85 research outputs found
Environmental regulation of yeast prions.
<p>Prionogenesis is a multistep process in which the prion determinant protein undergoes changes in its secondary structure to form intermediate species and then prion (amyloid) fibrils; this process relies on other cellular machinery to drive these changes. Thermal stress results in the relocalization of heat-shock factor 1 (Hsf1) from the cytoplasm to the nucleus; here it binds to the heat-shockelement (HSEs) of heat-shockāprotein genes, activating their transcription. Consequentially, a diverse group of heat-shock proteins (HSPs) are synthesized. Many HSPs (molecular chaperones) play important roles in prion formation and propagation, including Hsp104, Hsp70-Ssa, and Hsp40-Sis1. In a similar manner, general stresses including oxidative, osmotic, and heat stresses, activate a separate pathway in which Msn2,4 binds to the stress-response element (STREs) of stress-response genes, thereby activating their transcription. Some HSP genes also contain one or more STREs at their 5ā²-regulatory regions. Deletion of the <i>MSN2</i> gene results in a drastic increase of the frequency of [<i>PSI</i><sup>+</sup>] formation, suggesting that some stress-response proteins are also involved in prion formation. However, the identity of the Msn2,4 targets that are involved in prionogenesis remain elusive. Note: for simplicity, only the two major stress-response pathways that are regulated by Hsf1 and Msn2,4 are shown.</p
Yeast prions are āinfectious.ā
<p>A) A sexual cross of [<i>PRION</i><sup>+</sup>] and [<i>prion<sup>ā</sup></i>] cells of opposite mating types results in a [<i>PRION</i><sup>+</sup>] diploid, which can give rise to fourspores that are all [<i>PRION</i><sup>+</sup>] after sporulation. Note: in the case of weak [<i>PSI</i><sup>+</sup>] and [URE3], [<i>PRION</i><sup>+</sup>]Ć[<i>prion</i><sup>ā</sup>] crosses do not always give a 4ā¶0 segregation in progeny. Some other random, non-Mendelian segregation ratios of progeny can be seen, such as 1ā¶4, 1ā¶3, 3ā¶1, 4ā¶0, as well as 2ā¶2, due to their meiotic instabilities. B) Mating a [<i>PRION</i><sup>+</sup>] donor with a [<i>prion</i><sup>ā</sup>] recipient carrying a <i>kar1</i> mutation (which prevents nuclear fusion of the mating partners) will result in formation of a pseudodiploid carrying a mixed cytoplasm of the two mating partners. The pseudodiploid will give rise to haploid cytoductants containing either the donor or recipient nucleus. Shown is a cytoductant containing the recipient nucleus with a <i>kar1</i> mutation. C) Transformation of [<i>prion</i><sup>ā</sup>] spheroplasts (yeast with cell wall removed) with amyloid fibers assembled from recombinant prion protein can result in <i>de novo</i> formation of heritable [<i>PRION</i><sup>+</sup>] in the transformed cells. A <i>URA3</i> plasmid (green circle) was used as a selection marker for the transformation. Solid red color indicates the soluble, diffused prion-determinant protein, whereas red dots indicate the prion protein is in an aggregated prion conformation.</p
Spreading of a Prion Domain from Cell-to-Cell by Vesicular Transport in <i>Caenorhabditis elegans</i>
<div><p>Prion proteins can adopt self-propagating alternative conformations that account for the infectious nature of transmissible spongiform encephalopathies (TSEs) and the epigenetic inheritance of certain traits in yeast. Recent evidence suggests a similar propagation of misfolded proteins in the spreading of pathology of neurodegenerative diseases including Alzheimer's or Parkinson's disease. Currently there is only a limited number of animal model systems available to study the mechanisms that underlie the cell-to-cell transmission of aggregation-prone proteins. Here, we have established a new metazoan model in <i>Caenorhabditis elegans</i> expressing the prion domain NM of the cytosolic yeast prion protein Sup35, in which aggregation and toxicity are dependent upon the length of oligopeptide repeats in the glutamine/asparagine (Q/N)-rich N-terminus. NM forms multiple classes of highly toxic aggregate species and co-localizes to autophagy-related vesicles that transport the prion domain from the site of expression to adjacent tissues. This is associated with a profound cell autonomous and cell non-autonomous disruption of mitochondrial integrity, embryonic and larval arrest, developmental delay, widespread tissue defects, and loss of organismal proteostasis. Our results reveal that the Sup35 prion domain exhibits prion-like properties when expressed in the multicellular organism <i>C. elegans</i> and adapts to different requirements for propagation that involve the autophagy-lysosome pathway to transmit cytosolic aggregation-prone proteins between tissues.</p> </div
Sup35 prion domain aggregation is oligorepeat lengthādependent.
