Cortical Contractility Research Articles

Distinct roles for twoC. elegansanillins in the gonad and early embryo

Anillins are conserved proteins that are important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here, we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, ANI-2 and ANI-3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. By contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical ruffling and pseudocleavage, contractile events that occur in embryos prior to mitosis. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2-depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.

Two billion years of actin. Meeting on cytoskeletal dynamics: from cell biology to developmental disease.

This meeting began with personal retrospective presentations from two of the founding fathers of European cytoskeletal research—V. Small (Vienna, Austria) and U. Lindberg (Stockholm, Sweden)—to commemorate the thirtieth anniversary of the first definitive publication to establish the existence of an actin cytoskeleton in non‐muscle cells (Lazarides & Weber, 1974). ### Ancient cytoskeletons Whereas other meetings often feature an occasional talk on the prokaryotic cytoskeleton, this meeting devoted a whole session to several aspects of our present knowledge of these ancient systems. This was well received and served as a useful reminder that the cytoskeleton is not just the domain of eukaryotic cell biologists. J. Errington (Oxford, UK) introduced the topic of the bacterial cytoskeleton and reviewed earlier studies that showed the filamentous nature of the ancient bacterial actin proteins MreB and Mbl (MreB‐like). As he pointed out, although we have known about actin filaments for 30 years, they have almost certainly existed for more than two billion years. Many early studies on bacterial cell shape indicated that the cell wall alone conveyed the information that was required for morphology. However, the finding that some of the genes that had been identified in mutational cell‐shape screens encoded protein products that resided in the cytosol meant that this idea had to be revisited. Errington and colleagues are now investigating the functions of the Mbl protein, which is one of three MreB homologues in Bacillus subtilis . They have used fluorescence recovery after photobleaching (FRAP), which is a technically demanding technique in an organism as small as a bacterium, to show that the helical Mbl cables are dynamic structures in vivo (Fig 1). The kinetics of recovery indicate that this is achieved through turnover from unbleached parts of the cell. These studies also imply that the organization of the cables is not likely to be …

Direct, high-resolution measurement of furrow stiffening during division of adherent cells.

It is unclear whether cell division is driven by cortical relaxation outside the equatorial region or cortical contractility within the developing furrow alone. To approach this question, a technique is required that can monitor spatially-resolved changes in cortical stiffness with good time resolution. We employed atomic force microscopy (AFM), in force-mapping mode, to track dynamic changes in the stiffness of the cortex of adherent cultured cells along a single scan-line during M phase, from metaphase to cytokinesis. Video microscopy, which we used to correlate the AFM data with mitotic events identified by light microscopy, indicated that the AFM force-mapping technique does not perturb dividing cells. Here we show that cortical stiffening occurs over the equatorial region about 160 seconds before any furrow appears, and that this stiffening markedly increases as the furrow starts. By contrast, polar relaxation of cells does not seem to be an obligatory event for cell division to occur.

Induction of cortical oscillations in spreading cells by depolymerization of microtubules.

Actomyosin-based cortical contractility is a common feature of eukaryotic cells but the capability to produce rhythmic contractions is found in only a few types such as cardiomyocytes. Mechanisms responsible for the acquisition of this capability remain largely unknown. Rhythmic contractility can be induced in non-muscle cells by microtubule depolymerization. Spreading epithelial cells and fibroblasts in which microtubules were depolymerized with nocodazole or colcemid underwent rhythmic oscillations of the body that lasted for several hours before the cells acquired a stable, flattened shape. By contrast, control cells spread and flattened into discoid shapes in a smooth and regular manner. Quantitative analysis of the oscillations showed that they have a period of about 50 seconds. The kinase inhibitors, HA 1077 and H7, and the more specific rho-kinase inhibitor, Y 27632, caused the oscillations to immediately cease and the cells to become flat. Transient increases in cytoplasmic calcium preceded the contractile phase of the oscillations. Wrinkle formation by cells plated on elastic substrata indicated that the contractility of colcemid-treated cells increased in comparison to controls but was drastically decreased after HA 1077 addition. These data suggest that an intact microtubular system normally prevents pulsations by moderating excessive rho-mediated actin myosin contractility. Possible mechanistic interactions between rho-mediated and calcium activated contractile pathways that could produce morphological oscillations are discussed.

