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

<p>Abstract</p> <p>Background</p> <p>IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The <it>Drosophila </it>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 <it>ik2 </it>regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since <it>spn-F </it>mutants display oocyte defects similar to those of <it>ik2 </it>mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2.</p> <p>Results</p> <p>We 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.</p> <p>Conclusion</p> <p>We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during <it>Drosophila </it>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.</p

Localization of the Drosophila Rad9 Protein to the Nuclear Membrane Is Regulated by the C-Terminal Region and Is Affected in the Meiotic Checkpoint

Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the C-terminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint

O impacto, sobre estudantes brasileiros, de uma linguagem visual para aprender a aprender conjuntamente

Resumo Um dos temas mais importantes na aprendizagem colaborativa apoiada pelo computador é a autorregulação da aprendizagem sem o apoio de professores. A autorregulação da colaboração pode ser definida como o conjunto dos processos sociais que os alunos usam para coordenar o seu esforço conjunto em uma atividade. Este trabalho apresenta um estudo de caso brasileiro que examina o impacto da plataforma computacional Metafora para apoiar a regulação da colaboração entre os estudantes brasileiros. Nosso objetivo é investigar se o uso da linguagem visual Metafora ajuda os alunos a aprenderem a aprender em conjunto (learn to learn togueter – L2L2). L2L2 abrange o desenvolvimento da capacidade de coordenação da colaboração. Para perseguir esse objetivo, são fornecidas evidências de mecanismos de coordenação e as respostas emocionais subjacentes ao uso, pelos alunos, da ferramenta de planejamento Metafora. Os resultados deste estudo de caso demonstram que as interações dos alunos, ao usarem a ferramenta de planejamento Metafora, influenciaram o seu desenvolvimento de L2L2 de maneira natural e envolvente. A ferramenta de planejamento Metafora proporciona aos alunos um ambiente amigável para a regulação dos processos de grupo e tem potencial para modificar os pensamentos dos estudantes com respeito à coordenação de processos colaborativos

Multimodal dialogue in small-group mathematics learning

In this paper, we combine dialogic and embodied theories of learning to create a unified analytic lens. Embodied cognition is a theoretical approach operating under the premise that thinking and communication are multimodal activities. Under this premise, dialogue between learners needs to be conceptualized using a multimodal lens. We identify multimodal voices as speech and movement bundles situated within a learning context and describe a phenomenon that we call Multimodal Dialogue – multimodal interaction between different multimodal voices. To demonstrate this phenomenon, we analyze a learning sequence by two third-grade students who participated in a mathematics lesson aimed to foster embodied learning of proportion. Our analysis zooms in on the phenomenon of a multimodal voice as a speech-and-movement bundle situated within a learning context. We further show how multimodal dialogic gaps – differences between multimodal voices within and between modalities – drive communication and eventual changes in voices

Multimodal Dialogue in Small-Group Mathematics Learning

In this paper, we combine dialogic and embodied theories of learning to create a unified analytic lens. Embodied cognition is a theoretical approach operating under the premise that thinking and communication are multimodal activities. Under this premise, dialogue between learners needs to be conceptualized using a multimodal lens. We identify multimodal voices as speech and movement bundles situated within a learning context and describe a phenomenon that we call Multimodal Dialogue – multimodal interaction between different multimodal voices. To demonstrate this phenomenon, we analyze a learning sequence by two third-grade students who participated in a mathematics lesson aimed to foster embodied learning of proportion. Our analysis zooms in on the phenomenon of a multimodal voice as a speech-and-movement bundle situated within a learning context. We further show how multimodal dialogic gaps – differences between multimodal voices within and between modalities – drive communication and eventual changes in voices.Space Systems Egineerin

Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in mammalian

<p>Human Embryonic Kidney 293 <b>(HEK293).</b> Confocal images of cells expressing (A) GFP-DmHus1, (D) GFP-DmRad1, and (G) GFP-DmRad9A. (B, E and H) Antibody staining of the NUP 414 protein, which recognizes several nucleoporins. (C, F and I) are merged images of (A–B), (D–E), and (G–H), respectively.</p

Identification of the DmRad9A nuclear localization signal.

