/ . Embryol. exp. Morph. Vol. 65, pp. 1-25, 1981
Printed in Great Britain © Company of Biologists Limited 1981
REVIEW ARTICLE
Cytoskeletal coordination and
intercellular signalling during metazoan
embryo genesis
By J. B. TUCKER1
From the Department of Zoology, St Andrews University, Fife
SUMMARY
This article draws attention to certain recently discovered features of cell surface organization and cytoskeletal deployment that may be revealing a new basis for intercellular signalling
during metazoan embryogenesis. It is a signal mode that could coordinate many aspects of
'Entwicklungsmechaniic' by spatiotemporal integration of the cytoskeletal/motor network
throughout developing tissues. Evidence that this is achieved by ' intercellular cytoskeletal/
plasma membrane connecting systems' which coordinate the spatial organization of microtubules, microfilaments, and intermediate filaments in developing animal tissues is critically
examined. It is argued that this system does operate but that it is not used to transmit
positional information in embryonic fields. However, it probably responds to such information and might play an important part in establishing field boundaries during the very earliest
stages of embryogenesis.
Certain aspects of cell surface organization in contemporary protozoans reveal ways in
which the Protozoa could have been pre-adapted for the employment of cytoskeletal/cell
surface signalling during the advent of multicellularity. In marked contrast, such signalling
does not appear to be exploited during plant morphogenesis. The extent to which cytoskeletal
organization might be coordinated in sisier cells by transmission of spatial instructions during
cell division in both animal and plant tissues is also considered.
INTRODUCTION
Specification of tissue shape and size is crucial for successful embryogenesis.
Control of the growth and form of developing animal tissues involves the
spatially coordinated shaping, orientation, polarization, locomotion, division
and positioning of their member cells (Grant, 1978). It transpires that all these
activities have a common feature. They are influenced by the spatial layout and
mechanics of intracellular cytoskeletons (Wessels et al. 1971; Goldman, Pollard
& Rosenbaum, 1976; Burgess & Schroeder, 1979). There is also evidence for
cytoskeletal involvement during certain types of cell-cell adhesion (Phillips,
1
Author's address: Department of Zoology, The University, St Andrews, Fife
KYI6 9TS, U.K.
J. B. TUCKER
Fig. 1. Portions of a glial cell process and an adjacent neuron, that is migrating
along the process and is wrapped partly around it, showing the alignment of
microtubules in these two cell types during foetal monkey brain-cortex development. Based on the micrographs and diagrams of Rakic (1972).
Jennings & Edwards, 1980) and matrix secretion (Ehrlich & Bornstein, 1972),
but whether such participation in these two tissue-shape-related activities is
widespread remains to be ascertained.
Since cytoskeletons play a large part in defining the shapes and positions of
individual tissue cells during embryogenesis it follows that some intercellular
spatial coordination of intracellular cytoskeletons may be required, and that
some form of intercellular signalling additional to those currently recognized
has evolved to effect it. These important possibilities and their main implications for cell and developmental biologists are pursued below.
Tissue cytoskeleton spatiomechanics
If cytoskeletal coordination contributes significantly to tissue shape control
it should be possible to detect 'supracellular' cytoskeletal alignments and
lattices that correlate spatially with tissue axes and topography. Several arrays
that meet these criteria have been described. Some of them are difficult to
account for unless cytoskeletal coordination is effected to at least a certain
extent by intercellular signalling.
The alignment of actin and myosin filaments in adjacent striated muscle cells
has long represented a striking instance of a cytoskeletal arrangement that is
spatially coordinated throughout a tissue. Equally pronounced is the alignment
of microtubules in adjacent cells during the elongation of closely juxtaposed
myoblasts (Warren, 1974) and axons (Yamada, Spooner & Wessels, 1971) as
muscles and nerve connexions, respectively, are formed. There are also instances
of microtubule alignment in different cell types. For example, this occurs as
Cytoskeletal coordination during embryogenesis
Fig. 2. Schematic diagram showing a tube-shaped portion of epithelium, such as that
which forms part of an ovarian follicle or the epidermis of a portion of a developing
leg, in certain insects. The alignment of subsurface microtubules is indicated
by the thin lines, and cell boundaries by the thicker lines.
neurons migrate along glial cell processes in the cerebral walls of foetal monkeys
(Rakic, 1972) (Fig. 1). However, in all these cases alignment need not necessarily
be effected by direct transmission of signals between the interiors of the cells
in question. They may simply arise because adjacent cells, cell processes, and
their cytoskeletons become aligned as a result of orientational responses to
local environmental conditions. Alignments might be coordinated, for example,
by chemotactic gradients or surface contact responses to previously oriented
cells or matrix fibrils. A similar argument applies to cytoskeletal alignments in
many epithelia; especially to those in which microtubules are aligned at right
angles to the plane of the cell sheet (for example, Burnside, 1971). Such alignment could be set up as a consequence of apicobasal cell polarities induced by
different conditions on opposite sides of epithelia.
