Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study.

New methods of visualizing subcortical actin filament bundles, or fibrils, in Characean internodes confirm that they are associated with chloroplasts at the surface facing the streaming endoplasm, and reveal that they are continuous over long distances. With the scanning electron microscope, an average of four to six fibrils are seen bridging a file of chloroplasts. The same configuration appears in negatively stained preparations of large blocks of chloroplast files connected by actin fibrils. Few branches of the subcortical fibrils are evident. These findings are discussed with respect to the mechanism of cytoplasmic streaming in Characeae.

Migratory cell locomotion versus nerve axon elongation: differences based on the effects of lanthanum ion.

The effects of lanthanum ions (La(+++)) on the locomotion and adhesion of g lial cells and elongating nerve axons are reported. La(+++) increases adhesion of both glia and of nerve growth cones to a plastic substratum. La(+++) also markedly reduces glia locomotion, but it does not inhibit nerve elongation. Electron-opaque deposits are seen on the cell surface and within cytoplasmic vesicles of glia and nerves cultured in a La(+++)-containing medium. Possible modes of action for La(+++) are discussed, particularly the possibilities that Ca(++) fluxes or Ca(++) involvement in adhesion are altered by La(+++). The results are consistent with the hypothesis that cell migration and nerve axon elongation differ in mechanism, with respect to both adhesive interactions and the activity of microfilament systems.

Microfilaments and cell locomotion.

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro. Such cells are found to contain two types of microfilament systems: First, a sheath of 50-70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes. Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell. The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network. Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited. Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis. Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity. The microfilament network is, therefore, indispensable for locomotion.

Ultrastructure and function of growth cones and axons of cultured nerve cells.

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.

Expression of an epidermal antigen used to study tissue induction in the early Xenopus laevis embryo.

A monoclonal antibody (Epi 1) has been produced that recognizes an antigen expressed in epidermal cells of Xenopus laevis embryos. The Epi 1 antigen appears in embryonic epidermis at the end of gastrulation and is not expressed in nonepidermal structures derived from ectoderm (for example, neural tube or cement gland). The capacity to express the Epi 1 antigen is restricted to cells of the animal hemisphere prior to the midblastula stage of development (stage 8), and tissue interactions during gastrulation inhibit the expression of the Epi 1 antigen in neural ectoderm. This epidermal antigen will be a valuable marker for studies of ectodermal commitment.

Microfilaments in cellular and developmental processes.