The role of pre- and post-anaphase microtubules in the cytokinesis phase of the cell cycle

The cytokinesis phase, or C phase, of the cell cycle results in the separation of one cell into two daughter cells after the completion of mitosis. Although it is known that microtubules are required for proper positioning of the cytokinetic furrow [1] [2], the role of pre-anaphase microtubules in cytokinesis has not been clearly defined for three key reasons. First, inducing microtubule depolymerization or stabilization before the onset of anaphase blocks entry into anaphase and cytokinesis via the spindle checkpoint [3]. Second, microtubule organization changes rapidly at anaphase onset as the mitotic kinase, Cdc2-cyclin B, is inactivated [4]. Third, the time between the onset of anaphase and the initiation of cytokinesis is very short, making it difficult to unambiguously alter microtubule polymer levels before cytokinesis, but after inactivation of the spindle checkpoint. Here, we have taken advantage of the discovery that microinjection of antibodies to the spindle checkpoint protein Mad2 (mitotic arrest deficient) in prometaphase abrogates the spindle checkpoint, producing premature chromosome separation, segregation, and normal cytokinesis [5] [6]. To test the role of pre-anaphase microtubules in cytokinesis, microtubules were disassembled in prophase and prometaphase cells, the cells were then injected with anti-Mad2 antibodies and recorded through C phase. The results show that exit from mitosis in the absence of microtubules triggered a 50 minute period of cortical contractility that was independent of microtubules. Furthermore, upon microtubule reassembly during this contractile C-phase period, approximately 30% of the cells underwent chromosome poleward movement, formed a midzone microtubule complex, and completed cytokinesis.

A two-component cytoskeletal system of Xenopus laevis egg cortex: concept of its contractility.

The cortex of Xenopus laeviseggs comprises two components: the plasma membrane with underlying microfilaments (external layer) and the cytoplasmic matrix with embedded pigment granules (internal layer). Both components of the egg cortex are capable of contracting under the influence of calcium ions. The cortex of the fully grown oocyte does not have the ability to contract, but acquires it during progesterone-stimulated maturation, when the oocyte is transformed into an egg. It has been proposed, on the basis of the data on the cortex cytoskeletal organization, that the submembranous microfilamentsform an anisotropic network in the oocytes, which is transformed into an isotropic, randomly organized network in the egg. The latter is capable of contractile acts. Reorganization of the cytoskeleton in the internal cortex layer leads to the formation of the actin contractile gel. Data are provided on the role of actin-associated proteins in changes of organization of the actin cortical cytoskeleton. Mechanisms underlying the different sensitivity of microfilaments of the internal and external layers to cytochalasin B, as well as the coordinated (in time) development of the contractility in these layers, are discussed. The model proposed for development of the cortical contractility during oocyte maturation (Ryabova et al., 1994a) is considered on the basis of a two-component cytoskeletal system.

Development of cortical contractility in the Xenopus laevis oocyte mediated by reorganisation of the cortical cytoskeleton: a model.

As the amphibian oocyte becomes the fertilisation-competent egg an actin-myosin network assembles in the cortex which provides for the cortical contraction that accompanies fertilisation. A number of recent investigations provide data for development of a model detailing the structural changes which should accompany the development of this contractile network as well as the signalling mechanisms which regular assembly and contraction.

Contractile proteins and nonerythroid spectrin in oogenesis of Xenopus laevis.