Certain insect epithelia provide particularly clear examples of microtubule
alignment in the plane of a cell sheet (Fig. 2). Microtubules positioned close to
the outer surfaces of ovarian follicle cells in certain insects are oriented in the
plane of the follicle surface so that they curve around the spheroidal follicle
and run at right angles with respect to its longitudinal (polar) axis (Tucker &
Meats, 1976). A similar situation occurs during evagination of leg imaginal
discs in Calliphora. Microtubules just below the outer surfaces of epidermal
cells are oriented around the elongating leg (Figs. 2, 3, 4). Microfilamentous
actin cables are also sometimes aligned in neighbouring cells and lie in the
plane of a sheet of such cells. Reasonably extensive arrays of this sort have been
demonstrated for cell groupings in tissue culture (Albrecht-Buehler, 1979; Lo &
Gilula, 1980) (Fig. 5). Whether, like the microtubule arrays described above,
such configurations are ever present in the cell layers and sheets of intact
embryos and organisms remains to be ascertained. The fact that they are
established in tissue culture is probably an indication that this is also the case
J. B. TUCKER
Cytoskeletal coordination during embryogenesis
5
for some tissues in situ. It is an important issue in the present context because
arrays of actin cables (or stress fibres as they are sometimes called) and microtubules are included in mechanochemical systems that alter cell shape by active
contraction or tension transmission and by promoting the elongation of whole
cells or cell processes. The supracellular alignment of these cytoskeletal arrays
that run in the plane of an epithelium is such that they are especially favourably
deployed to exert a major influence on spatial variations in the contraction,
spreading, curvature, and thickness of a cell sheet or layer of cells and hence
on the shaping of tissues that include sheets and layers of cells (i.e. most tissues).
There is evidence that cytoskeletons really do provide intercellular motor
networks for control of tissue shaping.
The positioning of filaments and microtubules in adjacent cells on either side
of desmosomes (Figs. 6,7) provide striking instances of direct structural coupling
between the cytoskeletons of adjacent tissue cells. It is generally agreed that
they probably transmit mechanical forces on an extensive intercellular basis
to modify and/or maintain tissue shape in a variety of situations. These include
desmosome/terminal web complexes in intestinal epithelia (Hull & Staehelin,
1979), alignment and attachment of cardiac muscle actin filaments on either
side of desmosomal intercalated discs (Fawcett & McNutt, 1969), and a neurectodermal desmosome/microfilament-ring lattice that acts as a coordinated
intercellular contractile system during neurulation (Karfunkel, 1974). Some
of the 'intercellularly-aligned' insect follicle cell microtubules (Fig. 2) are
attached to desmosomes where adjacent cells interdigitate (Fig. 7). Fine structural and experimental analyses indicate that they form part of a cytoskeletal
system that transmits tension right around a follicle to promote anisometric
epithelial expansion and elongation of the enclosed oocyte (Tucker & Meats,
1976; Went, 1978). The oocyte fails to elongate if enclosure by the follicle is
prevented although other cytoskeletally associated events such as nuclear
division and migration, and blastoderm formation, sometimes apparently
proceed normally.
If some form of intercellular signalling exists so that adjacent tissue cells
can interact directly for the purposes of spatiotemporal modulation of cytoskeletal arrangement and mechanochemical activity this would represent a
valuable facility for control and coordination of cell and tissue shaping during
embryogenesis. Greater architectural complexity and versatility would presumably be possible than in a system that relied entirely on chemotactic gradients or
Fig. 3. Transmission electron micrograph of a thin section grazing through the apical
surface of an epidermal cell in the third tarsomere of a metathoracic leg in the
dipteran Calliphora erythrocephala during pupal cuticle secretion. Dense cuticular
material borders the surface profile of the cell. Most of the sub-surface microtubules (see Fig. 2) are oriented at right angles with respect to the longitudinal axis
of the developing leg (the orientation of this axis is shown by the arrow), x 27000.
Fig. 4. As Fig. 3. x 32000.
J. B. TUCKER
Fig. 5. Microfilamentous actin cables (thin lines) are aligned in some neighbouring
cells within small monolayer groupings of cultured epithelial Pt Kl cells that
migrate together across the substratum. Based on micrographs of AlbrechtBuehler (1979).
the pre-existing orientation of cells and matrix fibrils for cytoskeletal coordination. At a cellular level tissue architecture is very complex and versatile. The
fine structure and behaviour of certain regions where cells make contact in
tissues and tissue cultures provide substantial indications that surface interactions between adjacent cells actually do make a major contribution to intercellular cytoskeletal coordination.
Cytoskeletons and cell surface contacts
Diffusible signals could not ensure that the two 'halves' of a cell junction are
exactly positioned with respect to each other and groupings of microtubules
(Fig. 7) or filament bundles (Fig. 6) in cell neighbours unless generated on a
very highly localised basis. This must also be so in some cases of contact
inhibition; specialized plasma membrane regions and attached groupings of
filaments are rapidly (20 sec) established on either side of points where cell
surfaces initially make contact (Heaysman & Pegrum, 1973). Indeed, cell
shaping and locomotion are sensitive to the orientation of other cells and
extracellular matrix fibrils during a whole range of other surface-contact-mediated phenomena such as contact-following and substrate-guidance (Rakic, 1972;
Dunn & Ebendal, 1978; Lofberg, & Ahlfors, 1978). The cytoskeletal reorganizations that ensue are almost certainly induced by surface-bound signals rather
than diffusible ones. The possibility that surface contact interactions could
promote cytoskeletal alignments with respect to the orientation of pre-existing
materials has already been considered. However, this possibility does not exclude
another. Namely, one in which surface contact interactions are exploited as
part of a mechanism for transmission of signals between cell interiors and the
setting up of cytoskeletal alignments and lattices with new orientations. For
example, some cells produce a variety of cell extensions such as filopodia and
lamellipodia that effect what appear to be exploratory sorties in which the
Cytoskeletal coordination during embryogenesis
Fig. 6. Attachment desmosomes and associated intermediate filaments joining an
outer epidermal cell (towards the left of the micrograph) to a basal cell in a newt
tadpole {Triturus helveticus). x 72000.
