Cytoplasmic filaments - Investigative Ophthalmology & Visual Science

Cytoplasmic filaments: their role in
motility and cell shape
M.oving cells are fascinating to watch.
Today, 300 years after cells and their movements
were first observed with the primitive
microscopes of van Leeunwenhoek, cell biologists
are beginning to understand how
cells move. New fluorescence and electron
microscopic techniques are now being used
in eye research to study cell movements
which occur in corneal wound healing,
phagocytosis by the retinal pigment epithelium
(RPE), morphogenetic cell shape
changes which occur at the lens bow, and
other biologically important cell shape
changes.
Filamentous structures within the cytoplasm
are involved in cell motility and cell
shape. Three major size classes of cytoplasmic
filaments or tubules are present in
higher eukaryotic, nonmuscle cells. They
are microfilaments (actin filaments), intermediate
filaments, and microtubules.
Microfilaments
Microfilaments, the smallest of the three
types are approximately 50 to 75 A in diameter
and of variable length and are composed
of globular monomers of the protein
actin, aligned to form double-helical filaments.
The actin of microfilaments is similar
to muscle actin, and this protein is present
in all eukaryotic cells examined to date.
(For reviews see refs. 1 and 2.)
Actin filaments can be localized intracellularly
by a specific histochemical technique
which uses fragments of myosin,
either heavy meromyosin (HMM) or the
smaller subfragment-one (S-l). Both HMM
and S-l retain the active ATPase head, and
when they penetrate cells whose mem-
1081
branes are made permeable by glycerination,
the fragments bind specifically to
actin, forming arrowheads, visible by electron
microscopy, along the actin filaments. 8
Two light microscopic techniques for actin
localization have been developed; both use
actin-specific protein ligands labeled with
fluorescent dyes. One method employs fluorescent
actin antibodies 4 and the other fluorescence-tagged
HMM. 5 These methods
show that actin filaments are distributed in
two basic patterns in nonmuscle cells: (1)
cortical networks of actin filaments along
plasmalemmae, with individual filaments of
the network appearing to insert into the
membrane, and (2) parallel bundles of filaments
known as sheaths or stress fibers. The
latter occur along basal plasma membranes
of moving cells, in cleavage furrows of cytokinesing
cells, in microvilli of intestinal
epithelia, and in the acrosomal process of
some invertebrate sperm.
Myosin also appears to be present in nonmuscle
cells, although in lower concentration
than actin. The properties of specific
myosins from different cell types vary considerably,
1 but all have ATPase activity.
Antibodies to myosin of platelets and
smooth muscle have been prepared, and
their distribution studied with immunofluorescence
microscopy. Myosin distribution
appears to parallel actin filament distribution
in fibroblasts. G
Since actin filaments are associated with
motile cells (amoeba and moving cells in
culture) and with motile portions of cells
(i.e., cleavage furrows, microvilli of intestinal
epithelia) and because myosin is also
present in the cytoplasm of these cells, the

1082 Gipson
Invest. Ophthaltnol. Visual Set.
December 1977
general theory has arisen that an acto-myosin
system, similar in some respects to that
found in striated muscle cells, is involved in
generating force for cell motility. The actin
networks at cell surfaces are also believed to
contribute to cell shape. (For reviews see
refs. 1 and 2.)
With the use of S-l labeling, actin filament
distribution has been studied in two
ocular epithelia: corneal epithelia of rats 7
and RPE of monkeys and humans. s In
corneal epithelial cells of rats, actin filaments
form a cortical network under microplicae
of the superficial cells. Perhaps these
filaments form a cytoskeleton which holds
microplicae in their orderly array. Corneal
epithelial cells moving to cover an abrasion
have a dramatically different arrangement
of actin filaments in their cytoplasm;
bundles of parallel actin filaments extend
along the base of the cell and out into their
leading edges. In addition, inside the very
tip of the leading edge of the moving cell
there are thick networks of actin filaments.
The bundles may play an important role in
generating the force required for movement
of these cells across the denuded basement
membrane.
It has been long known that local anesthetics
inhibit corneal epithelial wound
healing. Although it was assumed that these
drugs act directly at the level of the plasma
membrane, recent reports indicate that the
tertiary amine local anesthetics (including
procaine) disrupt actin filaments along cell
membranes of endothelioid 3T3 cells. 9 Filament
arrangements within corneal epithelial
cells migrating to cover corneal abrasions
are also disrupted when corneas are cultured
in the presence of 1 mM proparacaine.
10 These findings indicate that the adverse
effects of local anesthetics on wound
healing are related to disruption of the contractile
filament system of the epithelial
cells.
In human and monkey RPE, actin filaments
are present in ordered arrays in the
apical processes which extend to surround
the distal disks of the photoreceptors. The
filaments appear to be attached or inserted
into the cell membrane, and it has been suggested
that they form a cytoskeleton which
supports the processes or perhaps they generate
an active force for the phagocytic
event. s
With the use of actin antibody, cortical
networks and large quantities of parallel
sheaths of actin filaments have been demonstrated
in sheets of cultured bovine lens
cells. 11 These filaments may play a role in
the cell movements and shape changes
which occur at the lens bow.
Recent reports indicate that cytochalasin
B reversibly reduces outflow resistance in
intact and disinserted monkey eyes. 12 Since
cytochalasin B causes disruption of microfilaments
in some cell types, it has been
suggested that microfilaments play a role
in aqueous outflow. It should be noted,
however, that cytochalasin B has numerous
effects on cells, including disruption of
hexose transport across cell membranes (for
review see ref. 13), and thus the conclusions
of these studies must be viewed with caution.
The effects of cytochalasin B on the
ultrastructure of the endothelial cells of the
trabecular meshwork and Schlemm's canal
have not yet been reported, but study of
these cells in normal and glaucomatous
eyes, with modern techniques for actin
localization, may provide new information
on the etiology and morphological basis of
open-angle glaucoma.
Investigations of the role that defects in
actin filaments may play in disease process
have only begun. Already two human diseases
are known that show defects in microfilament
distribution or in proteins associated
with actin; they are adenomatosis of
the colon and rectum and spherocytosis, respectively.
Fibroblasts of patients with the
inherited cancer of the colon have an altered
actin filament distribution 14 ; in spherocytosis,
spectrin, a membrane protein
which together with actin has a role in regulation
of cell shape, is altered. 15
Intermediate filaments
Intermediate filaments are distinct from
actin filaments and microtubules, and they,

