Mobility and Intranuclear Dynamics of Single Balbiani Ring mRNP Particles in Living Chironomus tentans Salivary Glands

The salivary glands of the dipteran Chironomus tentans provide an elegant model system for the analysis of specific messenger ribonucleoprotein particles, the Balbiani Ring (BR) mRNP. BR mRNP particles contain long RNA transcripts of roughly 35-40 kb in size which possess a highly repetitive sequence. The diameter of the granular BR mRNP particles is around 50 nm and their geometry was meanwhile characterised by Electron Tomography. Transcription and splicing of the BR mRNA and the formation of the BR Particles is genetically and biochemically thoroughly investigated, and in recent studies their intranuclear localisation in fixed glands was visualized by electron microscopy [1]. However, up to now little is known about the intranuclear dynamics and mobility of the BR mRNP particles in vivo. Using a direct kinetic approach [2,3] we analyse the intranuclear diffusion of BR particles by single particle tracking of fluorescence labelled BR mRNPs in living cells. Labelling is achieved in situ by nuclear microinjection of fluorescence labelled oligonucleotides, which are complementary to the highly repetitive sequence on the BR mRNA. This approach generates fluorescent particles in vivo. Injection of control oligos proves that this labelling is specific. Labelling could be inhibited by the transcription inhibitor DRB. Using high speed laser microscopy we analysed the intranuclear movement of the particles from the site of transcription towards the nuclear pore complexes and characterisezed their diffusion within the nucleosol. Furthermore we also investigated the influence of a known interaction partner on the intra-nuclear diffusion of the particles by co-injection of recombinant proteins or antibodies.