Fig. 7. Part of the outer surface of an ovarian follicle in the gall midge Heteropeza
pygmaea where two adjacent epithelial cells overlap, interdigitate, and are joined
by spot desmosomes. Aligned microtubules (arrows) (see Fig. 2) run alongside
the desmosomes and seem to be connected to them by strands of dense material.
From Tucker & Meats (1976) with the permission of the Rockefeller University
Press, x 63 000.
J. B. TUCKER
:.H4<**v..,.-'w
Fig. 8. A region where tw;o Ca///p/wratarsomericepidermal cells interdigitate. Aligned
sub-surface microtubules^(compare Figs. 3, 4) extend into the interdigitation and
run alongside it. x 145000.
Fig. 9. Tarsomeric epidermal cells in Calliphora. Basal portions of two cell bodies
are situated to either side of the micrograph. As pupal cuticle secretion starts
these cell bodies are closely juxtaposed at their apical extremities (not shown) and
joined by several types of cell junctions. They also make contact at levels just above
the basement membrane (arrow) via filopodial cell processes that extend across
the spaces between the basal portions of cell bodies and overlap each other, x 55000. .
surfaces of neighbouring cells and substrates are contacted and examined
(Gustafson & Wolpert, 1967; Albrecht-Buehler, 1976). Such behaviour may
be an indication that cells and their cytoskeletons monitor, and respond to,
materials bound to the surfaces of other cells as well as to noncellular substrata.
Further indication that this is so is provided by the complex topography often
present where the plasma membranes of adjacent cells apparently make close
(10-20 nm) 'contact'. This appears to be excessive if coupling via gap junctions
and/or other modes of diffusible signal transmission can account for all forms
of cell coordination.
Epithelial cell surface interdigitation is especially common (Figs. 7, 8)
(Lawrence & Green, 1975; Schliwa, 1975; Tucker & Meats, 1976; Meier, 1978).
Cytoskeletal coordination during embryogenesis
9
Filopodia and pseudopodia sometimes interconnect portions of cell bodies
where they are separated by substantial extracellular spaces although such
cells are closely juxtaposed at other levels (Figs. 9, 10, 11) (Baker, 1965). In
some of these instances the extravagance of interdigitation and 'podial' contact
(Figs. 8, 9) is difficult to reconcile only with a need for exchange of diffusible
signals, cell adhesion, and locomotory requirements. Their presence is reasonable if cytoskeletal organization near a plasma membrane region in one cell
locally influences that in a closely contacted portion of an adjacent cell. If this
is the case, it is to be expected that cells might locally increase the frequencies
and areas of such contacts, and the opportunities for concentrating certain
cytoskeletal combinations at particular surface loci. This is especially the case
in situations like that found in the epithelium illustrated in Figs. 10 and 11,
where it is clear from the shapes of individual cells that the cytoskeletal configurations controlling shape must differ at various levels in the cells but be more
or less in register with each other through the thickness of the epithelium. There
are numerous fine filopodial cell processes (arrow) that interconnect long
widely spaced cell extensions. These extensions run between the closely packed
cell bodies and the basal portions of the cells which are spread over the basement
lamina. The filopodia may play a part in coordinating cytoskeletal arrangement
and cell shape at different levels in the epithelium as well as increasing its mechanical integrity.
All this evidence for intercellular coordination of cytoskeletal organization,
although circumstantial, strongly suggests that interactions between the cytoskeletons of adjacent cells might take place via their plasma membranes in
regions of close cell contact. This possibility is particularly attractive because
evidence for a range of structural associations between cytoskeletal and plasma
membrane components has recently emerged that could, at least in theory,
provide the molecular basis for such interactions.
Cytoskeletal/plasma membrane interactions
The rather substantial cytoskeletal/desmosome complexes that occur in
certain tissues (Figs. 6,7) (Kelly, 1966; Lentz & Trinkaus, 1971; Burnside, 1971;
Friedman, 1971) provided the first really tangible indications that intercellular
cytoskeletal coordination might be effected via certain cell surface regions
where cytoskeletal/plasma membrane associations occur. The diversity of
associations now documented (Weihing, 1979) provides a considerable 'vocabulary' for the cytoskeletons of adjacent cells to potentially indulge in fairly
detailed cell-surface-mediated 'conversations' concerning spatial coordination.