<p>(A) Schematic representation of Sup35p and Sup35NM constructs. The yeast prion protein Sup35 comprises three regions, the aminoterminal (N), the middle (M), and the carboxyterminal (C) domain. N consists of a glutamine/asparagine (Q/N)-rich region (aa 1ā40) and oligopeptide repeats (OR) (aa 41ā97). The prion domain NM, NM with a deletion of oligorepeats number 2ā5 (RĪ2-5), and NM with 2x extended oligorepeat number 2 (R2E2) were fused to YFP under the control of a BWM-specific promoter (<i>unc-54p</i>). (B) Collapsed confocal z-stack images of <i>C. elegans</i> lines stably expressing the indicated transgene. Pictures were taken at displayed stages during nematode development (embryo/L2 larvae/adult). Scale bar: 10 Āµm. (C) Total lysates of nematodes expressing RĪ2-5m::YFP, NMm::YFP, and R2E2m::YFP. Proteins were detected using an anti-GFP antibody. Anti-Ī±-tubulin was used to demonstrate loading of comparable protein amounts. Arrows indicate full-length protein. Lower bands represent NM degradation products. YFP alone was not detected indicating that the tag was not cleaved off. (D) Detergent-solubility assay of lysates of nematodes expressing RĪ2-5m::YFP, NMm::YFP, and R2E2m::YFP. Proteins were detected using an anti-GFP antibody. SNā=āsupernatant, Pā=āpellet.</p
R2E2 co-localizes with autophagosomes, amphisomes, and autolysosomes.
<p>(AāD) Confocal images of BWM cells of <i>C. elegans</i> expressing R2E2m::RFP together with the indicated vesicle markers. Note that LMP-1 localizes to both tubular shaped vesicles (arrows) and round vesicles. Scale bars: 10 Āµm.</p
NMm::YFP and R2E2m::YFP aggregates are highly toxic.
<p>(A) Motility of 1-day-old adults was determined as speed in % relative to the wild-type N2 control. (B) Confocal images of control N2 (I) and R2E2m::YFP (II) expressing nematodes were stained with rhodamine-phalloidin to reveal actin fibers. Scale bars: 10 Āµm. (C) The average amount of eggs laid within 2.5 hours by the indicated <i>C. elegans</i> lines. The developmental stage of synchronized embryos was determined after 72 h (displayed as % of eggs laid). (D) 20 L1 larvae of <i>C. elegans</i> lines expressing the indicated transgene were grown at 20Ā°C for 4 days before light micrographs were taken. Within this time wild-type N2 animals become adults and have progeny, some of which in turn would have developed into young adults, as seen with RĪ2-5, but not with NM and R2E2 transgenic animals. (E) DIC images of control (I), and R2E2m::YFP (IIāIV) expressing animals. Arrows indicate abnormal gonad (II), old cuticle that has failed to shed and remains attached indicating molting defects (III) and disrupted intestinal cells bordering the distended lumen (IV).</p
Overview of different aggregate types, vesicular structures, and phenotypes in the <i>C. elegans</i> prion model.