In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile. In all of the cases that have been examined so far, microfilaments of some type are observed in the cells; furthermore, those filaments are at points where contractility could cause the respective phenomenon. The correlations from the cytochalasin experiments greatly strengthen the case; microfilaments are present in control and "recovered" cells and respective biological phenomena take place in such cells; microfilaments are absent or altered in treated cells and the phenomena do not occur. The evidence seems overwhelming that microfilaments are the contractile machinery of nonmuscle cells. The argument is further strengthened if we reconsider the list of processes insensitive to cytochalasin (Table 2). Microtubules and their sidearms, plasma membrane, or synthetic machinery of cells are presumed to be responsible for such processes, and colchicine, membrane-active drugs, or inhibitors of protein synthesis are effective at inhibiting the respective phenomena. These chemical agents would not necessarily be expected to affect contractile apparatuses over short periods of time, they either do not or only secondarily interfere with the processes sensitive to cytochalasin (Table 1). It is particularly noteworthy in this context that microtubules are classed as being insensitive to cytochalasin and so are not considered as members of the "contractile microfilament" family. The overall conclusion is that a broad spectrum of cellular and developmental processes are caused by contractile apparatuses that have at least the common feature of being sensitive to cytochalasin. Schroeder's important insight (3) has, then, led to the use of cytochalasin as a diagnostic tool for such contracile activity: the prediction is that sensitivity to the drug implies presence of some type of contractile microfilament system. Only further work will define the limits of confidence to be placed upon such diagnoses. The basis of contraction in microfilament systems is still hypothetical. Contraction of glycerol-extracted cells in response to adenosine triphosphate (53), extraction of actin-like or actomyosin-like proteins from cells other than muscle cells (54), and identification of activity resembling that of the actomyosin-adenosine triphosphatase system in a variety of nonmuscle tissues (40, 54) are consistent with the idea that portions of the complex, striated muscle contractile system may be present in more primitive contractile machinery. In the case of the egg cortex, calcium-activated contractions can be inhibited by cytochalasin. If, as seems likely, microfilaments are the agents activated by calcium, then it will be clear that they have the same calcium requirement as muscle. Biochemical analyses of primitive contractile systems are difficult to interpret. Ishikawa's important observation (31), that heavy meromyosin complexes with fine filaments oriented parallel to the surface of chondrocytes and perpendicular to the surface of intestinal epithelial cells, implies that both types of filaments are "actin-like" in this one respect. Yet, it is very likely that these actin-like filaments correspond respectively to the cytochalasin-insensitive sheath of glial and heart fibroblasts and the core filaments of oviduct microvilli. No evidence from our studies links contractility directly to these meromyosin-binding filaments. Apart from this problem, activity resembling that of the myosin-adenosine triphosphatase has been associated with the microtubule systems of sperm tails and cilia (55), but those organelles are insensitive to cytochalasin in structure and function. Clearly, a means must be found to distinguish between enzymatic activities associated with microfilament networks, microfilament bundles, microtubules, and the sheath filaments of migratory cells. Until such distinctions are possible, little of substance can be said about the molecular bases of primitive contractile systems. Three variables are important for the control of cellular processes dependent upon microfilaments: (i) which cells of a population shall manufacture and assemble the filaments; (ii) where filaments shall be assembled in cells; and (iii) when contractility shall occur. With respect to distribution among cells, the networks involved in cell locomotion are presumed to be present in all cells that have the potential to move in cell culture. In this respect, the networks can be regarded as a common cellular organelle in the sense that cytoplasmic microtubules are so regarded. In some developing systems, all cells of an epithelium possess microfilament bundles (7, 13), whereas, in others, only discrete subpopulations possess the bundles (5, 6). In these cases the filaments can be regarded as being differentiation products associated only with certain cell types. These considerations may be related to the fact that microfilament networks are associated with behavior of individual cells (such as migration, wound healing, and cytokinesis), whereas the bundles are present in cells that participate in coordinated changes in shape of cell populations. With respect to placement in cells, two alternatives are apparent, namely, localized or ubiquitous association with the plasma membrane. Microfilament bundles of epithelial cells are only found extending across the luminal and basal ends of cells. In this respect they contrast with desmosomal tonofilaments and with microtubules, each of which can curve in a variety of directions through the cell. The strict localization of microfilament bundles probably rests upon their association with special junctional complex insertion regions that are only located near the ends of cells. In the case of mitotically active cells, the orientation of the spindle apparatus may determine the site at which the contractile ring of microfilaments will form (4, 56); this raises the question of what sorts of cytoplasmic factors can influence the process of association between filament systems and plasma membranes. In contrast to such cases of localized distribution, contractile networks responsible for cell locomotion are probably found beneath all of the plasma membrane, just as the network of thrombosthenin may extend to all portions of the periphery of a blood platelet. This ubiquitous distribution probably accounts for the ability of a fibroblast or glial cell to establish an undulating membrane at any point on its edge, or of an axon to form lateral microspikes along its length. The third crucial aspect of control of these contractile apparatuses involves the choice of when contraction shall occur (and as a corollary the degree or strength of contraction that will occur). In the simplest situation, contraction would follow automatically upon assembly of the microfilament bundles or networks. In cleavage furrows of marine embryos (4), for instance, microfilaments are seen beneath the central cleavage furrow and at its ends, but not beyond, under the portion of plasma membrane that will subsequently become part of the furrow. This implies that the furrow forms very soon after the contractile filaments are assembled in the egg cortex. In other cases, microfilaments are apparently assembled but not in a state of (maximal?) contraction. Thus, networks are seen along the sides of migratory cells, although such regions are not then active as undulating membrane organelles. Similarly, microfilament bundles occur in all epithelial cells of the salivary gland (13), or pancreatic anlage (7), although only the ones at discrete points are thought to generate morphogenetic tissue movements. Likewise, bundles begin to appear as early as 12 hours after estrogen administration to oviduct, although visible tubular gland formation does not start until 24 to 30 hours. Finally, streaming in plant cells can wax and wane, depending upon external factors such as auxin (57). All of these cases imply a control mechanism other than mere assembly of the microfilament systems and even raise the possibility that within one cell some filaments may be contracting while others are not. In discussing this problem, it must be emphasized that different degrees of contraction or relaxation cannot as yet be recognized with the electron microscope. In fact, every one of the cases cited above could be explained by contraction following immediately upon some subtle sort of "assembly." Inclusive in the latter term are relations between individual filaments, relations of the filaments and their insertion points on plasma membrane, and quantitative alterations in filament systems. Furthermore, the critical role of calcium and high-energy compounds in muscle contraction suggest that equivalent factors may be part of primitive, cytochalasinsensitive systems. The finding that calcium-induced contraction in the cortex of eggs is sensitive to cytochalasin strengthens that supposition and emphasizes the importance of compartmentalization of cofactors as a means of controlling microfilaments in cells.