For example, it is becoming apparent that microtubules, microfilaments and
intermediate filaments can all probably become attached to certain cell junctions
- most especially to desmosomes. In certain situations: microtubules appear to
be connected to spot (Fig. 7) and belt desmosomes and to desmosome-like
junctions, certain intermediate filaments (tonofilaments) to spot desmosomes
10
J. B. TUCKER
Cytoskeletal coordination during embryogenesis
11
(Fig. 6), microfilaments to spot and belt desmosomes and intermediate junctions,
and fine filaments of as yet unknown composition to tight junctions (Friedman,
1971; Burnside, 1971; Bernstein & Wollman, 1976; Tucker & Meats, 1976;
Staehelin & Hull, 1978; Hull & Staehelin, 1979). However, it is also becoming
apparent that microtubules and microfilaments may be joined to integral
membrane proteins at many points on a cell's surface and not just where
plasma membranes form part of distinct cell junctions or specialized substrate
attachment regions such as hemidesmosomes and focal adhesions. Indeed it
has been argued that the plasma membrane should be regarded as an integral
component of a cell's cytoskeletal framework (Ben-Ze'ev, Duerr, Solomon &
Penman, 1979) because a substantial proportion of this membrane appears to
remain more or less intact, and connected to the underlying cytoskeleton, after
certain cells have been subject to extraction with a non-ionic detergent. Some of
the membrane proteins in question form the proximal portions of glycoprotein
surface receptors. The evidence comes mainly from studies of surface receptor
mobility and its spatial relationship to sub-plasmalemmal cytoskeletal deployment for certain cells, particularly fibroblasts and lymphocytes, in tissue culture
(Nicholson, 1974; Ash, Louvard & Singer, 1977; Flanagan & Koch, 1978). For
example, certain receptors diffuse in the plane of the membrane up to ten times
more rapidly in directions that parallel sub-plasmalemmal stress fibres than
they do at right angles to them (Smith, Clark & McConnell, 1979). Actin and
tubulin, as well as receptors are sometimes transported into 'caps' when
capping and receptor cross-linkage are induced by the application of multivalent
ligands such as lectins and antibodies (Gabbiani, Chaponnier, Zumbe &
Vassalli, 1977). Drugs that promote disassembly of microtubules and microfilaments influence receptor mobility in the plane of the plasma membrane in a
variety of ways (depending on the system studied) which seem to indicate that
these fibres anchor and/or propel surface receptors under certain conditions
(Weihing, 1979). Microfilaments may also interact with another type of cell
surface glycoprotein, fibronectin, that influences cell spreading and adhesion
(Yamada & Olden, 1978). In certain fibroblasts so exact is the alignment of
sub-surface microfilaments with fibronectin filaments projecting from the
Fig. 10. Tarsomeric epidermal cells in Calliphora. As pupal cuticle secretion is
completed much larger intercellular spaces than previously present intervene
between extensively narrowed elongate portions of cells. Filopodia (arrow) interconnect cells at these levels although cell bodies make direct contact with those
of their neighbours at their wider apices (towards the top of the figure) and where
the basal portions of cells spread across the basement membrane (towards the
bottom of the figure). Part of a haemocyte that is adhering to the inner haemocoelic
surface of this membrane is also shown. Living material mounted in saline (King,
Rubinson & Smith, 1956); interference contrast microscopy, x 1100.
Fig. 11. As Fig. 10 showing a more extensive set of filopodial interconnexions in an
inter-tarsomeric region, x 1400,
J. B. TUCKER
Fig. 12. Portions of three epidermal cells in the peripodial membrane of an eyeantennal imaginal disc in Drosophila melanogaster. A microtubule is connected by a
distinct bridge to one of the plasma membranes and two regions (arrows) where the
outer surfaces of plasma membranes approach to within 20 nm of each other are
shown. Previously unpublished micrograph courtesy of Mr A. A. E. Pyott. x 420000
plasma membrane's outer surface that direct connexion between them by some
transmembrane intermediary seems likely (Singer, 1979).
The precise ways in which cytoskeletal fibres connect to integral membrane
proteins and the substantial aggregates of membrane-bound materials that
make up the bulk of cell junctions and specialized substrate attachment regions
remains to be ascertained. It is already clear that many, perhaps all, connexions
to integral membrane proteins are not direct ones. Proteins such as ankyrin,
and perhaps a-actinin and vinculin, are included in molecular complexes that
join actinoid microfilaments to the plasma membrane in certain cells (Lux,
1979; Geiger & Singer, 1979; Geiger, Tokuyasa, Dutton & Singer, 1980;
Lloyd, 1980). Connexion of microtubules is sometimes effected by the bridges
that join them to adjacent portions of plasma membranes (Fig. 12) (Roberts,
1974; Tucker & Meats, 1976; Dentler, Pratt & Stephens, 1980) and resemble
intertubule arms and links. Bridges might attach to, and account for, the
(mysteriously) substantial quantities of tubulin that is tightly bound to certain
plasma membranes (Bhattacharyya & Wolf, 1976; Stephens, 1977; Matus,
1978; Weihing, 1979). Such bridging may be more common than is generally
appreciated. Portions of microtubules are sometimes situated within a ' bridge'slength' (up to about 40 nm) of the plasma membrane in many types of tissue
cells. It is only in cases where bridges are closely concentrated together along
most of the lengths of such tubule portions that they are likely to be detected.
