Expression of the 53 kD forked protein rescues F-actin bundle formation and mutant bristle phenotypes in Drosophila.

forked mutations affect bristle development in Drosophila pupae, resulting in short, thick, gnarled bristles in the adult. The forked proteins are components of 200-300-microm-long actin fiber bundles that are present transiently during pupal development [Petersen et al., 1994: Genetics 136:173-182]. These bundles are composed of segments of 3-10 microm long, and forked protein is localized along the actin fiber bundle segments and accumulates at the junctions connecting them longitudinally. In the forked mutants, f(36a) and f(hd), F-actin bundles are greatly reduced in number and size, and bundle segmentation is absent. The p-element, P[w(+), falter] contains a 5.3-kb fragment of the forked gene that encodes the 53-kD forked protein [Lankenau et al., 1996: Mol Cell Biol 16:3535-3544]. Expression of only the 53-kD forked protein is sufficient to rescue the actin bundle and bristle phenotypes of f(36a) and f(hd) mutant flies. The 5.3-kb forked sequence, although smaller than the 13-kb region previously shown to rescue forked mutants [Petersen et al., 1994: Genetics 136:173-182], does contain the core forked sequence that encodes actin binding and bundling domains in cultured mammalian cells [Grieshaber and Petersen, 1999: J Cell Sci 112:2203-2211]. These data show that the 53-kD forked protein is sufficient for normal bristle development and that the domains shown previously to be important for actin bundling in cell culture may be all that are required for normal actin bundle formation in developing Drosophila bristles.     

Lifetime patterns of pairing success in male Ortolan Buntings Emberiza hortulana

Analyses of lifetime fitness in birds are typically based on estimates of breeding success, in particular the number of offspring fledged. Small and isolated bird populations often have a male‐skewed adult sex ratio, so that male lifetime productivity depends to a large degree on pairing success, but few studies have focused on patterns of lifetime pairing success. The Norwegian population of Ortolan Buntings Emberiza hortulana is strongly male‐skewed, such that in any year about half of all males are unpaired. Pairing success of first‐year males (16–44%) was significantly lower than for older males (52–89%). Lifetime pairing success was correlated with lifespan and was strongly skewed, with a majority of males being paired only once or never, and only 11% paired three or more times despite a stable lifetime annual survival rate of 63%. Males that were paired in one year were more likely to be paired the next year than males that were unpaired in the previous year. The shortage of females caused even the older males to have a substantial probability of becoming unpaired, and 49% of long‐lived males (known as adults for at least 4 years) were unpaired after years in which they were paired. Pairing success in the Ortolan Bunting therefore follows similar age‐related and lifetime patterns in breeding success documented in other species. However, even the older males ran a high risk of not being paired, contrasting with earlier distinctions between pre‐breeding and breeding lifespans. I discuss the importance of knowledge of pairing success for the management of endangered and declining populations.     

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.     

The pairing of nucleolar patterns in an epithelium as evidence for a conserved nuclear skeleton

The nucleoli in epidermal cells of fifth instar Calpodes and Manduca larvae undergo three cycles of unravelling into necklaces with condensation back to a dense particle or particles. Mitosis occurs before the first cycle and at the beginning of the third cycle. In spite of the formation and condensation of these nucleolar necklaces the nucleoli in the intercycle condition all have paired patterns. Adjacent pairs of nuclei have nucleoli that resemble one another in the number of their component particles, the shape and size of the particles and sometimes in their arrangement as mirror images. The paired patterns begin at mitosis and reform after necklace elongation and condensation. The patterns are presumed to reflect a nuclear skeleton that is paired from mitosis and conserved until the next mitosis. The nuclear pairing is related to the retention of mid bodies by sibling cells so that the epidermis is a collection of minimal two-cell syncytia.     

Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization

Most animal cells use a combination of actin-myosin–based contraction and actin polymerization– based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389–399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53–62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401–410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.     