Volume 16
Number 12
Cytoplasmic filaments 1083
too, are ubiquitous in eukaryotic cells.
These 100 A diameter filaments have been
called tonofilaments, neurofilaments, glial
filaments, and endothelial filaments, depending
on the cell of origin. Little is
known about the biochemistry of these filaments
and even less of their cellular function.
Moreover, it is not clear whether these
filaments from different tissue sources are
related chemically or functionally.
One of the most characteristic features of
tonofilaments is their insertion into desmosomes,
structures along plasma membranes
that form attachments between cells or between
cells and basement membranes. Tonofilaments
occur in loose networks in epithelial
cells and are especially plentiful in
the basal cells of the epidermis. Some evidence
indicates that they play a role in
keratinization in epidermis, and these intermediate
filaments have therefore been
termed keratin fibers. Proteins of keratin
filament-enriched preparations from epidermis
separate into four to seven major
bands termed a-keratins on sodium dodecyl
sulfate (SDS) electrophoresis. 1 ' 1
Neural and glial filaments have been isolated
by cell fractionation, and a major
component of both filaments appears to be
a protein of about 54,000 to 56,000 m.w.
Antibodies to neurofilament protein will
cross-react with glial filament protein, and
immunofluorescence studies show that neurofilament
antibodies will stain 100 A filaments
of endothelial cells. The intermediate
filaments of various cell types and species
may be related, but biochemical evidence
of this is lacking.
Ocular tissues which have an abundance
of intermediate filaments are the corneal
and conjunctival epithelia, RPE, and all
neurons. Little direct evidence is available
concerning the function of the intermediate
filaments at these sites. Generally, they are
considered to be cytoskeletal structures.
Microtubules
Largest of the three types of cytoplasmic
filaments is the microtubule. It is made up
of 13 protofilaments arranged concentrically
to form hollow circular tubes 180 to 250 A
in diameter. The protofilaments are made
of two electrophoretically separable protein
chains (a- and /3-tubulin) of identical
molecular weight but different amino acid
composition (for review see ref. 17). Microtubules
are present in most cell types, and
they are most obvious in mitotic spindles,
cilia, flagella, and neurons.
Microtubules contribute to formation and
maintenance of cell shape. Neurotubules of
axons maintain the shape of these important
long cytoplasmic processes. Microtubules
are often found as components of biological
motility machines, e.g., sperm, flagella, and
mitotic spindles. The drug colchicine specifically
depolymerizes tubulin and inhibits
microtubule formation. By the criterion of
colchicine sensitivity, microtubules are
known to contribute to pigment granule
movement in a variety of cell types and
axonal vesicle transport, as well as chromosome
movement.
Morphological evidence suggests that in
epithelial cells at the lens bow microtubules
elongate to force these cells to form lens
fibers. Other ocular cells in which microtubules
are prominent are the cilia of photoreceptors,
all neurons, and RPE cells. Microtubules
are associated with phagocytic
vacuoles of RPE, and it has been suggested
that they play a role in movement of the
vacuoles into the cell. ls Microtubules are
also evident in mitotic spindles of the corneal
epithelium and in the cytoplasmic extensions
that spread during corneal wound
healing. Colchicine inhibits epithelial motility
during wound healing.
At least one human disorder is known to
be caused by defective microtubules: congenital
immotile-cilia syndrome. 19 Defects
in the dynein arms along microtubules of
cilia result in chronic airway infections, and
men have immotile spermatozoa. A deficiency
of microtubules has also been implicated
in Chediak-Higashi syndrome. 20
In summary, it is clear that there are distinct
differences between the major cytoplasmic
filaments, both in their morphology
and biochemistry. It is also clear that cell

1084 Gipson Invest. Ophthalmol. Vistial Sci.
December 1977
biologists are just beginning to sort out the
functions of these filamentous structures
within cells. Investigations of the role they
may play in disease processes have only
just begun. We are rather ignorant of disease
at the cell organelle level; a sick organelle
may be the very basis for a sick
organ. Investigating the cell biology of filament
systems of eye tissues may prove to be
a fruitful avenue of research on normal and
abnormal function of cells of the eye.
Ilene K. Gipson
Department of Ophthalmology
University of Oregon Health
Sciences Center
Portland, Oregon
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