Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.     

Pollen performance before and during the autotrophic-heterotrophic transition of pollen tube growth

For species with bicellular pollen, the attrition of pollen tubes is often greatest where the style narrows at the transition between stigmatic tissue and the transmitting tissue of the style. In this region, the tubes switch from predominantly autotrophic to predominantly heterotrophic growth, the generative cell divides, the first callose plugs are produced, and, in species with RNase-type self-incompatibility (SI), incompatible tubes are arrested. We review the literature and present new findings concerning the genetic, environmental and stylar influences on the performance of pollen before and during the autotrophic-heterotrophic transition of pollen tube growth. We found that the ability of the paternal sporophyte to provision its pollen during development significantly influences pollen performance during the autotrophic growth phase. Consequently, under conditions of pollen competition, pollen selection during the autotrophic phase is acting on the phenotype of the paternal sporophyte. In a field experiment, using Cucurbita pepo, we found broad-sense heritable variation for herbivore-pathogen resistance, and that the most resistant families produced larger and better performing pollen when the paternal sporophytes were not protected by insecticides, indicating that selection during the autotrophic phase can act on traits that are not expressed by the microgametophyte. In a study of a weedy SI species, Solanum carolinense, we found that the ability of the styles to arrest self-pollen tubes at the autotrophic-heterotrophic transition changes with floral age and the presence of developing fruits. These findings have important implications for selection at the level of the microgametophyte and the evolution of mating systems of plants.     

Genetic selection for reciprocal translocation at chosen chromosomal sites in Saccharomyces cerevisiae.

We have constructed viable Saccharomyces cerevisiae strains containing a reciprocal translocation between the URA2 site of chromosome X and the HIS3 site of chromosome XV. Our methodology is an extension of the method originally developed to introduce an altered cloned sequence at the chromosomal location from which the parent sequence was derived (S. Scherer and R.W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76:4951-4955, 1979). It comprises three essential steps. First, a nonreverting ura2- strain was constructed by deleting a 3.7-kilobase fragment from the coding sequence of the wild-type URA2 gene. Second, part of the coding sequence of the wild-type URA2 gene (without promotor) was inserted at the HIS3 locus of the ura2- strain. Third, after several generations of growth on uracil-supplemented medium, ura2+ colonies were selected which resulted from mitotic recombination between the nonoverlapping deletions of URA2 located on chromosomes X and XV.     

POLLEN LOADS AND PROGENY VIGOR IN CUCURBITA PEPO: THE NEXT GENERATION

Previous research on the Black Beauty bush cv. of zucchini has documented a strong positive relationship between the size of the pollen load and the vigor (performance) of the progeny. Here we report the results of three studies designed to test the hypothesis that the previously observed differences in progeny vigor are heritable. Two studies examined the transmission of the pollen load effect to subsequent generations through the ovules (female role). The third study determined if there is genetic variation for pollen performance and if the pollen load effect could be transmitted to a subsequent generation through the pollen (male role). In each of these studies the vigor of the progeny from the subsequent generation was evaluated in the greenhouse and/or the field. The results of these studies reveal (1) that the ability to sire seeds does respond to selection imposed by high pollen loads, (2) that only 23 of the 35 total traits that we measured in the three studies of transmission to subsequent generations changed in the direction predicted by the pollen competition hypothesis, (3) that only 5 of the 35 traits were significantly affected by the size of the pollen load that produced the previous generation (but all 5 were in the direction predicted by the pollen competition hypothesis), and (4) that only one study produced an overall significant difference (MANOVA) attributable to the size of the pollen load that produced the previous generation (but it too was in the direction predicted by the pollen competition hypothesis). From these experiments we conclude that pollen competition appears to play a real but minor role in the production of differences in vigor between progeny arising from low versus high pollen loads. In Black Beauty bush cv. of zucchini, maternal effects, pollen-pistil interactions, or nonrandom patterns of seed abortion must play important roles as well.     

Linking extinction-colonization dynamics to genetic structure in a salamander metapopulation

Theory predicts that founder effects have a primary role in determining metapopulation genetic structure. However, ecological factors that affect extinction-colonization dynamics may also create spatial variation in the strength of genetic drift and migration. We tested the hypothesis that ecological factors underlying extinction-colonization dynamics influenced the genetic structure of a tiger salamander (Ambystoma tigrinum) metapopulation. We used empirical data on metapopulation dynamics to make a priori predictions about the effects of population age and ecological factors on genetic diversity and divergence among 41 populations. Metapopulation dynamics of A. tigrinum depended on wetland area, connectivity and presence of predatory fish. We found that newly colonized populations were more genetically differentiated than established populations, suggesting that founder effects influenced genetic structure. However, ecological drivers of metapopulation dynamics were more important than age in predicting genetic structure. Consistent with demographic predictions from metapopulation theory, genetic diversity and divergence depended on wetland area and connectivity. Divergence was greatest in small, isolated wetlands where genetic diversity was low. Our results show that ecological factors underlying metapopulation dynamics can be key determinants of spatial genetic structure, and that habitat area and isolation may mediate the contributions of drift and migration to divergence and evolution in local populations.     