The reorganization of microfilaments, centrosomes, and microtubules during in vitro small wound reendothelialization

The repair of small endothelial wounds is an important process by which endothelial cells maintain endothelial integrity. An in vitro wound model system was used in which precise wounds were made in a confluent endothelial monolayer. The repair process was observed by time-lapse cinemicrophotography. Using fluorescence and immunofluorescence microscopy, the cellular morphological events were correlated with the localization and distribution of actin microfilament bundles and vinculin plaques, and centrosomes and their associated microtubules. Single to four-cell wounds underwent closure by cell spreading while wounds seven to nine cells in size closed by initially spreading which was then followed at approximately 1 h after wounding by cell migration. These two processes showed different cytoskeletal patterns. Cell spreading occurred independent of centrosome location. However, centrosome redistribution to the front of the cell occurred as the cells began to elongate and migrate. While the peripheral actin microfilament bundles (i.e., the dense peripheral band) remained intact during cell spreading, they broke down during migration and were associated with a reduction in peripheral vinculin plaque staining. Thus, the major events characterizing the closure of endothelial wounds were precise in nature, followed a specific sequence, and were associated with specific cytoskeletal patterns which most likely were important in maintaining directionality of migration and reducing the adhesion of the cells to their neighbors within the monolayer.     

Analysis of early processes in wound-induced vascular regeneration using TED3 and ZeHB3 as molecular markers

Interruption of the vascular bundles of Zinnia internodes induced transdifferentiation of cells into tracheary elements (TEs) or sieve elements (SEs) within 4 d of wounding. The early stage of the regeneration processes was analyzed using two molecular marker genes, TED3 and ZeHB3, which are expressed specifically in TE precursor cells and immature phloem cells, respectively. An increase in the numbers of TED3 and ZeHB3 mRNA-expressing cells always preceded an increase in the numbers of TEs and SEs formed. The earliest sign of vascular differentiation was the appearance 24 h after wounding of a layer(s) of TED3 mRNA-expressing cells in the inter- and intrafascicular cambial-like regions along the severed vascular bundles. In contrast, the number of ZeHB3 mRNA-expressing cells decreased dramatically along the severed bundles 24 h after wounding, and increased again 36 h after wounding. These results clearly indicate that xylem and phloem differentiation are not synchronized during vascular regeneration. Treatment with 10(-3) M colchicine abolished the expression of ZeHB3 mRNA in pith parenchyma, but not TED3 mRNA; this suggests that cell division is a prerequisite for the transdifferentiation of pith parenchymal cells into immature phloem cells expressing ZeHB3. In contrast, transdifferentiation of pith parenchymal cells to TE precursor cells does not require preceding cell division. However, the inhibition of cell division prevented the formation of both radial files of TEs and the cambial-like layer(s) of TED3 mRNA-expressing cells, and, ultimately, vascular regeneration altogether. These results imply that wound-induced cambial-like activity in and between severed vascular bundles is essential for vascular regeneration.     

Processes induced by tau expression in Sf9 cells have an axon-like microtubule organization

We have indirectly analyzed the role of tau in generating the highly organized microtubule (MT) array of the axon. Axons contain MT arrays of uniform polarity orientation, plus ends distal to the cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-673). Surprisingly, these MTs do not radiate from a single discrete nucleating structure in the cell body (Sharp, G. A., K. Weber, and M. Osborn. 1982. Eur. J. Cell Biol. 29: 97-103), but rather stop and start at multiple sites along the length of the axon (Bray, D., and M. B. Bunge. 1981. J. Neurocytol. 10:589-605). When Sf9 ovarian cells are induced to express high levels of tau protein, they develop cellular processes which are similar in appearance to axons and which contain dense arrays of MTs (Knops, J., K. S. Kosik, G. Lee, J. D. Pardee, L. Cohen-Gould, and L. McConlogue. 1991. J. Cell Biol. 114:725-734). We have analyzed the organization of MTs within these arrays, and determined it to be similar, but not identical, to the organization of MTs within the axon. The caliber, MT number, and MT density vary significantly from process to process, but on average are manyfold higher in the tau-induced processes than typically found in axons. Greater than 89% of the MTs in the processes are oriented with their plus ends distal to the cell body, and this proportion is even higher in the processes that are most similar to axons with regard to caliber, MT number, and MT density. Similar to the situation in the axon, MTs are discontinuous along the length of the tau-induced processes, and do not emanate from any observable nucleating structure in the cell body. We have also identified bundles of MTs throughout the cell bodies of the Sf9 cells induced to express tau. Similar to the MT arrays in the processes, these MT bundles are not visibly associated with any other cytological structures that might regulate their polarity orientation. Nevertheless, these bundles consist of MTs most (greater than 82%) of which have the same polarity orientation. Collectively, these results suggest that tau may play a fundamental role in generating MT organization in the axon. In particular, a key property of tau may be to bundle MTs preferentially with the same polarity orientation.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

