Uri Abdu

Oocyte development and polypeptide dynamics during ovarian maturation in the red-claw crayfish Cherax quadricarinatus

Summary Ovarian development in the red claw crayfish Cherax quadricarinatus is divided into two major phases, namely primary- and secondary-vitellogenesis. Two anatomically distinctive types of primary-vitellogenic ovary are present: one contains uniform milky white oocytes (in the chromatin, chromatin-nucleolus, early-perinuclear and late-perinuclear stages), while the other contains two diversely colored oocyte populations, the smallest oocytes being uniform milky white and the largest being yellow to orange (i.e., at the lipid stage). Secondary-vitellogenic ovaries are characterized by the presence of a synchronously growing large oocyte group together with oocytes of all the first four, primary-vitellogenic stages. The synchronous group develops from the yolk stage via the prematuration stage into the maturation stage. Polypeptides of relatively low molecular masses (65–95 kDa) are abundant in the primary-vitellogenic ovary containing chromatin to lipid-stage oocytes. Polypeptides of relatively higher molecular masses (<100kDa) were detected both in the secondary-vitellogenic ovary (composed mainly of yolk-stage oocytes) and in newly laid eggs. During secondary vitellogenesis, the high-density lipoprotein (HDL) fraction of the hemolymph contains four secondary vitellogenic-specific polypeptides (208, 196, 177 and 80 kDa) that are not present in the hemolymph of the primary vitellogenic female. In this study we have elucidated the relationship between the progress of gonad maturation, oocyte development and the sequence of appearance of specific polypeptides in the ovary and the hemolymph of C. quadricarinatus.

Effect of Methyl Farnesoate on Late Larval Development and Metamorphosis in the Prawn Macrobrachium rosenbergii (Decapoda, Palaemonidae): A Juvenoid-like Effect?

Methyl farnesoate (MF), the unepoxidated form of insect juvenile hormone III, was detected in larvae of the freshwater prawn Macrobrachium rosenbergii, which metamorphose to post-larvae following 11 larval stages. The possible role of MF as a morphogen was studied by administering the compound to M. rosenbergii larvae via an Artemia vector. Higher MF levels caused earlier retardation of late larval growth, and the highest dose retarded larval development. Furthermore, MF significantly affected the patterns of metamorphosis and the appearance of intermediate individuals exhibiting both larval and post-larval morphology and behavior. Three intermediate types were defined, two of which were found only at the MF-treated groups and one that was exclusive to the higher dose treatments. The relative abundance of intermediate specimens increased from 2% in the control to 32% in the high MF concentration, which suggests that MF has a juvenoid-like effect in this decapod crustacean.

Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its interaction with scaffold proteins

The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a “fishing rod” molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed.

Physiological effects of methyl farnesoate and pyriproxyfen on wintering female crayfish Cherax quadricarinatus

Methyl farnesoate (MF), the predominant juvenile hormone-like compound of crustaceans, was found in the hemolymph of female Cherax quadricarinatus crayfish. Administration of MF to C. quadricarinatus females during their winter reproductive arrest period had no effect on reproduction; however, it did have a tendency to accelerate molting. However, since MF caused increased mortality (∼47% survival in the high MF treatment), we were not able to draw definitive conclusions regarding its physiological affect. In contrast, administration of pyriproxyfen, a juvenile hormone analog, did not cause significant mortality (95% survival in the high pyriproxyfen treatment), although it accumulated in high quantities in the hepatopancreas and, to a lesser extent, muscle tissue, ovaries and gills. The highest dose of pyriproxyfen used in this study, 20 μg/gram animal body weight/week, caused a delay in spawning, which became statistically significant from the seventeenth week. This dose of pyriproxyfen caused a tendency of acceleration of molting without any effect on molt increment. The results of this study show that pyriproxyfen does not seem to be toxic to the crayfish, even in relatively high doses, and might affect the energetic balance between molt and reproduction.

spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila.

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfα-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. We found that the ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. We cloned spn-F and found that it encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. We also show that Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show that we have identified a novel protein that affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis.