Population‐level thermal performance of a cold‐water ectotherm is linked to ontogeny and local environmental heterogeneity

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Neuroethology of Melibe leonina Swimming Behavior

The nudibranch Melibe leonina swims by rhythmically flexingits body from side to side at a frequency of 1 cycle every 2–5sec. Melibe swim spontaneously, when they are dislodged fromthe substrate, or when they come in contact with predatory seastars,such as Pycnopodia helianthoides. Intracellular recordings obtainedfrom semi-intact swimming Melibe reveal a population of 15 swimmotoneurons (SMNs) in each pedal ganglion. In general, SMNsin one pedal ganglion fire out-of-phase with SMNs in the oppositepedal ganglion, resulting in rhythmic side-to-side bending movements.In isolated brains, recordings from SMNs yield similar results,indicating the existence of a swim central pattern generator(CPG). There is no evidence for synaptic interactions betweenSMNs and either inhibiting or exciting SMNs has no impact onthe swim pattern. The SMNs are driven by a CPG consisting of4 interneurons; 2 in the cerebropleural ganglia and 1 in eachpedal ganglion. Appropriate bursting activity in the swim interneuronsis necessary for swimming to occur. Either hyperpolarizationor depolarization of any of the 4 CPG interneurons disruptsthe normal swim pattern. Swimming behavior, and the fictiveswim motor program expressed by the isolated brain, are inhibitedby light and nitric oxide donors. NADPH-diaphorase stainingand nitric oxide synthase (NOS) immunocytochemistry of Melibebrains suggests the source of nitric oxide might be a pair ofbilaterally symmetrical cells located in the cerebropleuralganglia.     

Endogenous rhythms of locomotion in the American horseshoe crab, Limulus polyphemus

American horseshoe crabs (Limulus polyphemus) exhibit clear circadian rhythms of visual sensitivity in the laboratory and in the field they exhibit seasonal patterns of mating behavior that are closely associated with the tides. Recent reports suggest that Limulus locomotor activity may be controlled by endogenous circadian and/or circatidal clocks and that light:dark (LD) cycles may affect the rhythmic output of both of these clocks. In this study, we examined locomotor behavior in the laboratory to determine the extent of this endogenous activity and to examine the influence of LD cycles on these rhythms. Thirty-three L. polyphemus were captured during the breeding season and their activity was monitored with activity boxes and “running wheels” in seawater kept at constant temperature and salinity. Activity patterns were analyzed using visual inspection of actograms and Chi-square and Lomb-Scargle periodograms. Overall, 36% of the animals was significantly more active during L, while only 12% was more active during D (52% showed no preference). Circatidal rhythms were observed in LD in 67% of the horseshoe crabs. Surprisingly, LD cycles appeared to synchronize these rhythms at times. In DD, the majority of animals tested (63%) exhibited circatidal rhythms that persisted for at least seven days. Overall, the results demonstrate that an endogenously controlled tidal rhythm of locomotion operates during, and significantly after, the breeding season in this species. In addition, the present results are consistent with the presence of circalunidian oscillators controlling these rhythms.     

The role of the atrial diverticulum in the copulatory apparatus of the terrestrial flatworm Platydemus manokwari de Beauchamp (Tricladida: Terricola)

Diverticula are present in the copulatory organs of many species of the Terricola. In some species these structures are copulatory bursae or resorptive vesicles. The function of diverticula present in the copulatory organs in species of Dolichoplana and Platydemus (Rhynchodemidae), in Australoplana (Geoplanidae) and in some other caenoplaninid genera has been an enigma. From histochemical and other investigations of the ventro-posterior copulatory diverticulum in Platydemus manokwari de Beauchamp, it is evident that this structure is neither a copulatory bursa, resorptive vesicle nor a uterus. It is an adhesive gland producing a viscid, collagen-like fibrous glycoprotein derived from two secretory elements present in the gland. The secretion is expelled during cocoon laying to adhere the cocoon onto the substratum. The diverticulum appears late in the development of the copulatory organs, and is present when the gonopore is patent; this has implications for the taxonomy of platydemid flatworms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and localization of catecholamines in the nervous system of Limulus polyphemus

The concentrations of various catecholamines in the nervous system of the horseshoe crab Limulus polyphemus have been determined by high-performance liquid chromatography with electrochemical detection. Dopamine, norepinephrine, epinephrine, and their precursor L -Dopa were present in appreciable quantities in discrete regions of the central nervous system and cardiac ganglion. The catecholamines were localized more precisely by use of the glyoxylic-acid-histofluorescence technique of de la Torre and Surgeon (1976). Catecholamine fluorescence appeared in protocerebral and tritocerebral neuropile, including regions of the central body and optic medulla. Posterior to these brain areas, tracts extended through the circumesophageal ganglionic ring and laterally out each of the pedal ganglia. Small clusters of large fluorescent somata were present in the protocerebrum. No fluorescence was observed in the corpora pedunculata.