Nitrate Fluxes and Nitrate Reductase Activity of Suspension-Cultured Tobacco Cells (Effects of Internal and External Nitrate Concentrations)

Cell suspensions of tobacco (Nicotiana tabacum L., cv KY14) were used to determine the responses of NO3- uptake and NO3- reductase activity (NRA) to exogenous NO3- levels in the absence of long-distance NO3- transport. Tobacco cells grown with complete Murashige and Skoog medium for 7 d were subcultured for 3 d with NH4+-free media containing 0, 5, 10, 20, 30, and 40 mM NO3-. Cell NO3-, in vitro NRA, NO3- influx, and efflux of cell NO3- were determined. The NRA increased as cell NO3- increased. Cell NO3- efflux values increased as cell NO3- level increased. Cells with low intracellular NO3- had greater NO3- influx than cells with high intracellular NO3-. Woolf-Augustinsson-Hofstee transformations of the NO3- influx kinetic data revealed patterns characteristic of a high- and low-affinity two-component NO3- uptake system. Apparent Vmax values generally decreased and Km values increased as cell NO3- concentration increased. The NRA of cells supplied with 10 and 20 mM NO3- after 3-d growth in N- free medium increased about 5-fold within 2 h and then remained constant for the next 2 h, whereas NRA of cells supplied with 5 mM NO3- increased only 2-fold during the 4-h period. Intracellular NO3- and other N metabolites associated with cell NO3- levels exerted differential effects on the NO3- influx activity and NRA of tobacco cells cultured in suspension. Expression of high NRA was correlated with both high external and intracellular NO3-, whereas maximum NO3- influx activity required a low (depleted) level of cell NO3-.     

Monoclonal antibody to intermediate filament antigen cross-reacts with higher plant cells

The monoclonal antibody (anti-IFA) raised (Pruss et al., 1981, Cell 27:419-428) against an intermediate filament antigen, which is widespread throughout phylogeny, has been shown here to cross-react with higher plants. On immunoblotting, anti-IFA cross-reacted with proteins in homogenates of carrot suspension cells and of meristematic cells from onion root tips. A 50-kD cross-reactive protein was enriched in a fraction that consisted of detergent-insoluble bundles of 7-nm fibrils from carrot protoplasts (Powell et al., 1982, J. Cell Sci. 56:319-335). By use of indirect immunofluorescence, anti-IFA stained formaldehyde-fixed onion meristematic cells and carrot protoplasts in patterns approximating those obtained with monoclonal anti-tubulins. That anti-IFA was not recognizing plant tubulins was established by use of immunoblots of two-dimensional gels on which the proteins that comprised isolated fibrillar bundles and taxol-purified carrot tubulins had been separated. The two groups of proteins had different positional coordinates: anti-IFA recognized the fibrillar bundle proteins, and anti-tubulins recognized plant microtubule proteins with no cross-reaction to the heterologous proteins. Likewise, formaldehyde-fixed taxol microtubules from carrot cells could be stained with anti-tubulin but not with anti-IFA. It is concluded that an epitope common to intermediate filaments from animals co-distributes with microtubules in higher plant cells.     

Limitation of substratum size alters cytoskeletal organization and behaviour of Swiss 3T3 fibroblasts

Cell spreading and adhesion formation in Swiss 3T3 cells was studied on circular adhesive islands of size 400-500 microns 2 made by evaporating palladium through a mask onto an underlying non-adhesive surface. Cell spreading was limited since focal contacts were restricted to the palladium. On islands less than 2000 microns 2, focal contacts and actin bundles were arranged at the cell periphery. On islands less than 1000 microns 2, the size and number of focal contacts were reduced. Focal contacts may be important regulators of proliferation, but they do not seem to form a deterministic link between substratum contact and proliferative stimulus.     

Morphological evidence for cyclic AMP-induced reverse transformation in vole cells infected with avian sarcoma virus.

