Cleavage Furrow Establishment A Preliminary to Cylindrical Shape Change

Transcription

1 AMER. ZOOL., 13: (1973). Cleavage Furrow Establishment A Preliminary to Cylindrical Shape Change RAYMOND RAPPAPORT Department of Biological Sciences, Union College, Schenectady, New York and The Mount Desert Island Biological Laboratory, Salsbury Cove, Maine SYNOPSIS: The predictable pattern of cell shape changes characterizing animal development must be a consequence of control mechanisms that are at least analogous to those operating in dividing cells. When cells change shape by an internal mechanism, it is implied that they also contain systems that will activate and deactivate the mechanism, localize it within the cell, insure proper timing, and impart the proper vectoral qualities. Experimental investigations designed to elucidate similar processes in dividing cells reveal that the physical mechanism that accomplishes cytokinesis is established at or near the equatorial cell surface by the mitotic apparatus. The process is completed by late metaphase or early anaphase. In cleaving eggs a pair of asters can substitute for the intact mitotic apparatus. The nature of the stimulus which apparently passes from the mitotic apparatus to the surface is presently unknown. It moves toward the surface at about 6 microns per minute and requires about 1 minute to establish the mechanism. The resulting equatorial contractile activity is initially isotropic but becomes anisolropic at the beginning of visible constriction. The embryonic cell's ability to alter its shape by application of intrinsic physical force at a predictable time and in a predictable manner strongly suggests the existence of control systems which have hardly yet been subjected to experimental analysis. Activity of this kind implies the existence of a system for establishing a force-producing mechanism in a localized cell region at a time consistent with or dictated by an overall developmental time table. The mechanism must be activated and deactivated and some means must exist for insuring that the force has the proper vectoral qualities. Investigations concerning the mechanisms of animal cell division have dealt with similar mechanisms for about 100 years, and perhaps a brief account of some of the thought and technique that has evolved in studies of cytokinesis may prove useful in studies of morphogenesis. Three long-range goals have historically determined the direction taken by investigations on cytokineses. The first goal is elucidation of the nature of the force that accomplishes division. The second is loca- The original investigations described in this report were supported by giants from the National Science Foundation. 941 tion of the force-producing mechanism within the cell, and the third goal is an understanding of the process that establishes the mechanism. Simple observation (regardless of elegance or magnification) has rarely furnished conclusive information on any of these subjects. Observations of division do not usually permit discrimination between cause and effect and may not reveal whether a structure or phenomenon present during division is causally related to it. That cell division depends upon the operation and interaction of many cell structures and processes has been evident since the process was first clearly viewed. Its complex nature means that unequivocal interpretation of experimental results is possible only when the cell's basic fabric is intact and functioning. The constraints thus placed upon experimental design are severe. Progress has been accomplished by stepwise, systematic elimination of hypothetical alternatives rather than by heroic breakthroughs. For reasons of experimental convenience, cleaving eggs and blastomeres are the most commonly used experimental material for studies of cytokinesis. However, investigations in which the responses to experimentation of cleaving eggs and adult tis-

2 942 RAYMOND RAPPAPORT FIG. 1. (left) Manipulation chamber. E, egg; M, microscope objective; P, micropipette; W, wall made of coverslip. (right) Relation between position of mitotic figure and cleavage plane, a-c, Successive stages of cleavage when the spindle is removed: a, before removal of the spindle; b, after removal; c, furrow appears at a predetermined position, d-f, Successive stages of cleavage when sue cells were compared (Rappaport and Rappaport, 1968) revealed no fundamental differences. The outstanding morphological differences between these two kinds of cells are the relatively greater size of the asters during the cleavage divisions and the more irregular form of adult tissue cells. The location of the cleavage mechanism was determined by experiments in which regions and organelles were removed, displaced, or specifically disorganized. Persistence of division despite such localized disruption implies that the affected area makes no essential contribution to the process. When the mitotic apparatus, comprising the spindle, asters, and chromosomes, is removed at late metaphase or later, while the sea urchin is still spherical, division ensues and a normallooking cleavage furrow bisects the egg into a pair of enucleated blastomeres (Fig. 1) (Hiramoto, 1956). Hiramoto (1965) also showed that after anaphase the mitotic apparatus and immediately surrounding cytoplasm could be displaced by a large oil droplet or disrupted by injection of the mitotic figure is displaced by the removal of a part of egg protoplasm: d, before the displacement of the mitotic figure; e, after displacementastral rays of the lower aster are much elongated; f, furrow appears at a predetermined position. In both series, cleavage planes are independent of shifted position of asters. (From Hiramoto, 1956.) isotonic sucrose solutions and sea water without blocking cleavage. In other similar experiments, cleavage of sand dollar eggs was unaffected when the cytoplasm in the equatorial plane was vigorously stirred with a needle (Fig. 2) (Rappaport, 1966). Vertebrate tissue cells behave in similar fashion, as newt kidney cells continue to divide while the mitotic apparatus and associated cytoplasm are pushed back and forth through the equatorial plane (Rappaport and Rappaport, 1968). These experiments, and others, performed on cleaving cells indicate that the furrowing process in animal cells is in no way physically dependent upon the arrangement or presence of any structures in the subsurface cytoplasm. They have been interpreted as indicating that the physical mechanism that accomplishes division is in or attached to the surface. Although the mitotic apparatus plays no physical role in furrowing, it appears to be the structure that determines where the furrow will appear. One of the earliest clear demonstrations of this relationship