The distribution of contractile proteins, actin and myosin, and an actin-binding protein, spectrin, was studied in oogenesis of Xenopus laevis. These proteins are present in oocytes already at the previtellogenic stages, which are characterized by their diffuse distribution. The localization of proteins changed with the beginning of vitellogenesis. At all vitellogenic stages, including the fully grown oocyte, animal-vegetal differences were noted in localization of actin and myosin: in the animal hemisphere they appear as fibrillar-like structures, while in the vegetal one they are localized around the yolk platelets. By the end of the oocyte's growth, a cortical gradient appeared: predominant localization of actin and myosin in the cortical area. As the oocyte maturation proceeded, the distribution of actin and myosin again became diffuse and nonuniform, so that a cortical gradient appears. At the beginning of vitellogenesis spectrin is distributed as a network all over the ooplasm, while in the fully grown oocyte it is localized mostly in the subcortical area of the animal hemisphere and, as individual inclusions, in other regions of the oocyte. No spectrin is found by the end of maturation. Actin, myosin, and spectrin are also present in the oocyte's nuclei. Changes in the distribution of contractile proteins and spectrin during oocyte maturation are discussed with respect to the development of cortical contractility, as well as to the changes in spatial distribution of yolk platelets and regional sensitivity of the maturing oocyte to cytochalasin B.

Simulation testing of mechanisms for inducing the formation of the contractile ring in cytokinesis.

There is persuasive evidence that the role of the mitotic apparatus (MA) in cytokinesis is to control the location of the cleavage furrow. The geometric aspects of this interaction between the MA and the cortex are complex and, thus, computer simulation can be a useful means for testing hypotheses about the induction process. White and Borisy (1983. J. Theor. Biol. 101:289-316) used computer simulations to show that long-range signals from the asters, varying inversely as various powers of distance, produce summed effects that are minima at the equator of spherical cells. Their results have seemed to support the "polar relaxation" class of hypotheses, in which the effect of the asters is to weaken cortical contractility so that contraction becomes maximized at the equator because it is least inhibited there. However, the experimental studies of Rappaport and Rappaport (1988. J. Exp. Zool. 247:92-98) indicate that the asters actually strengthen cortical contractility. In this paper, we use computer simulation to determine how signals from the MA will need to vary in effect as functions of distance to cause cortical contractility to become maximized where the furrows are to be induced. Although we confirm that inverse power inhibitory signals could induce equatorial furrows in spherical cells, we also find that this ability is destroyed by flattening, constricting, or distorting cells into cylinders, geometries for which Rappaport's experiments show furrows form (1986. Int. Rev. Cytol. 105:245-281). We then show that stimulatory signals of the right kind would induce furrows at the locations observed, in spherical cells as well as cells distorted by experimental manipulation. These signals must be constant out to a threshold distance but decrease abruptly beyond that distance. We also show that this ability depends on having the "drop-off" threshold occur at just the right distance relative to the dimensions of the cell and separation of the asters.

The origin of cleavage forces in dividing eggs: A mechanism in two steps

The mechanical behavior of sea urchin eggs is examined from soon after fertilization up to the time that the cleavage furrow begins to form. The procedure involves compressing eggs between parallel plates so that tensile forces in the egg surface can be calculated. Results show that eggs of Strongylocentrotus purpuratus at 12–13 °C undergo a strong cortical contraction which significantly precedes and is independent of mitosis and which persists through anaphase, the time when the cleavage stimulus from the mitotic asters differentiates the cortex for cleavage. The cleavage furrow begins to form when cortical tension is high, although there is no obvious incremental increase in tension which specifically correlates with the cleavage stimulus. It is therefore concluded as a tenable hypothesis that there are two separate but coordinated steps which occur while the egg is still spherical and which then lead to cleavage. The cleavage stimulus, instead of causing contraction as often believed, elicits relaxation of the polar cortex after the entire cortex has first contracted by an independent mechanism. Accordingly, the global contraction-polar relaxation hypothesis explains that the motive force for the cleavage furrow actually originates from the prior general contraction and, in a strict sense, is independent of mitosis. The mitosis-linked step is brought about by the cleavage stimulus and upsets the prior balance of high tensile forces by relaxing the poles; this channels the cortical contractility into the equator, resulting in the formation of the cleavage furrow. These ideas are contrasted with alternate hypotheses of cleavage initiation.