Normal fibroblasts of the vole displayed moderately spread or flattened, spindle-shaped, or polygonal morphologies and attached firmly to a substrate. Topographic features of these cells included sparse microvilli, ruffles, and filopodia. Microfilament bundles, intermediate filaments, and long microtubules generally parallel to each other, and the long axis of the cell or its extensions were present in the cytoplasm. Fibronectin was abundant, and fibronectin fibrils often formed junctions at the cell membrane with microfilament bundles. Transformation with avian sarcoma virus converted 90% of the cells to spheres 5 to 10 microns in diameter. In contrast to the normal vole cells, microfilament bundles were absent, microtubules were short and randomly arranged, and fibronectin was no longer visible. Exposure to dibutyryl cyclic AMP and testololactone caused a majority of the spherical cells to stretch and flatten, a process referred to as reverse transformation. Microtubules radiated out to the cell periphery and became parallel in cell extensions, while long microfilament bundles appeared in the cytoplasm. Parallel intermediate filaments were arranged throughout the cell. This ultrastructural analysis of reverse transformation in avian sarcoma virus-transformed vole cells detailed the status of the cytoskeletal system and showed agreement with earlier findings (Puck et al., J. Cell. Physiol. 107:399-412, 1981) using indirect immunofluorescence.     

Myogenic programs of mouse muscle cell lines: expression of myosin heavy chain isoforms, MyoD1, and myogenin

Different mouse muscle cell lines were found to express distinct patterns of myosin heavy chain (MHC) isoforms, MyoD1, and myogenin, but there appeared to be no correlation between the pattern of MHC expression and the patterns of MyoD1 and myogenin expression. Myogenic cell lines were generated from unconverted C3H10T1/2 cells by 5-azacytidine treatment (Aza cell lines) and by stable transfection with MyoD1 (TD cell lines) or myogenin (TG cell lines). Myogenic differentiation of the newly generated cell lines was compared to that of the C2C12 and BC3H-1 cell lines. Immunoblot analysis showed that differentiated cells of each line expressed the embryonic and slow skeletal/beta-cardiac MHC isoforms though slow MHC was expressed at a much lower, barely detectable level in BC3H-1 cells. Differentiated cells of each line except BC3H-1 also expressed an additional MHC(s) that was probably the perinatal MHC isoform. Myogenin mRNA was expressed by every cell line, and, with the exception of BC3H-1 (cf., Davis, R. L., H. Weintraub, and A. B. Lassar. 1987. Cell. 51:987-1000), MyoD1 mRNA was expressed by every cell line. To determine if MyoD1 expression would alter the differentiation of BC3H-1 cells, cell lines (termed BD) were generated by transfecting BC3H-1 cells with MyoD1 under control of the beta-actin promoter. The MyoD1 protein expressed in BD cells was correctly localized in the nucleus, and, unlike the parental BC3H-1 cell line that formed differentiated MHC-expressing cells, which were predominantly mononucleated, BD cell lines formed long, multinucleated myotubes (cf., Brennan, T. J., D. G. Edmondson, and E. N. Olson. 1990. J. Cell. Biol. 110:929-938). Despite the differences in morphology and MyoD1 expression, BD myotubes and the parent BC3H-1 cells expressed the same pattern of sarcomeric MHCs.     

Increase in actin contents and elongation of apical projections in retinal pigmented epithelial cells during development of the chicken eye

The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections. The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with approximately 12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca2+, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.     

Social discrimination in lambs: the role of indirect familiarization and methods of assessment

Familiarization with individuals resulting from direct exposure is the primary mechanism mediating social recognition in many vertebrates. However, discernible phenotypic similarity among close kin may provide an additional basis for recognizing previously unencountered individuals. We investigated the existence of such a mechanism of indirect familiarity among artificially reared twin lambs, Ovis aries, that were separated shortly after birth. When they were 2-3 weeks old, we tested their recognition of familiar penmates, their unfamiliar twin and twins of familiar penmates in two-choice and paired tests. Lambs responded discriminatively to familiar penmates. When allowed to choose between their twins or twins of penmates versus an unfamiliar unrelated individual, lambs did not show any preference. Nevertheless, when paired with a twin or penmate’s twin, lambs bleated less than did those paired with an unfamiliar unrelated partner, which suggests that twins and twins of penmates were recognized. Discrimination between twin and unrelated individuals could be based either on early learning of the twin's phenotypic traits (template) before separation or on a process of indirect familiarization (comparison with familiar self cues). However, indirect familiarization was clearly implicated in the discrimination of twins of penmates; that is, lambs learned the characteristic phenotypic traits of (unrelated) penmates and detected a resemblance between those familiar individuals and their twins. Overall, the two-choice and paired tests were differentially effective procedures depending on the research question being addressed, that is, relatively simple or subtle social discrimination. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.      