3 CLEAVAGE FURROW ESTABLISHMENT 943 FIG. 2. Cleavage in a sand dollar egg with a moving needle inserted through the cleavage plane. The needle was swept back and forth during the period between the photographs. (From Rappaport, 1966.) was that of Conklin (1917) who produced abnormally large polar bodies by centrifuging the meiotic apparatus to the center of the egg before metaphase. By manipulating the cell's geometry before the furrow is established, it has been shown that, in cleaving eggs, the asters of the mitotic apparatus can substitute for the intact mitotic apparatus (Rappaport, 1961; Hiramoto, 1971). After a spherical sand dollar egg is reshaped into a torus by forcing a glass ball completely through it, the first cleavage results in a horseshoe-shaped binucleate cell (Fig. 3). As the egg enters the second cleavage cycle, the two mitotic apparatuses form in the arms of the horseshoe and extend toward its bend. Two furrows form in the arms of the horseshoe adjacent to the spindle of the intact mitotic apparatus and almost simultaneously a third furrow forms in the bend of the horseshoe between two asters that were never joined by a spindle. Other experiments have shown that furrows will form between cytasters (Wilson, 1901) and sperm asters and between combinations of amphiasters and sperm asters (Sugiyama, 1951). Although the dispensability of the spindle has been clearly shown in cleaving eggs, none of the results thus far achieved eliminate the possibility that the spindle may share the asters' capacity for furrow establishment, even though it may normally remain unused by reason of geometrical circumstances. No comparable analysis of the role of asters in furrow establishment in tissue cells has yet been accomplished, and the relatively larger spindle in these cells lies closer to the equatorial surface. I have speculated elsewhere (Rappaport, 1971) that the important components of the mitotic apparatus for this function may be its linear elements. Whether linear elements of the asters or spindle were primarily responsible for furrow establishment could depend upon their relative sizes and the geometrical relations obtaining in the cell. Since it is customary to consider the furrowing mechanism as a modified portion of the cell surface, a brief discussion of the meaning of the term "surface" in this context is pertinent. In echinoderm eggs, the plasma membrane is underlain by a cytoplasmic region which has for some years been termed the cortex. The cortex was demonstrated in echinoderm eggs by microdissection (Chambers, 1921) and by centrifugation (Marsland, 1939). Its dimensions were carefully determined by Hiramoto (1957). It comprises a S-4/i thick layer that is inseparable from the plasma membrane and is denser than the cytoplasm lying immediately beneath it. In dividing cells, the cortex is thickest at the base of the furrow. Pigment granules may be held within it and dense strands of cytoplasm may be pulled from its undersurface with needles. Unfortunately no ultrastructural basis for these cortical properties has been clearly demonstrated, and nearly all studies on the cell cortex have

4 944 RAYMOND RAPPAPORT FIG. 3. Cleavage of a torus-shaped cell. Condition of the mitotic apparatus is shown in line drawings. The position of the spindle is shown by a double line. Note synchrony with controls. Initial temperature 19.5 C. Timing begins at fertilization. Upper left: immediately before furrowing (69 min). Upper right: first cleavage completed, producing a binucleate cell (79 min). Lower left: concerned marine invertebrate eggs. However, it appears that the cleavage mechanism is established in what must be considered the cortical region in those cells where the mechanism has apparently been observed (Schroeder, 1968; Arnold, 1968, 1969). More information concerning the structure, behavior, and constitution of the cortex is highly desirable. The mitotic apparatus apparently determines the position of the furrow by essecond cleavage; two cells have divided from the free ends of the horseshoe and the binucleate cell, and the binucleate cell is dividing between the polar regions of the asters of the second division (142 min). Lower right: division completed; each cell contains one nucleus (144 min). (From Rappaport, 1961.) tablishing the division mechanism in a localized portion of the cell surface. Any part of the surface can be changed into furowing mechanism, and the mitotic apparatus could accomplish its role by altering the constitution of the subsurface cytoplasm. The alteration, or stimulation, might result from materials added to or subtracted from the local area, or from changes that move by propagation rather than transport (Rappaport, 1965). The in-