The Free-Living Trypanoplasms: Descriptions of Three Species of the Genus Procryptobia n. g., and Redescription of Dimastigella trypaniformis Sandon, with Notes on Their Relevance to the Microscopical Diagnosis of Disease in Man and Animals

VICKERMAN, K. 1978. The free-living trypanoplasms: descriptions of three species of the genus Procryptobia n. g. and redescription of Dimastigella trypaniformis Sandon, with notes on their relevance to the microscopical diagnosis of disease in man and animals. Trans. Amer. Micros. Soc., 97: 485-502. Detailed light microscopical descriptions of four free-living trypanoplasm-like flagellates are given and their position in the order Kinetoplastida Honigberg, 1963 ascertained. In gross morphology, members of the new genus Procryptobia resemble members of the genus Cryptobia Leidy, 1846 but differ in being phagotrophic and in lacking cortical contractility. The trypanocide 4'.6-diamidino-2-phenylindole (DAPI) used as a DNA-specific vital stain in conjunction with fluorescence microscopy demonstrates the presence of a single prominent kinetoplast in the trophic phase of Procryptobia spp; the three species described here-P. vorax n. sp., P. tremulans n. sp., and P. glutinosa n. sp. -differ in the shape of this structure. All form mononucleate thin walled cysts; in P. glutinosa, however, several kinetoplast nucleoids are present in the mature cyst and in the metacystic flagellate. The genus Dimastigella Sandon, originally described as an akinetoplastic trypanoplasm, is shown here to differ from Cryptobia and Procryptobia in the arrangement of the basal bodies of its flagella, and from Cryptobia in being phagotrophic. DAPI-staining, moreover, reveals that D. trypaniformis has multiple minute kinetoplast nucleoids present throughout the mitochondrion in the trophic flagellate; this polykinetoplastic condition is maintained in the thick walled cyst. Scanning electron microscopy shows marked homologies between the oral and flagellar pocket regions of Procryptobia, Dimastigella, and Cryptobia; but in the phagotrophic genera the rostrum and cytostomal lip are developed for seizing prey. Procryptobia tremulans was isolated from a contaminated human urine sample, where

DNA Throughout the Single Mitochondrion of a Kinetoplastid Flagellate: Observations on the Ultrastructure of Cryptobia vaginalis (Hesse, 1910)

SYNOPSIS. Cryptobia vaginalis (Hesse 1910) occurs as long thin and short broad forms in the vagina of the gnathobdelliform leeches Haemopis sanguisuga (Linnaeus) and Hirudo medicinalis Linnaeus. Cytochemical staining for DNA and transmission electron microscopy of sectioned material indicate that in the thin forms the kinetoplast DNA (kDNA) is dispersed irregularly through the mitochondrial network (pankinetoplastic condition) rather than concentrated in the adbasal region of the mitochondrion (eukinetoplastic condition) as in trypanosomatids and most other kinetoplastid flagellates. Light‐microscopic studies on the rare broad forms, however, suggest that these have conventional adbasal location of the kinetoplast. Binary fission appears to occur in the thin forms, suggesting that the dispersed kinetoplast is either highly polyenergid or lacks a genetic function. In other features of its microanatomy, C. vaginalis is a conventional kinetoplastid. The flagellate has an incomplete corset of pellicular microtubules which may have a role in the cortical contractility characteristic of the genus Cryptobia. Feeding is by pinocytosis of vaginal colloids through a microtubule‐lined cytopharynx, possibly after binding to a prominent filament‐coated preoral ridge. A pulsatile (contractile) vacuole is present and appears to be responsible for defecation as well as osmoregulation. Some individuals have elongate bacterial epibionts attached to the body in parallel with the cortical microtubules. All individuals have 2–8 spheroplast‐like endobiotic bacteria in the prenuclear cytoplasm.