So far there is no evidence for connection of intermediate filaments to the
plasma membrane except at desmosomes. However, lateral cross-bridge associations between microtubules and intermediate filaments (Yamada et al. 1971;
Albertini & Anderson, 1977; Rice, Roslansky, Pascoe & Houghton, 1980) and
microtubules and microfilaments (Griffith & Pollard, 1978) raises the possibility
that intermediate filaments might interact with plasma membranes generally
by virtue of connexion to other types of cytoskeletal fibres.
Cytoskeletal coordination during embryogenesis
13
Could some of these cytoskeletal/plasma membrane associations link the
cytoskeletons of adjacent tissue cells and transmit signals for cytoskeletal
coordination?
Transmembrane molecules and signal transmission
Four years ago Edelman (1977) suggested that surface receptors and associated cytoskeletal components (surface modulating assemblies) might be major
transmembrane control elements responsible for coordinating cell movement,
division, and interaction, if cell-associating molecules from different cells
interact directly with each other. In view of more recent developments this is an
attractive hypothesis for intercellular cytoskeletal coordination that merits more
detailed attention.
Structural interactions between cytoskeletal fibres, surface receptors, and
other components projecting from plasma membranes could provide a structurally contiguous intercellular signalling system for effecting cytoskeletal
coordination if certain receptors of adjacent cells bind to each other on a
specific complementary basis (Fig. 13). Intercellular cytoskeletal coordination
could be achieved if: the affinity with which these receptors bind to each other
depends on pre-existing connexions to cytoskeletal fibres (or the lack of them),
cytoskeleton/receptor attachments are sensitive to the state of receptorreceptor binding, and cytoskeletal organization is influenced by association
with receptors. Desmosomes, synapses, and other cytoskeletally associated cell
junctions may simply be very compact, highly specialized examples of a more
general, less readily detectable, intercellular coupling phenomenon.
This hypothesis is supported by a number of observations. It has been established for some cell types that the distribution of certain surface receptors is
sensitive to the spatial state of the underlying cytoskeleton which, in turn, is
modified when lectins and antibodies bind to surface receptors (Albertini &
Anderson, 1977; Thorn, Cox, Safford & Rees, 1979; Damsky, Wylie & Buck,
1979). Aggregated human blood platelets remain firmly attached to each other
after extraction with the non-ionic detergent Triton X-100; there are indications
that attachment is effected and maintained because transmembrane glycoproteins interconnect the cytoskeletons of adjacent platelets and that this mode
of structural coupling survives the extraction procedure (Phillips et al. 1980).
Cohesion of aggregating cellular slime mould amoebal cells is apparently
facilitated by the binding of complementary surface-bound lectin-like molecules
and receptor-like glycoproteins; a similar procedure may operate in certain
vertebrate tissues (Newell, 1977; Barondes, 1978). There are also indications
that binding of substrate-attached lectin-like molecules to surface receptors is
part of a mechanism for effecting cytoskeletal reorganization during cell
attachment to, and spreading on, certain substrata (Rees, Lloyd & Thorn, 1977;
Grinnell, 1978).
Studies of chemotaxis provide additional support for the notion that surface
14
J. B. TUCKER
Fig. 13. Schematic diagram summarizing spatial relationships and possible modes
of interconnexion between cytoskeletal fibres and integral membrane glycoproteins
(unshaded) where the plasma membranes of adjacent cells contact each other. The
circular cross-sectional profiles of microtubules, intermediate filaments, and
microfilaments (blocked in black) have been drawn to scale (diameters 24 nm, 10 nm,
6 nm, respectively), cross-bridges between them are stippled; molecules and molecular
complexes that bridge them to membrane proteins are cross-banded. The glycoproteins represent complementary sets of surface receptors and junctional proteins
that bind to each other at their distal extremities or are interconnected, perhaps in
some cell junctions, via other macromolecular intermediates. This representation
is not intended to exclude the possibility that a much greater range of receptors than
indicated above may participate. For example, each member of a complementary
pair of receptors might connect to a different type of cytoskeletal fibre. Several
different species of receptors might connect to the same type of cytoskeletal fibre
and modify its potential for connection to other cytoskeletal components and
receptors in a correspondingly varied range of ways. Similarly, a greater variety
of bridges than indicated above may be involved to joinfibresand receptors together.
This would also increase the range of structural interactions available for intercellular
signalling.
Cytoskeletal coordination during embryogenesis
15
receptors can mediate control of cytoskeletal deployment. Concentration
gradients of peptides and cyclic AMP chemotactically induce polarised orientation and locomotion of leucocytes (Zigmond, 1977) and cellular slime mould
amoebae (Newell, 1977), respectively. A certain amount of cytoskeletal reorientation and repolarization is involved as these cells effect up-gradient
directed locomotion (Malech, Root & Gallin, 1977; Spilberg, Mandell &
Hoffstein, 1979). Binding of chemotactic agents to surface receptors is apparently the first step for signalling cytoskeletal reorganization (Zigmond, 1977;
Williams, Snyderman, Pike & Lefkowitz, 1977; Hewitt, 1978). It would make
good sense in terms of design and material economy if the same basic system
monitors diffusible signals as well as bound signals presented to it by the
surfaces of nearby cells and noncellular substrates.