The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. Here we show that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. We show that pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. We further show that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells.     

Non-homologous chromosome pairing and crossover formation in haploid rice meiosis

While many studies have provided significant insight into homolog pairing during meiosis, information on non-homologous pairing is much less abundant. In the present study, fluorescence in situ hybridization (FISH) was used to investigate non-homologous pairing in haploid rice during meiosis. At pachytene, non-homologous chromosomes paired and formed synaptonemal complexes. FISH analysis data indicated that chromosome pairing could be grouped into three major types: (1) single chromosome paired fold-back as the univalent structure, (2) two non-homologous chromosomes paired as the bivalent structure, and (3) three or more non-homologous chromosomes paired as the multivalent structure. In the survey of 70 cells, 65 contained univalents, 45 contained bivalents, and 49 contained multivalent. Moreover, chromosomes 9 and 10 as well as chromosomes 11 and 12 formed non-homologous bivalents at a higher frequency than the other chromosomes. However, chiasma was always detected in the bivalent only between chromosomes 11 and 12 at diakinesis or metaphase I, indicating the pairing between these two chromosomes leads non-homologous recombination during meiosis. The synaptonemal complex formation between non-homologs was further proved by immunodetection of RCE8, PAIR2, and ZEP1. Especially, ZEP1 only loaded onto the paired chromosomes other than the un-paired chromosomes at pachytene in haploid.     

Kinetochore behavior during generative cell division inTradescantia virginiana

Summary Previous observations indicate that division of the generative cell inTradescantia virginiana is characterized by several unusual features, including persistence of surrounding microtubule (Mt) bundles during karyokinesis, lack of a distinct metaphase plate and direct contribution by mitotic Mts to the cytoskeleton of young sperm. We have further probed karyokinesis in these cells using additional antitubulin and chromosome staining, as well as kinetochore visualizations with CREST serum. The CREST antibodies reveal kinetochores as paired and single fluorescent dots similar to those seen in other species stained with this preparation. Double localizations show that the dots are located at the ends of Mt bundles previously identified as kinetochore fibers (Palevitz and Cresti 1989). Before anaphase, paired kinetochores are distributed along the length of the cell. They also tend to be located at the cell periphery or are directly connected to peripheral Mt bundles by their kinetochore (K)-fibers. Twelve pairs of dots can be counted per cell, equal to the expected number of chromosomes. During anaphase, kinetochore separation starts at various positions along the length of the cell, producing single, relatively uniformly distributed kinetochores in the crotches of forks formed by K-fiber trunks and elongating Mt branches attached to the base of the trunks. Eventually, K-fibers with attached kinetochores aggregate in stepwise fashion on thick Mt bundles at both ends of the cell. This pattern is reflected in the cytoskeleton of young sperm. These results further document the unusual distribution of chromosomes and kinetochores inTradescantia generative cells and the origin of the Mt cytoskeleton in sperm cells.Abbreviations CREST Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia - K-fiber kinetochore fiber - Mt microtubule Dedicated to the memory of Professor Oswald Kiermayer     

Tip Transformation in Tetrahymena: A Morphogenetic Response to Interactions Between Mating Types*

SYNOPSIS. In Tetrahymena thermophila subline B, a morphogenic alteration of the anterior end of cells of mating types III and VII results from a cellular interaction which precedes and is a prerequisite for pairing. Cell pairing begins 1 h after starved cells of complementary mating type are mixed. The 1 h-long lag period is characterized by an actinomycin D-sensitive inductive interaction in the first 30 min, followed by a maturation period. Tip transformation begins during the maturation period and continues after pairing. Scanning electron microscopy of deciliated cells reveals ridges which form a chevron meeting in a midline seam between the oral apparatus and the anterior tip. During transformation, the seam broadens until the ridged surface is completely smooth. Melding of the ridges also occurs at the tip of the cell resulting in its blunted appearance. Cells of complementary mating types join in the region of this modified surface, which eventually becomes a specialized cell junction perforated by cytoplasmic bridges. Thus, pursuant to an inductive interaction the structure of the tip of cells is modified in anticipation of the pairing event. Rate of cell pairing might be limited by rate of tip transformation.