No abstract

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria. Using time-lapse microscopy and a microfluidic set-up, we have assessed in Escherichia coli the vertical transmission of the amyloidosis caused by the synthetic bacterial model prionoid RepA-WH1 at single cell resolution within their lineage context. We identify in vivo the coexistence of two strain-like types of amyloid aggregates within a genetically identical population and a controlled homogeneous environment. The amyloids are either toxic globular particles or single comet-shaped aggregates that split during cytokinesis and exhibit milder toxicity. Both segregate and propagate in sublineages, yet show interconversion. ClpB (Hsp104) chaperone, key for spreading of yeast prions, has no effect on the dynamics of the two RepA-WH1 aggregates. However, the propagation of the comet-like species is DnaK (Hsp70)-dependent. The bacterial RepA-WH1 prionoid thus provides key qualitative and quantitative clues on the biology of intracellular amyloid proteinopathies.

The nucleoskeleton of plants contains a peripheral lamina (also called plamina) and, even though lamins are absent in plants, their roles are still fulfilled in plant nuclei. One of the most intriguing topics in plant biology concerns the identity of lamin protein analogues in plants. Good candidates to play lamin functions in plants are the members of the NMCP (nuclear matrix constituent protein) family, which exhibit the typical tripartite structure of lamins. This paper describes a bioinformatics analysis and classification of the NMCP family based on phylogenetic relationships, sequence similarity and the distribution of conserved regions in 76 homologues. In addition, NMCP1 in the monocot Allium cepa characterized by its sequence and structure, biochemical properties, and subnuclear distribution and alterations in its expression throughout the root were identified. The results demonstrate that these proteins exhibit many similarities to lamins (structural organization, conserved regions, subnuclear distribution, and solubility) and that they may fulfil the functions of lamins in plants. These findings significantly advance understanding of the structural proteins of the plant lamina and nucleoskeleton and provide a basis for further investigation of the protein networks forming these structures.

This study provides evidence that nuclear and chromosome remodelling has taken place in sugarcane, a vegetative crop with a complex genome derived from interspecific hybridizations between Saccharum officinarum and S. spontaneum. Detailed knowledge on the chromosomal compositions of the three clones analysed was acquired. (1) All hybrid cultivars were found to be aneuploid, affecting both parental genomes (having chromosomes in addition to full genomes), with chromosome numbers from 2n=102-106 in My5514 and up to 2n=113-117 in C236-51. (2) Comparative in situ hybridization showed that about 16% of these chromosomes are inherited from S. spontaneum and less than 5% are recombinant or translocated chromosomes containing sequences of both S. officinarum and S. spontaneum. (3) Differences between the observed DNA contents (estimated by flow cytometry) and those expected from the number of chromosomes, allowed the introgression of additional S. spontaneum or S. officinarum DNA pieces into the B42231 and C236-51 cultivars to be estimated. (4) Size heterogeneity between S. officinarum homologous chromosomes carrying the 18S-5.8S-25S and 5S ribosomal genes (identified by FISH with pTa71 and pTa794, respectively) confirms remodelling occurred by chromosomal interchange events, at least in these homologous chromosomes. (5) Simultaneous visualization of nucleoli and NORs showed that all 18S-5.8S-25S loci were potentially functional in the three clones, independent of their origin and size.

Upon binding to short specific dsDNA sequences in vitro, the N-terminal WH1 domain of the plasmid DNA replication initiator RepA assembles as amyloid fibres. These are bundles of single or double twisted tubular filaments in which distorted RepA-WH1 monomers are the building blocks. When expressed in Escherichia coli, RepA-WH1 triggers the first synthetic amyloid proteinopathy in bacteria, recapitulating some of the features of mammalian prion diseases: it is vertically transmissible, albeit non-infectious, showing up in at least two phenotypically distinct and interconvertible strains. Here we report B3h7, a monoclonal antibody specific for oligomers of RepA-WH1, but which does not recognize the mature amyloid fibres. Unlike a control polyclonal antibody generated against the soluble protein, B3h7 interferes in vitro with DNA-promoted or amyloid-seeded assembly of RepA-WH1 fibres, thus the targeted oligomers are on-pathway amyloidogenic intermediates. Immuno-electron microscopy with B3h7 on thin sections of E. coli cells expressing RepA-WH1 consistently labels the bacterial nucleoid, but not the large cytoplasmic aggregates of the protein. This observation points to the nucleoid as the place where oligomeric amyloid precursors of RepA-WH1 are generated, and suggests that, once nucleated by DNA, further growth must continue in the cytoplasm due to entropic exclusion.