Accurate assessments of the distances separating the plasma membranes of
neighbouring cells in regions of close contact are essential for elucidating
whether the mechanism for cytoskeletal coordination proposed above (Fig. 13)
could operate. Unfortunately, this information is not available because of
uncertainty of the extent to which plasma membrane separation is altered during
preparation of cell aggregates and tissues for electron microscopy. The plasma
membranes of adjacent cells often appear to approach to within 10-20 nm of
each other even in regions that do not contribute to obvious junctional specializations (Fig. 12) (Heaysman & Pegrum, 1973; Radice, 1980). If these values
represent the situation in vivo then the surface receptors of one cell could bind
directly to those of a neighbour, because it is not unreasonable to suppose that
receptors project for 5-10 nm from the outer lipid bilayer surfaces of plasma
membranes (Fig. 13).
The scheme suggested here (Fig. 13) is an extreme representation of one of a
range of related ways in which intercellular cytoskeletal coordination may be
mediated by membrane proteins. It does not exclude the possibility that
coordination is also promoted by local changes in membrane permeability and
ion levels induced by receptor interactions (Wang, Heggeness & Singer, 1978),
although these would provide less stringent spatial information than structurally
contiguous coupling (Fig. 13) of cytoskeletal fibres across cell boundaries.
Signal transduction and the assembly and repositioning of cytoskeletal components
Although structural interactions between cytoskeletal fibres, surface receptors,
and cell junctions might locally influence fibre orientation and positioning (and
determine whether fibres elongate, shorten, or break down completely), some
of the cytoskeletal remodelling required for spatial coordination will involve
the assembly of new components. Could interactions between the plasma
membranes of adjacent tissues cells promote such assembly?
Actinoid microfilament assembly appears to be nucleated at sites attached
to plasma membranes in some situations (Gordon & Bushnell, 1979; Tilney,
1979). It is not yet clear if, or how, this is influenced by plasma-membrane
16
J. B. TUCKER
Fig. 14. Schematic diagrams showing the two possible distributions indicated by
immunofluorescent studies (Weber & Osborn, 1979) for microtubules in certain
cells assuming for simplicity that only one central pericentriolar MTOC (stippling)
is present, (a) Proximal portions of all microtubules remain associated with the
MTOC that nucleated their assembly as their distal portions elongate and ramify
around the cell surface, (b) Some tubules do not maintain this association, or
alternatively, were nucleated at the cell surface.
organization. However, in terms of proximity these sites are favourably located
for interaction with surface receptors that might promote or inhibit site formation and nucleating activities. Little is known about the initiation of intermediate filament assembly, although it is suspected that assembly of some types
may be nucleated by sites attached to desmosomes (Lazarides, 1980). On the
other hand, if cell surface modulation of microtubule initiation occurs, it must
often be of a very indirect nature. This is because in many tissue cell types
assembly of most, perhaps all, microtubules is nucleated by microtubuleorganizing centres (MTOCs). These MTOCs are situated several microns
from the nearest cell surface region and are commonly in the centre of the
cell close to the nucleus (Schliwa, 1978; Tucker, 1979; Raff, 1979). When
microtubules grow out from these MTOCs some of them extend towards and
Cytoskeletal coordination during embryogenesis
17
then run closely beneath the cell surface (Weber & Osborn, 1979; Brinkley,
Fistel, Marcum & Pardue, 1980). However, it is not yet clear whether they all
retain an association with an MTOC (Fig. 14#) (Warren, 1974), or if some of
them detach from the MTOC that nucleated their assembly and migrate out to
the cell surface (Fig. 14&). Nor can the possibility that the assembly of some
subsurface microtubules is nucleated by sites attached to the plasma membrane
be ruled out. The important point is that the spatial details of subsurface microtubule arrangement are unlikely to be organized by centrally positioned MTOCs.
Arrangement more probably depends on conditions, such as the deployment of
other cytoskeletal elements and receptors, at the cell surface. Hence, plasmamembrane interactions could play a considerable part in coordinating the
microtubule layout in adjacent cells even if they have little direct impact on the
production of new microtubules.
However, if one considers that subsurface microtubule arrangement may be
sensitive to the deployment of other plasma-membrane-associated fibres such as
microfilaments, then indications that microtubules are sometimes involved in
defining the positions and orientations of nearby microfilament bundles and
intermediate filaments (Lloyd, Smith, Woods & Rees, 1977; Wang & Goldman,
1978; Berlin, Caron & Oliver, 1979) cannot be ignored. They raise the question
of what organizes what, and whether there is an organizational hierarchy?
Microtubules, actin cables, and bundles of intermediate filaments often exhibit
distinctly different overall layouts within individual cells and sometimes display
a variety of orientations with respect to each other in different regions of the
same cell (Lazarides, 1978; Weber & Osborn, 1979; Blose, 1979; Henderson &
Weber, 1979). Thus there is no evidence for any species of 'master fibre' that
dictates the arrangement of all others in its vicinity under all physiological
conditions. It seems more likely that all the main cytoskeletal fibre types
interact reciprocally, perhaps by lateral cross-bridge associations, in ways that
are sensitive to a wide spectrum of local physicochemical conditions and
structural associations. Hence, surface-receptor-induced alterations in the
organization of any one type might less directly influence that of others in its
vicinity.
