Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: I. Ultrastructure and effects of cytochalasin B and colchicine

Formation of polar lobe constrictions and cleavage furrows in fertilized eggs of the marine mudsnail, Ilyanassa obsoleta, is associated with localized concentrations of microfilaments in the cortical cytoplasm. These microfilaments disappear after treatment with cytochalasin B, with concomitant regression of polar lobe constrictions and cleavage furrows. Microtubules are present in the noncortical cytoplasm of the polar lobe and disappear after treatment with colchicine. Colchicine application early in lobe development inhibits both polar lobe formation and cytokinesis; however, drug treatment later in lobe development, although still inhibiting cytokinesis, fails to inhibit the continued constriction of the polar lobe neck and subsequent lobe resorption. The data therefore suggest that separate colchicine-sensitive steps are required for the complete constriction of the polar lobe neck and for initiation of cytokinesis. Polar lobe necks constrict at two markedly different rates in contrast to the smooth, single-phase progression of cleavage furrows. As with cytokinesis, polar lobe formation may occur by the contraction of a microfilament ring whose polymerization or activity is regulated by microtubules.     

Contractile ring formation in Xenopus egg and fission yeast

How actin filaments (F-actin) and myosin II (myosin) assemble to form the contractile ring was investigated with fission yeast and Xenopus egg. In fission yeast cells, an aster-like structure composed of F-actin cables is formed at the medial cortex of the cell during prophase to metaphase, and a single F-actin cable(s) extends from this structure, which seems to be a structural basis of the contractile ring. In early mitosis, myosin localizes as dots in the medial cortex independently of F-actin. Then they fuse with each other and are packed into a thin contractile ring. At the growing ends of the cleavage furrow of Xenopus eggs, F-actin at first assembles to form patches. Next they fuse with each other to form short F-actin bundles. The short bundles then form long bundles. Myosin seems to be transported by the cortical movement to the growing end and assembles there as spots earlier than F-actin. Actin polymerization into the patches is likely to occur after accumulation of myosin. The myosin spots and the F-actin patches are simultaneously reorganized to form the contractile ring bundles. The idea that a Ca signal triggers cleavage furrow formation was tested with Xenopus eggs during the first cleavage. We could not detect any Ca signals such as a Ca wave, Ca puffs or even Ca blips at the growing end of the cleavage furrow. Furthermore, cleavages are not affected by Ca-chelators injected into the eggs at concentrations sufficient to suppress the Ca waves. Thus we conclude that formation of the contractile ring is not induced by a Ca signal at the growing end of the cleavage furrow.     

Effect of hexylene glycol-altered microtubule distributions on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta

Some effects of gravity on early morphogenesis are correlated with microtubule locations within cells. During first cleavage in Ilyanassa obsoleta embryos, a transitory polar lobe constriction forms and then relaxes, allowing the polar lobe to merge with one daughter cell. If the polar lobe is equally divided or removed, morphogenesis is severely disrupted. To examine microtuble locations during early Ilyanassa development, eggs were fixed and stained for polymerized alpha-tubulin during first cleavage. The mitotic apparatus assembles at the animal pole. The cleavage furrow forms between the asters, constricting to a stabilized intercellular bridge encircling midbody-bound microtubules, whereas the polar lobe constriction forms below and parallel to the spindle, constricting to a transitory intercellular bridge encircling no detectable microtubules. At metaphase an alpha-tubulin epitope is distributed throughout the spindle, whereas a beta-tubulin epitope is present predominantly in the asters. Incubation in hexylene glycol, a drug that increases microtubule polymerization, during mitosis causes the polar lobe constriction to tighten around polymerized alpha-tubulin and remain stably constricted. If hexylene glycol is removed, alpha-tubulin staining disappears from the polar lobe constriction, which relaxes, whereas microtubules remain in the cleavage furrow, which remains constricted. These observations suggest that asymmetric distribution of microtubules affects early Ilyanassa cleavage patterns, and that continued presence of microtubules extending through an intercellular bridge is important for stabilization of the bridge constriction prior to completion of cytokinesis. These data provide the basis for further analysis of the role of microtubules in possible microgravity disruptions of Ilyanassa development.     

Membrane potential of fertilized eggs of Ilyanassa obsoleta during polar lobe formation and cytokinesis

The membrane potential of fertilized eggs of Ilyanassa obsoleta does not change significantly during the cell shape changes involved in formation and resorption of the third polar lobe and in cytokinesis. The membrane potential is predominantly K⁺-dependent.     

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.     

Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four-cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second clevage in SDS-equalized embryos, and the four-cell stage consisted of four equal cells with reduced cell contacts. SDS inrreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS-equalized embryos had one or more lobe-dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe-dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS-treated embryos are discussed.     

Cell division in Dictyostelium with special emphasis on actomyosin organization in cytokinesis

This study focuses on the dynamic reorganization of actin and myosin ("conventional" myosin, or myosin-II) during cytokinesis in D. discoideum. This is the first study identifying the birefringence of the spindle microtubules as well as three sets of microfilamentous structure in Dictyostelium. The change of organization in these fibrillar structures was followed in real-time with video microscopy, using a Universal Polarizing Microscope equipped with polarized-light (POL) and differential interference contrast (DIC) optics combined with digital image processing. High-frequency mitotic cells were obtained by semi-synchronous culture, and high-resolution observations were made by utilizing the agar-overlay method (Yumura et al.: Journal of Cell Biology 99:894-899, 1984). The molecular identity of the birefringent structures was determined by fluorescence microscopy. Through-focus observations were performed with an axial resolution of 0.3 micron depth of field. The actomyosin fibrils show a dramatic reorganization throughout mitosis. The fibrils at the leading lamellipodia disappear, and there is a striking assembly of the cortical actomyosin in pro-metaphase, which is accompanied by a decrease in cell volume. The cortical actomyosin gradually increases through anaphase. After late anaphase, very active polar lamellipodia, with an average life of less than 1 minute, are formed. We confirmed that the polar lamellipodia include actin, but not myosin-II. At the cleavage furrow, the microfilaments form two distinctive structures: circular contractile ring at the equator, and a cortical filament array parallel to the polar axis. Myosin is localized in the contractile ring, but not associated with the axial array of F-actin. Actomyosin in the contractile ring gradually transforms into cortical network at the posterior region of daughter cells. The constriction of the furrow is accompanied by a drastic efflux of water as evidenced by highly active contractile vacuole formation and turbulent motion of minute vesicles connected to the furrow. This study demonstrates the presence of a new microfilament structure, as well as the dynamic property of the contractile ring, and sheds new light on the contractile mechanisms underlying cytokinesis.     

The role of microtubules in contractile ring function.

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.     

Arrangement of actin filaments and myosin-like filaments in the contractile ring and of actin-like filaments in the mitotic spindle of dividing HeLa cells

We used a glutaraldehyde-tannic acid-saponin fixative to improve the preservation of actin filaments in dividing HeLa cells during preparation for thin sectioning. The contractile ring in the cleavage furrow is composed of a parallel array of actin filaments that circle the equator. We show that many of these actin filaments are arranged in small bundles. These bundles consist of about 25 filaments throughout cytokinesis. For comparison, filopodia on these cells have about 23 actin filaments packed at a higher density than the filaments in the contractile ring bundles. Some of the contractile ring actin filaments appear to radiate out from electron-dense sites on the plasma membrane. The contractile ring also has a large number of short filaments 13 nm in diameter that closely resemble filaments formed from purified human cytoplasmic myosin. These thick filaments are aligned circumferentially and interdigitate with the actin filaments, as expected for a sliding filament mechanism of tension generation. There are no long actin filaments in the mitotic spindle, but there are a large number (400 to 1000 per μm ³) of very short filaments identical in appearance to actin filaments in other parts of these cells. These short filaments may account for the reported staining of the mitotic spindle with fluorescent antibodies to actin and with fluorescent myosin fragments.     

Analysis of actin and microfilament-associated proteins in the mitotic spindle and cleavage furrow of PtK2 cells by immunofluorescence microscopy: A critical note

The distribution of actin and the microfilament-associated proteins myosin and tropomyosin was studied in mitotic PtK2 cells. Using fluorescent heavy meromyosin and two different antibodies against actin we have found no evidence for increased accumulations of actin in the mitotic spindle but have found increased levels of actin in the cleavage furrow and the contractile ring. Short, thin microfilament pieces remain detectable in the cytoplasm throughout mitosis. Purified antibodies against myosin and tropomyosin also revealed no increased levels of these proteins in the spindle region, although both proteins were found in the contractile ring and areas of the cytoplasm close to the intercellular bridge. These data are in agreement with functional and ultrastructural studies involving a role for actin and microfilament-related proteins in cytokinesis. They do not support models in which microfilament-related proteins are assumed to be a major constituent of the mitotic spindle.     