<p>Confocal images of S₂R+ cells expressing DmRad9A mutated in suspected NLS sequences. (A) DmRad9A mutated at position 287 – 289 (NLS1). (D) DmRad9A mutated Position 300–302 (NLS2). (G) DmRad9A mutated Position 314–316 (NLS3). (B, E and H) stained with anti-lamin antibodies, which mark the nuclear membrane, in red. (C) Merged image of (A) and (B). (F) Merged image of (D) and (E). (I) Merged image of (G) and (H).</p

Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in S₂R+ and follicle cells.

<p>A–G, Confocal images of S₂R+ cells, I–K, Confocal images of follicle cells from egg chambers. (A) S₂R+ cells expressi ng HA-DmHus1 and stained with anti-HA antibodies in red. (B) S₂R+ cells expressing GFP-DmRad1. (C) S₂R+ cells expressing GFP-DmRad9A. (F) S₂R+ cells expressing DmRad9B-GFP. (D) and (G) Staining with anti-lamin antibodies, which mark the nuclear membrane, in red. (E and H) are merged image of (C with differential interference contrast (DIC) image) and (F with a DIC image), respectively. (I) Egg chamber from HA-DmHus1::<i>CY2Gal4</i> transgenic flies. (J) Egg chamber from GFP-DmRad1::<i>CY2Gal4</i> transgenic flies. (K) Egg chamber from FLAG-DmRad9A::<i>CY2Gal4</i> transgenic flies. In both S₂R+ and follicle cells, DmHus1 is found in the cytoplasm, DmRad1 is found throughout the cell and Dm DmRad9A is localized to the nuclear membrane. DmRad9B is localized to the nucleus in S₂R+ cells.</p

Effects of meiotic checkpoint activation on DmRad9A oocyte nuclear membrane localization.

<p>Confocal images of stage 7 egg chambers. (A, E, I and M) are stained for DNA (blue, arrows mark oocyte nucleus DNA, karyosome); arrows mark the oocyte nucleus (karyosome). (B, F, J and N) GFP-DmRad9A (green). (C, G, K and O) are stained with anti-lamin antibodies, which mark the nuclear membrane, in red. (Inset in H, L and P), represents a schematic description of the oocyte nucleus. Red-lamin, green-GFP-DmRad9A and blue-karyosome. (A–H) GFP-DmRad9A:: <i>nosGal 4-VP16</i> egg chamber, E–H are enlargement of the oocyte region from A–D, respectively. (I-L) BAF3A:: GFP-DmRad9A:: <i>nosGal 4-VP16</i> egg chamber. M–P, GFP-DmRad9A:: <i>nosGal 4-VP16; okr^AA</i>/<i>okr^RU</i>.</p

Physical interaction between DmRad9, DmRad1 and DmHus1.

<p>DmRad9 was co-expressed in S2 cells with DmRad1 and DmHus1. A total lysate of S2 cells was extracted and subjected to immunoprecipitation. (A) DmRad1 was immunoprecipitated using anti-GFP antibodies. Anti-HA antibodies were used to detect DmHus1. (B) The same blot as in (A) was probed for FLAG-DmRad9 using anti-FLAG antibodies. (C–F) Confocal images of S₂R+ cells expressing FLAG-DmRad9, GFP-DmRad1 and HA-DmHus1. (G–J) Confocal images of follicle cells from transgenic FLAG-DmRad9::HA-DmHus1::GFP-DmRad1::<i>CY2Gal4</i> flies expressing egg chamber. (C) Staining with anti-FLAG antibodies detecting Flag-DmRad9. (D) Staining with anti-HA antibodies detecting HA-DmHus1. (E) GFP-DmRad1. (F) Merged (C–E). (G) Staining with anti-FLAG antibodies detecting Flag-DmRad9. (H) Staining with anti-HA antibodies detecting HA-DmHus1. (I) GFP-DmRad1. (J) merged G–I. Total protein served as positive control while a sample treated with protein A alone (no beads) served as negative control.</p