescently
stained for microtubules with an
indirect rhodamine-conjugated
antibody technique. There are numerous microtubules radiating out
from the nuclear region (N) in
overlapping arcs (X3,8OO). Fig. 7.
TEM of a whole, critical-point
dried cell. Numerous granules and/
or vesicles (GV) are seen to concentrate around the nucleus (N).
Towards the periphery, the organization of filaments into stress fibers (arrows) is prominent (N-nucleus) (X5.3OO).
actin filaments appeared in bright stress fibers over a
darker fluorescent background (Fig. 5). These stress
fibers appeared in groups of dense parallel lines that
traversed all or part of the cell diameter, and concentrated along the periphery. Numerous microtubules
were seen to radiate from the nucleus to the periphery
in overlapping arcs (Fig. 6).
TEM observations on extracted, S-l labeled, and
critical-point dried cells revealed the organization of
all three major cytoskeletal elements (actin filaments,
microtubules, and intermediate filaments). Low power
views of the cells revealed the density of the total filament organization within the cell as well as the stress
fiber organization (Fig. 7). In addition, numerous
granules and vesicles were seen to concentrate around
the nuclear region. High power stereo image pairs re-
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vealed the complexity of the organization of the three
major cytoskeletal elements. Around the nucleus, this
organization was extremely dense due to the thickness
of the cell in this area (Fig. 8). However, larger elements,
such as microtubules and stress fibers, could be distinguished. At the thinner periphery, all three cytoskeletal
elements could be distinguished. Actin filaments appeared in bundles of stress fibers as well as in a fine
meshwork of single filaments between the stress fibers
(Fig. 9). At the periphery of cells, individual actin filaments were observed with the "barbed" end of the
S-l label directed outwards (Fig. 10). Between these
dense stress fibers, numerous intermediate filaments
were observed (Fig. 9). They appeared as smooth and
straight filaments with a thickness of 10-12 nm. Microtubules, with their parallel dense lines and 20-25
No. 9
TRABECULAR CELL CYTOSKELETON: I. GENERAL CONSIDERATIONS / Ryder and Weinreb
1309
Fig. 8. High power TEM stereo pair
of the perinuclear region of a whole,
Triton-extracted and S-l labeled criticalpoint dried cell. Due to the thickness of
the cell in this region, the cytoskeleton
appears very dense and complex. However, numerous microtubules (MT) can
be distinguished by their parallel dense
lines, and a stress fiber is seen (SF) (Nnucleus). Stereo pair taken at ±3° tilt
(X40,000). Fig. 9. High power TEM
stereo pair of the peripheral region of a
trabecular cell processed as in Figure 8.
At the thinner periphery, the major elements of the cytoskeleton are more
readily distinguishable, Intermediate
filaments (IF) appear as 10-12 nm
smooth, straight lines. Actin filaments
(AF) appear with their S-l myosin arrowheads (arrows—barbed end) attached along their entire length. They
are also seen to concentrate into stress
fibers (SF). Microtubules (MT) are also
readily distinguishable. Stereo pair taken
at ±3° tilt (X68,000). Fig. 10. High
power TEM of the periphery of a cell
processed as in Figure 8. Note how individual actin filaments (AF) radiate
outwards from the periphery. Along
these peripheral filaments, the barbed
ends of the S-1 arrowheads (arrows) are
directed outward from the cell body
<X62,000).
nm thickness, were also observed at the periphery
(Fig. 9).
Discussion
In this study, we have demonstrated the presence
and organization of the three major cytoskeletal elements (filamentous actin, microtubules, and intermediate filaments) in the cultured cynomolgus monkey
trabecular cells. These cells were selected because of
their morphologic similarity to human trabecular cells
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and the opportunity they provide for comparing our
in vitro data with in vivo physiologic studies. While
the substrate and extracellular environment of in situ
trabecular cells differ from those of cultured trabecular
cells, cells under both conditions may share common
morphologic21 and functional properties. Hence,
structure-function investigations of cultured cynomolgus monkey trabecular cells may provide insights
regarding these same relationships in in situ cynomolgus monkey trabecular cells.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1986
Although Nomarski observations of the cynomolgus
monkey trabecular cells revealed a more polygonal cell
than the spindle-like trabecular cells seen in human
culture, the conventional TEM features were identical
to those reported in human trabecular cells.21 These
identifying features included the growth of cells in thin
monolayers, the presence of microvillar-like projections
at the free cell surface, phagocytic inclusions, the presence of gap junctions, coated vesicles, coated pits at
the cell-to-cell interface, and, most characteristically,
the condensation of euchromatin into a spiked band
at the nuclear periphery with disappearance of the nuclear envelope when processed with non-reduced osmium. As with previous conventional TEM studies,
filamentous structures could be observed in short
lengths. However, the Triton X-100 extraction and
critical-point drying coupled with double-fluorescence
techniques gave a more complete view of the filamentous cytoskeletal structure.