Mechanism of the formation of contractile ring in dividing cultured animal cells. I. Recruitment of preexisting actin filaments into the cleavage furrow

Cytokinesis of animal cells involves the formation of the circumferential actin filament bundle (contractile ring) along the equatorial plane. To analyze the assembly mechanism of the contractile ring, we microinjected a small amount of rhodamine-labeled phalloidin (rh-pha) or rhodamine-labeled actin (rh-actin) into dividing normal rat kidney cells. rh-pha was microinjected during prometaphase or metaphase to label actin filaments that were present at that stage. As mitosis proceeded into anaphase, the labeled filaments became associated with the cortex of the cell. During cytokinesis, rh-pha was depleted from polar regions and became highly concentrated into the equatorial region. The distribution of total actin filaments, as revealed by staining the whole cell with fluorescein phalloidin, showed a much less pronounced difference between the polar and the equatorial regions. The sites of de novo assembly of actin filaments during the formation of the contractile ring were determined by microinjecting rh-actin shortly before cytokinesis, and then extracting and fixing the cell during mid-cytokinesis. Injected rhodamine actin was only slightly concentrated in the contractile ring, as compared to the distribution of total actin filaments. Our results indicate that preexisting actin filaments, probably through movement and reorganization, are used preferentially for the formation of the contractile ring. De novo assembly of filaments, on the other hand, appears to take place preferentially outside the cleavage furrow.     

The germinal centre kinase Don3 triggers the dynamic rearrangement of higher‐order septin structures during cytokinesis in Ustilago maydis

The dimorphic phytopathogenic fungus Ustilago maydis grows in its haploid phase by budding. Cytokinesis and separation of daughter cells are accomplished by the consecutive formation of two distinct septa. Here, we show that both septation events involve the dynamic rearrangement of septin assemblies from hourglass‐shaped collars into ring‐like structures. Using a chemical genetic approach we demonstrate that the germinal centre kinase Don3 triggers this septin reorganization during secondary septum formation. Although chemical inhibition of an analogue‐sensitive version of Don3 prevented septation, a stable septin collar was assembled at the presumptive septation site. Interestingly, the essential light chain of type II myosin, Cdc4, was already associated with this septin collar. Release of Don3 kinase inhibition triggered immediate dispersal of septin filaments and concomitant incorporation of Cdc4 into a contractile actomyosin ring, which also contained the F‐BAR domain protein Cdc15. Inhibition of actin polymerization or deletion of the cdc15 gene, did not affect assembly of the initial collar consisting of septin and myosin light chain. However, reassembly of septin filaments into a ring‐like structure was prevented in the absence of either F‐actin or Cdc15, indicating that septin ring formation in U. maydis depends on a functional contractile actomyosin ring.     

A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their “classical” contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole     

An activation mechanism for ATP cleavage in muscle

Evidence for a proposed activation mechanism is summarized. The low rate of ATP cleavage in the resting state of muscle is considered to result from the formation of a stable ring structure involving the two essential sulfhydryl groups on each myosin head and MgATP. Activation is thought to occur by interaction of actin in the vicinity of one of the essential sulfhydryl groups, Thus opening the stable ring leading to rapid dissociation of split products. This idea is consistent with the kinetic scheme of ATP cleavage developed recently by other workers and allows a prediction of the shift in population of intermediate states with changes in solvent conditions. It is also supported by our recent studies on the spatial geometry of the ring. The possibility that other nucleophilic groups may replace the sulfhydryl groups in other contractile systems is considered. The relevance of the ring structure to the tension generating event is discussed on the basis of recent measurements of the rate of contraction of modified (SH₁-blocked) actomyosin threads. Results indicate that the ability to form the ring structure is an essential requirement of the contractile process in these systems, and, moreover, that single, modified heads of myosin can act independently to produce the same rate of contraction as native myosin. This latter finding suggests that the myosin duplex exhibits some type of negative cooperativity in the contractile process.     

Cytokinesis without myosin II

The ability of substrate-anchored Dictyostelium cells to divide without myosin II has opened the possibility of analysing the formation of cleavage furrows in the absence of a contractile ring made of filamentous myosin and actin. Similar possibilities exist in mutants of budding yeast and, less strictly, also in drug-treated mammalian cells. Myosin-II-independent activities in Dictyostelium include the microtubule-induced programming of the cell surface into ruffling areas and regions that are converted into a concave furrow, as well as the translocation of cortexillins and cross-linked membrane proteins towards the cleavage furrow. A centripetal flow of actin filaments followed by their disassembly in the cleavage furrow is proposed to underlie the translocation.     