Expression of the Drosophila melanogaster GADD45 Homolog (CG11086) Affects Egg Asymmetric Development That Is Mediated by the c-Jun N-Terminal Kinase Pathway

The mammalian GADD45 (growth arrest and DNA-damage inducible) gene family is composed of three highly homologous small, acidic, nuclear proteins: GADD45α, GADD45β, and GADD45γ. GADD45 proteins are involved in important processes such as regulation of DNA repair, cell cycle control, and apoptosis. Annotation of the Drosophila melanogaster genome revealed that it contains a single GADD45-like protein (CG11086; D-GADD45). We found that, as its mammalian homologs, D-GADD45 is a nuclear protein; however, D-GADD45 expression is not elevated following exposure to genotoxic and nongenotoxic agents in Schneider cells and in adult flies. We showed that the D-GADD45 transcript increased following immune response activation, consistent with previous microarray findings. Since upregulation of GADD45 proteins has been characterized as an important cellular response to genotoxic and nongenotoxic agents, we aimed to characterize the effect of D-GADD45 overexpression on D. melanogaster development. Overexpression of D-GADD45 in various tissues led to different phenotypic responses. Specifically, in the somatic follicle cells overexpression caused apoptosis, while overexpression in the germline affected the dorsal–ventral polarity of the eggshell and disrupted the localization of anterior–posterior polarity determinants. In this article we focused on the role of D-GADD45 overexpression in the germline and found that D-GADD45 caused dorsalization of the eggshell. Since mammalian GADD45 proteins are activators of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signaling pathways, we tested for a genetic interaction in D. melanogaster. We found that eggshell polarity defects caused by D-GADD45 overexpression were dominantly suppressed by mutations in the JNK pathway, suggesting that the JNK pathway has a novel, D-GADD45-mediated, function in the Drosophila germline.

Asymmetric Microtubule Function Is an Essential Requirement for Polarized Organization of the Drosophila Bristle

ABSTRACT While previous studies have shown that microtubules (MTs) are essential for maintaining the highly biased axial growth of the Drosophila bristle, the mechanism for this process has remained vague. We report that the MT minus-end marker, Nod-KHC, accumulates at the bristle tip, suggesting that the MT network in the bristle is organized minus end out. Potential markers for studying the importance of properly polarized MTs to bristle axial growth are Ik2 and Spindle-F (Spn-F), since mutations in spn-F and ik2 affect bristle development. We demonstrate that Spn-F and Ik2 are localized to the bristle tip and that mutations in ik2 and spn-F affect bristle MT and actin organization. Specifically, mutation in ik2 affects polarized bristle MT function. It was previously found that the hook mutant exhibited defects in bristle polarity and that hook is involved in endocytic trafficking. We found that Hook is localized at the bristle tip and that this localization is affected in ik2 mutants, suggesting that the contribution of MTs within the bristle shaft is important for correct endocytic trafficking. Thus, our results show that MTs are organized in a polarized manner within the highly elongated bristle and that this organization is essential for biased bristle axial growth.

The Drosophila hus1 gene is required for homologous recombination repair during meiosis

The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Previously, we demonstrated that Drosophilahus1 is essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. In this study, we further analyzed the function of hus1 during meiosis and discovered that the synaptonemal complex (SC) disassembles abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, we showed that in hus1, DSBs that form during meiosis are not processed efficiently, and that this defect is not suppressed by a mutation in DmChk2. We found a genetic interaction between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, our results imply that hus1 is required for repair of DSBs during meiotic recombination.

The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis

BackgroundIkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2.ResultsWe found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization.ConclusionWe conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

Stable and dynamic microtubules coordinately determine and maintain Drosophila bristle shape.

Within interphase cells, microtubules (MTs) are organized in a cell-specific manner to support cell shape and function. Here, we report that coordination between stable and dynamic MTs determines and maintains the highly elongated bristle cell shape. By following MT-decorating hooks and by tracking EB1 we identified two MT populations within bristles: a stable MT population polarized with their minus ends distal to the cell body, and a dynamic MT population that exhibits mixed polarity. Manipulating MT dynamics by Klp10A downregulation demonstrates that MTs can initiate new shaft extensions and thus possess the ability to determine growth direction. Actin filament bundling subsequently supports the newly formed shaft extensions. Analysis of ik2 mutant bristles, established by elongation defects in the Drosophila ikkε homolog, led to the observation that stable and dynamic MT orientation and polarized organization are important for proper bristle elongation. Thus, we demonstrate for the first time that coordination between stable and dynamic MT sets that are axially organized yet differently polarized drives cell elongation.