Technical Reference #1707

1707.

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin Jan Peter Siebrassea; Roman Veitha; Akos Dobayb; Heinrich Leonhardtb; Bertil Daneholtc and Ulrich Kubitschecka;, Rheinische Friedrich Wilhelms University Bonn, PNAS, 105(1707), (2008)
Link To Paper

Abstract:
Messenger ribonucleoprotein particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus. To further understand mRNP trafficking we have studied the intranuclear movement of a specific mRNP the BR2 mRNP in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm which allows us to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled with microinjected oligonucleotides (DNA or RNA) complementary to BR2 mRNA or with the RNA-binding protein hrp36 the C. tentans homologue of hnRNP A1. Using high-speed laser microscopy we followed the intranuclear trajectories of single mRNPs and characterized their motion within the nucleoplasm. The Balbiani ring (BR) mRNPs moved randomly but unexpectedly in a discontinuous manner. When mobile they diffused with a diffusion coefficient corresponding to their size. Between mobile phases the mRNPs were slowed down 10-to 250-fold but were never completely immobile. Earlier electron microscopy work has indicated that BR particles can attach to thin nonchromatin fibers which are sometimes connected to discrete fibrogranular clusters. We propose that the observed discontinuous movement reflects transient interactions between freely diffusing BR particles and these submicroscopic structures.

Keywords:
Balbiani ring particles; in vivop labeling; mRNA trafficking; single-molecule fluorescence microscopy; sing-particle tracking

Materials & Methods:
Preparation of Salivary Glands. C. tentans larvae were raised in water-filled aerated plastic dishes and fed with fermented nettle powder (10). Salivary glands were isolated from rapidly growing fourth-instar larvae and kept in PBS during preparation. For microinjection and microscopy the glands were transferred onto a polylysine-coated coverslip embedded in a cell culture dish (MatTek). For incubation under the microscope either ZO medium or PBS was used. Usually it took5–10minto prepare theglandandanadditional5–10minto microinject 3–6 nuclei. Microinjection was carried out with an Eppendorf injection and micromanipulation setup using a holding pressure of 20 hPa and manual injection procedure. All injection solutions were diluted in transport buffer and centrifuged at 22000 g for 30 min at 4 °C.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
salivary glands