The role of unequal cleavage and the polar lobe in the segregation of developmental potential during first cleavage in the embryo of Chætopterus variopedatus

Summary The inequality of the first cleavage division of the Chætopterus embryo is caused by the production of a small polar lobe and the internal shifting of the first cleavage spindle. This division produces a two-celled embryo containing a small AB and a large CD blastomere. These blastomeres have different morphogenetic potentials. Only the larvae resulting from isolated CD blastomeres are able to form bioluminescent photocytes, eyes and lateral hooked bristles. The removal of the polar lobe during first cleavage does not have a great effect on development. These lobeless embryos display a normal pattern of cleavages through the time of mesentoblast formation. The resulting larvae are essentially normal, however they do not form functional photocytes. If the CD cell is isolated after the removal of the first polar lobe, the resulting larva is virtually identical to those formed by the intact CD cell except it lacks the photocyte cells. These results indicate that two separate pathways are involved in the segregation of developmental or morphogenetic potential which takes place during first cleavage. One set of factors, which are necessary for photocyte formation, are associated with the first polar lobe. Other factors that are necessary for the formation of the eyes and lateral hooked bristles are segregated by the unequal cleavage which results from an internal shifting of the cleavage spindle. The removal of a large portion of the vegetal region of the embryo during first cleavage leads to the production of larvae which display a decreased ability to form eyes and lateral hooked bristles. These embryos frequently display an abnormal pattern of cleavages. They do not form the primary somatoblast or the mesentoblast. These results indicate that the vegetal region of the CD cell of Chætopterus is analogous to polar lobes which have been studied in other species, and is therefore important in the specification of the D quadrant. These features of the first cleavage of Chætopterus are a combination of those displayed by forms with direct unequal cleavage and other forms which cleave unequally through the production of large polar lobes. The significance of these findings is discussed relative to the origins of these different types of unequal cleavage.     

Actomyosin organization during cytokinesis: reversible translocation and differential redistribution in Dictyostelium

Synchronized cultures of Dictyostelium discoideum were used to study organizational changes of the cytoskeleton during mitotic cell division. The agar-overlay technique (Yumura et al.: J. Cell Biol. 99:894-899, 1984) was employed for immunofluorescence localization and video microscopic observation of living mitotic cells. The mitotic phase was defined by changes in chromosome configuration by using a double stain with the fluorescent dye DAPI. This study showed that the actin- and myosin-containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase. Both actin and myosin filaments were dissociated from the cell cortex in prophase. Most of the actin and myosin was filamentous and remained in the endoplasm until telophase. Saltatory movements of organelles stopped suddenly, coincident with the breakdown of the cytoplasmic microtubule network. This change in the microtubule system was temporally coupled with the disappearance of actomyosin from the cortex. At the same time, the local vibrating movement of particles almost stopped, suggesting that the viscoelastic nature of the endoplasm was altered. In the late anaphase, actin and myosin relocalized to the cortical ectoplasm. Early in this phase, myosin filaments were localized specifically at the anticipated cleavage furrow region of the cleavage furrow, whereas actin filaments were redistributed more uniformly in the cell cortex, with an extremely large accumulation in the polar pseudopods. Subsequently the actin formed an orderly parallel array of cables along with myosin filaments in the contractile ring. The spatial segregation of actin and myosin in late anaphase was clearly demonstrated by multipolar cell division of artificially induced giant cells. Actin was relocalized in both the polar and the proximal constricting regions whereas myosin was only localized in the center of each pair of daughter microtubule networks where the cleavage furrow was formed. This study demonstrates that actin and myosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.     

Astral signals spatially bias cortical myosin recruitment to break symmetry and promote cytokinesis

BACKGROUND: After anaphase, the segregated chromosomes are sequestered by cytokinesis into two separate daughter cells by a cleavage furrow formed by the actomyosin-based contractile ring. The failure to properly position the contractile ring between the segregated chromosomes can result in aneuploidy. In both C. elegans embryos and human cells, the central spindle regulates division-plane positioning in parallel with a second pathway that involves astral microtubules. RESULTS: We combined genetic and pharmacological manipulations with live cell imaging to spatially separate the two division cues in a single cell. We demonstrate that the two pathways for furrow formation are mechanistically and genetically distinct. By following the distribution of green fluorescent protein (GFP)-tagged nonmuscle myosin, we have found that the astral pathway for furrow formation involves the negative regulation of cortical myosin recruitment. An asymmetrically positioned spindle induces the asymmetric cortical accumulation of myosin. This cortical myosin behaves as a coherent contractile network. If the cortical network is nonuniform over the cell, the cortical contractile elements coalesce into a single furrow. This coalescence requires interconnections among contractile elements. CONCLUSIONS: We conclude that the two pathways of cleavage-furrow formation are mechanistically distinct. In particular, we conclude that the astral pathway for cleavage-furrow formation involves the negative regulation of myosin distribution by astral cues.