Cytoskeletal coordination in embryonic fields
There is a tantalising possibility that cytoskeletal arrays (and the cytoskeletal/
plasma membrane interactions considered above) also influence tissue shaping
by providing a coordinate system that assigns positional values in embryonic
fields. The need for coordinate systems has often been considered but the
material basis involved remains elusive (Wolpert, 1978). Cytoskeletal arrays
have much to offer as potential candidates but also exhibit features incompatible
with this role. Neither their suitability, nor their drawbacks, have been emphasised before.
An intercellular signalling system that supplies positional information in
18
J. B. TUCKER
an embryonic field needs to transmit positional signals at velocities of up to at
least 4/*m min"1 (Wolpert, 1971). Elongation rates of up to 19/nnmin- 1
(Ockleford & Tucker, 1973) and 180/mimin- 1 (Tilney, Hatano, Ishikawa &
Mooseker, 1973) occur in vivo during microtubule and micron* lament bundle
assembly, respectively. During contact inhibition specialized plasma membrane
contact regions are established and filaments become oriented on either side
of them at points where cells meet each other within 60 sec of contact being
made (Heaysman & Pegrum, 1973). Hence, some changes in cytoskeletal/cell
surface organization proceed fast enough for them to transmit positional
information. Perhaps such modulations emanate from field boundaries to
generate patterns of scalar variation in some cytoskeletal component(s) that
assigns positional values? There are indications that the organization of a cell's
cytoskeleton and surface receptors is sensitive to, and influences, intracellular
levels of cyclic AMP in some instances (Glennie, Stevenson, Stevenson &
Virji, 1979; Kennedy & Insel, 1979; Dedman, Brinkley & Means, 1979). Hence
spatial modulation of cytoskeletal/plasma-membrane organization might (via
levels of cyclic nucleotides and enzyme activity) generate corresponding
variations in gene expression for cells in different field regions. Furthermore,
both microtubules and microfilaments have a polarized molecular structure
(Begg, Rodewald & Rebhun, 1978; Bergen & Borisy, 1980) and contribute to
cytoskeletal complexes that exhibit polarized mechanochemical activities
(Kersey, Hepler, Palevitz & Wessels, 1976; Tucker, 1978). Thus, cytoskeletal
modulation might also establish cell and tissue polarities within a field.
However, cytoskeletal organization is sensitive to local conditions. These
include intricate microenvironmental details, such as the surface topography of
adjacent cells and substrata, that are often presumably of purely parochial
significance so far as the assignation of positional values is concerned. The
cytoskeletal/plasma-membrane interactions considered above apparently form
part of a sensitive signalling system that enables a cell's cytoskeleton to be
modified subtly and appropriately in response to minor fluctuations in the
cell's immediate surroundings. As in government, conflicts between global and
local interests are bound to arise. It is important that cytoskeletal modifications
in response to certain local conditions should not blur the overall positional
'field of view'. Furthermore, in some cases of cell differentiation, such as during
the production of a bristle or a scale by an insect epidermal cell, assembly of a
radically different cytoskeletal array from that found in surrounding cells that
are not so committed occurs (Greenstein, 1972). All these involvements presumably put severe limitations on the extent to which cytoskeletons could be
exploited for transmitting positional information. So will the marked cytoskeletal
reorganisations that take place during cell division.
These arguments do not, however, exclude the possibility that cytoskeletal/
cell surface organization in oocytes, zygotes, and early blastomeric stages may
have important spatial consequences for the generation of positional informa-
Cytoskeletal coordination during embryogenesis
19
tion (Quatrano, Brawley & Hogsett, 1979; Zalokar, 1979). It may set up
spatial differences that subsequently provide a basis for the location of field
boundary regions. For this role, spatial responses to local conditions, such as
contact with adjacent cells and tissues, could be positively advantageous during
specification of embryonic axes. So would an ability to regulate in, for example,
response to the changes in surface contact consequent on the death or loss of
an adjacent blastomere.
If cytoskeletons do not transmit positional information then they must
surely be sensitive to it and provide the embryo with a supracellular motor
network that plays an important part in the interpretation of this information.
This would most obviously be the case during gastrulation, neurulation, and
the performance of other major morphogenetic movements. It would also
facilitate control of finer details during tissue construction; for example,
spatially coordinated cell shape modulations that promote cuticular ripple
patterns in Rhodnius (Lawrence, Crick & Munro, 1972; Wigglesworth, 1973)
and a cell elongation pattern in newt neural plate (Jacobson & Gordon, 1976).
Multice/lularity and signal evolution
Did the 'first metazoans' exploit intercellular cytoskeletal coordination from
'the beginning'? Is it a facility that is employed in plant tissues?
