ABSTRACT
Ultramicroscopy is a microscopical technique that allows optical sectioning and 3D reconstruction of biological and medical specimens. While in confocal microscopy specimen size is limited to several hundred micrometers at best, using ultramicroscopy even centimeter sized objects like whole mouse embryos can be reconstructed with micrometer resolution. This is possible by using a combination of a clearing procedure and the principle of lightsheet illumination. We present ultramicroscopic 3D reconstructions of whole immunohistochemically labelled mouse embryos and adult Drosophila, giving detailed insight into their anatomy. Its speed and simplicity makes ultramicroscopy ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.
Subject(s)
Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Animals , Drosophila/anatomy & histology , Embryo, Mammalian/anatomy & histology , Mice , Microscopy/instrumentation , Microscopy/methodsABSTRACT
Localization of bicoid (bcd) messenger RNA to the anterior pole of the Drosophila oocyte requires the exuperantia ( exu), swallow (swa) and staufen (stau) genes. We show here that Swa protein transiently co-localizes with bcd RNA in mid-oogenesis. Swa also localizes to the anterior pole of the oocyte in the absence of bcd RNA. This localization does not require Exu, but depends on intact microtubules. In mutant ovaries with duplicated polarity of microtubules, Swa and bcd RNA are ectopically localized at the posterior pole, as well as being present at the anterior pole. We identify dynein light chain-1 (Ddlc-1), a component of the minus-end-directed microtubule motor cytoplasmic dynein, as a Swa-binding protein. We propose that Swa acts as an adaptor for the dynein complex and thereby enables dynein to transport bcd RNA along microtubules to their minus ends at the anterior pole of the oocyte.
Subject(s)
Drosophila Proteins , Dyneins/metabolism , Homeodomain Proteins/genetics , Molecular Motor Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Trans-Activators/genetics , Animals , Biological Transport/genetics , Cell Polarity/physiology , Drosophila , Egg Proteins/genetics , Gene Expression Regulation, Developmental/physiology , Insect Proteins/genetics , Microtubules/genetics , Mutagenesis/physiology , Oocytes/cytology , Oocytes/physiology , Oogenesis/genetics , Protein Binding/genetics , Two-Hybrid System TechniquesABSTRACT
In the Drosophila embryo the nuclear localisation of Dorsal, a member of the Rel family, is regulated by an extracellular signal, which is transmitted to the interior of the egg cell by a cascade of proteins involving the novel protein Tube and the protein kinase Pelle. Here we analyse the activation mechanism of Tube and Pelle and the interaction between these two components. We show that both proteins, although having different biochemical activities, are activated by the same mechanism. Membrane association alone is not sufficient, but oligomerisation is required for full activation of Tube and Pelle. By deletion analysis we determined the domains of Tube and Pelle mediating the physical interaction and the signalling to downstream components. In order to investigate the link between Pelle and the target of the signalling cascade, the Dorsal/Cactus complex, we isolated and characterised the novel, but evolutionary conserved protein Pellino, which associates with the kinase domain of Pelle.
Subject(s)
Carrier Proteins/genetics , Drosophila Proteins , Insect Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription Factors , Animals , Blotting, Northern , Chromosome Mapping , Drosophila/embryology , Female , Genotype , Male , Models, Genetic , Phenotype , Phosphorylation , Phosphotransferases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant Fusion Proteins , Signal Transduction , Time FactorsABSTRACT
Pre-mRNA splicing is catalyzed by a multitude of proteins including serine/arginine-rich (SR) proteins, which are thought to play a crucial role in the formation of spliceosomes and in the regulation of alternative splicing. SR proteins are highly phosphorylated, and their kinases are believed to regulate the recruitment of SR proteins from nuclear storage compartments known as speckles. Recently, a family of autophosphorylating kinases termed CLK (CDC2/CDC28-like kinases) was shown to phosphorylate SR proteins and to influence alternative splicing in overexpression systems. Here we used endogenous CLK2 protein to demonstrate that it displays different biochemical characteristics compared with its overexpressed protein and that it is differentially phosphorylated in vivo. Furthermore, CLK2 changed its nuclear localization upon treatment with the kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. We have also identified a CLK2 autophosphorylation site, which is highly conserved among all CLK proteins, and we show by site-directed mutagenesis that its phosphorylation influences the subnuclear localization of CLK2. Our data suggest that CLK2 localization and possibly activity are influenced by a balance of CLK2 autophosphorylation and the regulation by CLK2 kinases and phosphatases.