Differential antigen adhesivity used to select spleen cells for the production of monoclonal antibodies to embryonic neurons.

Monoclonal antibodies specific for cell surface antigens on embryonic chick ciliary ganglion neurons (CG) have been obtained at high frequencies by fractionating spleen cells from immunized mice according to their adhesiveness for cell surfaces of the cultured neurons. Spleen cells from mice that had been immunized with live or lightly fixed (0.125% glutaraldehyde) CG neurons were selected for subsequent hybridization with myeloma cells after fractionation on lawns of CG neurons in tissue culture. Immunized spleen cells were cultured with the neurons for 4-7 days prior to fractionation. Three groups of spleen cells were selected for fusion with a myeloma cell line: a non-adherent population of spleen cells, a population of spleen cells that could be removed from the neuronal cells by shaking on a vibratory shaker for 1 h, and a population that could be removed from the neuronal cells only by treatment with low concentrations of trypsin. Of the 3 groups of spleen cells, the population that required trypsin treatment produced the greatest number of hybridomas specific for neurons and for neuronal cell surfaces. Fewer neuron-specific hybridomas resulted from fusion of the group of spleen cells that could be removed from the antigen lawn by shaking. None of these was specific for the CG neurons. No neuron-specific hybridomas resulted from the fusion of the cells that did not adhere to the neuronal cells, and at most only 1 neuron-specific hybridoma resulted from fusions of comparable groups of unselected spleen cells (spleen cells from immunized animals which were not selected on antigen lawns).

Cytochalasin B: effects upon microfilaments involved in morphogenesis of estrogen-induced glands of oviduct.

The administration of estrogen to immature chicks induces formation of tubular glands and differentiation of cells in the oviduct. As glands begin to form, organized bundles of 40-50 A filaments appear at the luminal end of the cells. These structures are not present in uninduced oviducts. Cytochalasin treatment of oviducts early in gland formation results in the disappearance of young glands already present and the inhibition of new gland formation. Furthermore, organized microfilaments are no longer present. When the oviducts are washed free of cytochalasin, however, organized bundles of microfilaments reappear. The correlations between the presence of filaments and formation and glands suggest that filaments are important agents in morphogenesis, presumably because of contractile properties which generate changes in cell shape and, consequently, tissue shape.

The early development of mystacial vibrissae in the mouse.

The initial generation of the pattern of mystacial vibrissae (whiskers) in the mouse is described. The maxillary process is present in 10-day embryos but has a relatively flat surface. Beginning at approximately 11.5 days, the first sign of vibrissal development is the formation of ridges and grooves on the maxillary and lateral nasal processes. The first vibrissal rudiment to form subsequently appears posterior to the most ventral groove on the maxillary process. It is the most ventral whisker of the posterior, vertical row. The next few rudiments appear: dorsal to the first, also in the vertical row; and anterior to the first, on the ventral-most ridge and in the groove beneath it. Formation of vibrissal rudiments continues in a dorsal and anterior progression usually by an apparent partitioning of the ridges into vibrissal units. The hypothesis that this patterning of mystacial vibrissae might be determined by the pattern of innervation in the early mouse snout was investigated. Nerve trunks and branches are present in the maxillary process well before any sign of vibrissal formation. Because innervation is so widespread there appears to be no immediate temporal correlation between the outgrowth of a nerve branch to a site and the generation of a vibrissa there. Furthermore, at the time just prior to the formation of the first follicle rudiment, there is little or no nerve branching to the presumptive site of that first follicle while branches are found more dorsally where vibrissae will not form until later. Thus, a one-to-one spatial correlation between nerve and follicle sites also appears to be lacking. The developmental changes in ultrastructure within the neurites of the trunks and branches as well as the apparent rearrangements of the nerve trunks suggest that early innervation of the snout is a labile phenomenon and that the vibrissal pattern begins to be established before the neural pattern is completely developed. The results indicate that vibrissal pattern formation is likely to be a complex process relying on the interplay of the cells and tissues involved, rather than on unidirectional instructions from neurons to other cell types.