Perhaps the most prominent element in the cytoskeleton, when observed in the critical-point dried cell,
was the actin microfilament. In non-muscle cells, such
as the trabecular cell, the actin filament has been implicated in a number of structural and motile functions.
In several cells, the stress fiber network has been implicated in determining and maintaining cell shape.26
Stress fibers were a prominent feature of cultured cells
in these studies, and were composed primarily of bundles of actin filaments (as revealed by S-l labeling and
fluorescence). In the in situ situation, these actin stress
fibers may maintain the flat shape and longitudinal
orientation of the trabecular cells upon the collagen
beams. Actin has also been implicated in the attachment of cells to substrate. In other non-muscle cells,
actin has been observed to form dense attachment
plaques along its basal surface at sites of adhesion.27'28
In the cultured cynomolgus monkey cell, stereoscopic
observations did not reveal such actin plaques at the
basal surface. However, the absence of such plaques
may be due to the type of attachment substrate used
in this study (glass or formvar). In the in situ situation,
such plaques may form along the basement membrane
and collagen complex to which the trabecular cells are
adherent.
Mediation of cell motility is another common role
for actin in non-muscle cells. Although myosin has
been localized in several non-muscle cells,15 an organized actin-myosin complex has yet to be demonstrated. Thus, the sliding filament model for motility
associated with muscle cells has not yet been applied
to the non-muscle cell. As a result, several other models
have been proposed for actin participation in cell motility. In other cells, for example, the three-dimensional
lattice of actin filaments has been observed to contract
and relax.29 Such a lattice of actin has been observed
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Vol. 27
between stress fibers29 and may be a viable mechanism
for movement of the trabecular cell. Actin polymerization at the periphery of the cell, which can possibly
push the overlying cytoplasmic membrane in the direction of movement, has also been ofFered as a means
of motility in non-muscle cells. This hypothesis is derived from two important observations. First, S-l labeled actin filaments orient with their barbed ends directed outwards30 and, second, actin polymerizes preferentially at this barbed end.31 In the current study,
this type of peripheral orientation of actin was also
observed. Thus, the trabecular cell may extend its processes using a similar actin polymerization mechanism.
Both microtubules and intermediate filaments were
also prominent in the monkey trabecular cells. In other
non-muscle cells, these two cytoskeletal elements have
been implicated in the maintenance of overall cell
shape, as well as the organization and intracellular
transport of various organelles.26 The possible role of
intermediate filaments and microtubules in the organization and transport of secretory granules (involved
in extracellular matrix synthesis), lysosomal granules,
and phagosomes (involved in both phagocytosis and
degradation of extracellular material) is of particular
interest in trabecular cells. Although these organelles
are intimately associated with microtubules and intermediate filaments26'32 in other non-muscle cells, such
an organelle-cytoskeleton relationship was difficult to
ascertain in the current study. This was most likely
related to the density of the cytoskeleton around the
organelle-rich nucleus and the effects on organelle
shape of the Triton extraction. However, further insights into the structure-function roles of microtubules
and intermediate filaments, as well as actin filaments,
can be obtained by treating the trabecular cells with
drugs known to have effects on the cytoskeleton.
Key words: cytoskeleton, actin, intermediate filaments, microtubules, trabecular cells
Acknowledgments
The authors gratefully acknowledge Dr. Mark Kirschner
for providing the antitubulin antibodies and Dr. Richard
Niederman for providing the S-l label. We also appreciate
the assistance of Donna Chesnut and Carol Fiuren in preparing the manuscript.
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