5 CLEAVAGE FURROW ESTABLISHMENT teraction between mitotic apparatus and surface appears to be completed by late metaphase or early anaphase, for thereafter the furrow develops and functions after the two are experimentally isolated from each other (Hiramoto, 1956). Establishment of the division mechanism in the cell surface imposes regional functional differentiation which persists for a relatively brief part of the cell cycle. It is logical to propose that the part of the surface influenced by the mitotic apparatus is altered in physical properties and behavior, and that these alterations precipitate division. Other parts of the cell surface would, presumably, play no essential physical role in the process. Although both the polar (Swann and Mitchison, 1958; Wolpert, 1960) and equatorial (Rappaport, 1965) surfaces have been suggested as the recipients of mitotic apparatus stimulation, present evidence supports the latter alternative. No essential geometrical relation between the mitotic apparatus and the polar surfaces can be shown to exist. For instance, cells that are extremely attenuated by attached weights long before the position of the furrow is determined cleave FIG. 4. Cleavage of a sand dollar egg attenuated by tensile stress. The dark sphere is a glass bead. (From Rappaport, 1960.) * 1 II 1II S 30- -r- * 1 i i i Interastrol distance (/x) FIG. 5. Summary of data from experiments in which the distance between the asters (interastral distance) and the distance from a line drawn between the astral centers to the equator (spindleto-surface distance) are varied. When the spindleto-surface distance is 35^ or more and the interastral distance is 35^t or more, furrowing almost invariably fails. If the spindle-to-surface distance is reduced to 20^ or less, furrowing occurs in conjunction with a 35fi interastral distance. Plus indicates a furrow formed adjacent to the asters; minus indicates no furrow formation in that location. (From Rappaport, 1969.) normally, although the distance from the mitotic apparatus to the polar surfaces is greatly increased (Fig. 4) (Rappaport, I960). On the other hand, relatively small alterations of the distances between the asters and the distances from the asters to the equatorial surface have profound effects on furrow formation (Fig. 5). When the distance between asters is increased, furrows do not form. Furrows will form, however, in surfaces located close to abnormally distant asters (Rappaport, 1969). The observation that a deficiency arising from increase in one dimension can be remedied by decreasing the other dimension lends support to the proposal that furrows may be established by the joint

