Search

To characterise the coiled bodies in meristematic nuclei of Saccharum officinarum, immunofluorescence labelling with antibodies against components of the splicing (U2B′′ and Sm core protein B) and pre-rRNA processing (fibrillarin) complexes was used in cells from the dormant root primordia and from roots at different times after activation to the steady state of proliferation. The number, size and distribution of coiled bodies varied in the meristematic tissue depending on cell activity. While G0 cells in the dry primordia and proliferating cells showed a similar number of coiled bodies attached to their nucleoli, the number of nucleoplasmic coiled bodies greatly increased after the primordia were stimulated to proliferate. Their number remained steady from the time the meristematic population reached the steady state of proliferation, as estimated by flow cytometry. Fractionation studies demonstrated that coiled bodies are a part of the underlying nuclear matrix. Comparison of immunocytochemical and cytochemical data from confocal and electron microscopical studies demonstrated that the nucleolar and nucleoplasmic coiled bodies detected by confocal microscopy shared many features, suggesting that they form a family of closely related structures. [ABSTRACT FROM AUTHOR]

Bacteria are the simplest cellular model in which amyloidosis has been addressed. It is well documented that bacterial consortia (biofilms) assemble their extracellular matrix on an amyloid scaffold, yet very few intracellular amyloids are known in bacteria. Here, we describe the methods we have resorted to characterize in Escherichia coli cells the amyloidogenesis, propagation, and dynamics of the RepA-WH1 prionoid. This prion-like protein, a manifold domain from the plasmid replication protein RepA, itself capable of assembling a functional amyloid, causes when expressed in E. coli a synthetic amyloid proteinopathy, the first model for an amyloid disease with a purely bacterial origin. These protocols are useful to study other intracellular amyloids in bacteria.

At present, two complementary approaches are used for in situ protein visualization in plant nuclei. Imaging of transformed fluorescent proteins is the election tool for the analysis of protein movement and interaction. However, this methodology presents several drawbacks for the identification/localization of endogenous nuclear factors, such as over-expression or mislocalization of transformed proteins. In contrast, immunocytochemistry with specific antibodies represents a powerful tool for the localization of endogenous nuclear proteins at their "native" nuclear sub-compartments. In plant cells, the cell wall hampers antibody accessibility during immunocytochemical analysis thereby reducing the effectivity of the technique, particularly in the case of lowly expressed proteins. To overcome this problem in nuclear protein immunodetection, we developed a method based on the in vitro incubation of isolated nuclei with specific antibodies followed by imaging by confocal fluorescence or electron microscopy. Here we describe the application of this methodology to the localization of Nuclear Matrix Constituent Proteins (NMCP), the plant analogs of lamins, of the monocot Allium cepa, using antibodies raised against highly conserved regions of the proteins.