Metazoans presumably evolved from protozoans. Contemporary protozoans
indulge in intercellular cytoskeletal/cell surface signalling and highly specific
cell surface recognition procedures, especially during feeding (Ockleford &
Tucker, 1973; Tucker, 1978) and mating (Preer, 1969). In trypanosomes the
flagellar membrane is attached to plasma membrane regions alongside the cell
body by desmosome-like junctions (Vickerman, 1969). Furthermore, the
arrangement of plasma-membrane-associated cytoskeletal components in ciliates
appears to be sensitive to positional information that may have a similar basis to
that which is transmitted in the embryonic fields of metazoans (Frankel, 1974;
Lynn & Tucker, 1976). Hence, there are plenty of indications that the prometazoan protozoans could have been well equipped to deal with new aspects
of intercellular cytoskeletal coordination that emerged with the advent of
multicellularity.
Although plant cell shaping is cytoskeletally coordinated (Marchant, 1979;
Gunning & Hardham, 1979; Lloyd, Slabas, Powell & Lowe, 1980) there are
no grounds for supposing that structurally and intercellularly contiguous
cytoskeletal/surface receptor complexes have evolved to effect coordination in
plant tissues. Outgrowth of motile cell extensions, cell migration, morphogenetic movements, actively contractile cell shortening, and hence a requirement for spatial coordination of the cytoskeletal activities associated with such
phenomena, are all lacking during plant tissue development. The entire range
of cell junctions that are so common in metazoan tissues are also lacking.
Plant tissue cells do not have the opportunity of exercising the dynamic
20
J. B. TUCKER
repertoire of surface contacts that is available in metazoans. In most cell surface
regions the plasma membranes of adjacent plant cells are separated by layers
of rigid extracellular wall material. Although the plasma membranes and
cytoplasm of neighbouring cells are usually continuous with each other via
the sometimes numerous plasmodesmata that traverse cell walls, there are as
yet no indications that plasmodesmata are structurally associated with cytoskeletal fibres (Gunning & Robards, 1976). Thus a dearth of cytoskeletal/
plasma membrane interactions for intercellular coordination may sunder the
plant kingdom from the animal kingdom as markedly as any of the other more
obvious distinctions.
There is a way of effecting intercellular cytoskeletal coordination that is
perhaps common to both animal and plant tissues. It is distinct from the
plasma-membrane-associated procedures considered so far. It is mainly confined
to coordination between cells of close clonal relation (most especially sister
cells) and is accomplished by the transmission of spatial instructions from one cell
generation to another during cell division. Indications that this is so are as
follows. It has been argued that cytoplasmic inheritance of specialized cortical
zones might provide a basis for specification of cortical microtubule arrangement in certain plant tissues (Gunning, Hardham & Hughes, 1978). Examinations of certain fibroblasts and neuroblastoma cells in tissue culture have
revealed that sister cells are often more or less identical twins, or alternatively
are sometimes mirror images of each other, in terms of cell shaping. For
fibroblasts this is also the case in terms of actin cable arrangement and patterns
of cell migration. Sister cells apparently inherit very similar sets of spatial
instructions concerning cytoskeletal deployment from a parent cell in these
instances (Albrecht-Buehler, 1977; Solomon, 1979). The coordination of
cytoskeletal layout in adjacent cells that results could play an important role
during tissue morphogenesis. For example, Solomon (1979) and Bate &
Grunwald (1981) have pointed out that it may help to set up certain mirrorimage axon configurations.
How might spatial instructions for cytoskeletal organization be transmitted
during metazoan tissue cell division? Pericentriolar MTOCs (Fig. 14) may be
involved (Solomon, 1980a). In many cases each sister cell receives one pericentriolar MTOC from the parent cell. These MTOCs replicate once prior
to cell division and are evenly segregated and cytoplasmically inherited during
division. Furthermore, there are indications that pericentriolar MTOCs exert
some control over the numbers and lengths of microtubules that grow out from
their surfaces (see Solomon, 1980&). Perhaps, in addition, a pericentriolar
MTOC can be switched into one of several modes and each provides a different
set of spatial instructions for control of microtubule orientation and/or the
extent to which microtubules elongate as they grow out in particular directions.
Some basal-body-associated MTOCs certainly do control microtubule orientation (see Tucker, 1979). Hence pericentriolar MTOCs might provide spatial
Cytoskeletal coordination during embryogenesis
21
programmes that influence cell shaping and locomotion via their impact on
cytoskeletal arrangements. No doubt, analyses of cortical basal body positioning and cytoskeletal assembly in certain 'doublet' ciliates that exhibit mirrorimage cell-surface patterns will continue to be illuminating in this context
(Jerka-Dziadosz & Frankel, 1979; Grimes, McKenna, Goldsmith-Spoegler &
Knaupp, 1980). A very sophisticated level of intercellular cytoskeletal coordination is displayed during metazoan embryogenesis. It is worth considering
whether its evolution has been facilitated because inherited central control by
pericentriolar MTOCs has been successfully integrated with cortical plasmamembrane-mediated control.
I thank Drs J. Cooke, C. W. Evans and M. J. Milner for valuable discussions, Mr J. B.
Mackie and Mrs M. Meats for assistance with electron microscopy, and the Science Research
Council (U.K.) for grant support.
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{Received 13 January 1981, revised 30 April 1981)