6 946 RAYMOND RAPPAPORT action of the asters on the equatorial surface. The stimulation process was not an early subject of speculation and experimentation, as was the physical nature of the division mechanism, and investigations concerning its nature are relatively recent. We have no information concerning the constitution of the stimulus, and detailed information will probably await a clearer conception of the physico-chemical basis of the functioning division mechanism. Experimentation has permitted a rough characterization of some aspects of the process. In many experiments bearing on this topic, the cell's shape is altered early in the mitotic cycle so that the furrow's position is established under unusual geometrical conditions. Frequently advantage is taken of the fact that in flattened cells the furrows appear in the margins and not in the flattened surfaces. In normal and experimentally altered cells the time and position of the furrow's first appearance is correlated with the position of the mitotic apparatus. When the apparatus is centered, as in typical sea urchin eggs, the furrow appears at the same time at all points on the equatorial circumference and it constricts symmetrically. In some eggs the nucleus is excentric. In these cases, typified by Astriclypeus manni, the furrow appears first at the surface closest to the mitotic apparatus so that the egg is temporarily heart-shaped (Dan and Dan, 1947). Subsequently, furrowing begins on the diametrically opposite side and then the entire equatorial surface engages in the division process. In cases of extreme nuclear excentricity, such as is found in coelenterate eggs, furrows form at the surface closest to the mitotic apparatus, and the diametrically opposite surface never actively participates. Furrowing is unilateral, as the furrow cuts through from one side only. These cleavage patterns are consequences of the geometrical relation between the mitotic apparatus and the surface; for eggs which normally form symmetrical furrows will form unilateral furrows and vice versa when the mitotic apparatus is shifted (Rappaport and Conrad, 1963). The observation that the time of the furrow's appearance is correlated with distance from the mitotic apparatus permits estimation of the rate of movement of the stimulus across the cell. In a flattened cell with an excentric nucleus, the furrow appears first at the equatorial surface closer to the mitotic apparatus, and later at the opposite, more distant, margin. All other things being equal, the time difference between the appearance of the two furrows should be proportional to the difference in distances between the two margins and the mitotic apparatus. In making the determinations, the distance from the spindle center to opposite equatorial margins was measured; a stopwatch was started when the furrow appeared in the nearer margin and stopped when it appeared in the more distant margin. In a series of determinations made on sand dollar eggs with varying degrees of nuclear excentricity the relation between time and distance was linear (Fig. 6), and the rate was calculated to be 6.3 ± 1.8 microns per min (Rappaport, 1972). This rate is slower than free diffusion (Swann, 1951), and the types of cytoplasmic streaming usually studied (Wolpert, 1965); it approximates the rate of microtubular growth (Bajer and Mole'- Bajer, 1972). These data may be correlated with the observations that the linear appearance of the astral rays is based primarily upon their microtubular content (Rebhun and Sander, 1967) and that the position of the furrow is established at late metaphase or early anaphase when the astral rays achieve maximum length (Wilson, 1895). The observation that a very excentric mitotic apparatus only establishes a furrow in the nearer equatorial margin has provided a method for estimating the time necessary for furrow establishment (Rappaport and Ebstein, 1965). After the furrow develops in the nearer region of a sand dollar egg with an experimentally displaced nucleus, a furrow can be established in the more distant margin if it is pushed toward the mitotic apparatus and held there. Under these circumstances, the total time

7 CLEAVAGE FURROW ESTABLISHMENT DISTANCE (microns) FIG. 6. The relation between the difference in distance between the spindle and the near and distant equatorial cell margin and the difference in time between the appearance of the furrow in the near and distant margins. (From Rappaport, 1972.) between pushing in the surface and the appearance of an active furrow is 3y min, regardless of the length of time the surface is held close to the mitotic apparatus. When the mitotic apparatus and surface are held together for 1 min, the surface upon release resumes its original contour beyond the limit of influence of the mitotic apparatus, and then 2i/ 2 min after release, develops a furrow. Exposure of surface to the mitotic apparatus for less than 1 min fails to produce a furrow. These results suggest that a stimulus period of about 1 min irreversibly alters the exposed surface; during the ensuing 2i/ 2 min latent period, the molecular reorganization essential for establishment of the functional division mechanism takes place. These observations permit the construction of a very approximate time table for events immediately preceding cytokinesis. If we use as an example an echinoderm egg with a 150 micron diameter, and assume that the stimulus originates near the centrosomes, and we determine that time 0 is the moment of appearance of the active furrow, the stimulus would begin to move toward the surface at about T = 12 min. At T =: S]/ m 2 in the stimulus reaches the surface and att = 2i/ 2 min the furrow's position is established. At some time between T = 2\/ 2 min and T = 0, equatorial contraction begins. Scott's (1960) study of the movement of cortical pigment granules revealed that the initial contraction is isotropic but becomes anisotropic and circumferential with the appearance of the furrow. His observation suggests that the cleavage stimulus may initially impart no vectoral information to the surface. We now have a general idea of what goes on in a cell immediately before and during division at a very complex level of organization. Several active cell components have been identified, as has the nature of their participation. It is reasonable to expect that analogous events occur in developing cells that change their form by application of intrinsic forces. A clear understanding of the mechanisms which enable such cells to control the time, location, and orientation of the internally applied stresses will require rarely used and, perhaps, refreshing experimental approaches to morphogenesis. FIG. 7. Egg outlines and corresponding pigment granules patterns during 4 stages of cleavage in an Arbacia egg. Patterns shrink two dimensionally in cells marked 13 and 14, but only circumferentially in 15 and 16. (From A. Scott, 1960.) REFERENCES Arnold, J. M Formation of the first cleavage furrow in a telolethical egg. Biol. Bull. 135: Arnold, J. M Cleavage furrow formation in a telolethical egg (Loligo pealii) I. Filaments in early furrow formation. J. Cell Biol. 41: Bajer, A. S., and J. Mole-Bajer Spindle dynamics and chromosome movement Int. Rev.

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