<p>Expression of R2E2 leads to a range of different foci that were detected with both the YFP and RFP tag (indicated by the orange color). Large foci were analyzed by FRAP and categorized into spherical mobile (containing slowly diffusing protein) and fibrillar immobile aggregates (containing non diffusing protein). Small foci were not assessed by FRAP and consist of presumably both, small aggregates and vesicles (that partially co-localized to LGG-1::GFP). None of the YFP-positive foci exhibited directed movement (very few small foci were moving irregular and undirected within a cell). Fragmented mitochondria were observed in muscle cells and non-expressing tissues by TEM, whereas the YFP signal was not detected outside of body wall muscle cells above the background autofluorescence. In contrast, RFP tagged R2E2 also localized to tubular structures that co-localized with LMP-1::GFP. These tubular structures containing R2E2m::RFP, exhibited directed movement within and between muscle cells (red color indicates vesicles that were only visible with the RFP tag). The RFP-tagged protein was also detected in vesicles of the intestine and coelomocytes, indicating that R2E2 is released from muscle cells and endocytosed by these tissues. Folding sensors are depicted in green. While the injection of recombinant fibrils led to a sequence-specific induction of RĪ2-5 aggregation, co-expression of R2E2 led to a cell autonomous and cell non-autonomous non-sequence specific aggregation of the folding sensors RĪ2-5 and polyQ44. No co-localization of aggregates was observed in both cases.</p
R2E2 induces widespread aggregation of RĪ2-5.
<p>(AāC) Co-expression of R2E2m::RFP promotes RĪ2-5m::YFP aggregation. Confocal images of (A) RĪ2-5m::YFP control and (B) RĪ2-5m::YFP co-expressed with R2E2m::RFP in BWM cells. Boxed area indicate representative region used for FRAP analysis. (C) FRAP analysis of RĪ2-5m::YFP foci in a line co-expressing RĪ2-5m::YFP and R2E2m::RFP. (DāF) Muscle-expressed R2E2m::RFP promotes intestinal RĪ2-5i::GFP aggregation. (D) Confocal image of control animal expressing RĪ2-5i::GFP in intestinal cells. (E) Confocal image of animals expressing R2E2m::RFP in BWM cells and RĪ2-5i::GFP in the intestine. Boxed areas indicate representative region analyzed by FRAP. (F) FRAP analysis of RĪ2-5i::GFP alone (1) and in animals co-expressing R2E2m::RFP and RĪ2-5i::GFP (2). RFIā=ārelative fluorescence intensity in [%]. Scale bars: 10 Āµm.</p
Sequence-specific seeding of RĪ2-5.
<p>(A, B) Confocal images of (A) animals expressing GFP-tagged RĪ2-5i::GFP in intestinal cells and (B) 24 h after the injection of Alexa-555-tagged (red) recombinant Sup35NM fibrils. Boxed areas in A and B are representative regions subjected to FRAP analysis. (C) Enlargement of overlay picture in B. (D) FRAP analysis of RĪ2-5i::GFP control (1) and RĪ2-5i::GFP aggregates (2) formed after injection of recombinant Sup35NM fibrils. RFIā=ārelative fluorescence intensity in [%]. (E) Confocal images of animals expressing RĪ2-5i::GFP in the intestine 24 h after injection of the indicated recombinant fibrils.</p
The prion domain spreads between cells and tissues in <i>C. elegans</i> by vesicular transport.
<p>(A) Nematode muscle organization: BWM cells are arranged in quadrants, four longitudinal bands that run along the entire worm. Each quadrant is formed by staggered pairs of striated mononucleated cells. (B). Collapsed confocal z-stacks of nematodes expressing R2E2m::RFP and CFP::RAB-5 in BWM cells. RAB-5 does not co-localize with R2E2 containing vesicles, but enables the visualization of distinct cells, which is difficult in R2E2m::RFP only expressing nematodes. Single BWM cells of two quadrants are outlined. Arrows indicate connections between cells of different quadrants. Although muscle arms are extended not only to motor neurons but also to distant muscle cells that could form such connections, gap junctions between these cells allow electrical coupling, but no exchange of cytosolic content. (C) Collapsed confocal z-stacks of nematodes expressing RFPm in BWM cells. Single BWM cells of two quadrants are outlined. (D, E) Collapsed confocal z-stack and brightfield images of <i>C. elegans</i> expressing R2E2m::RFP or RFPm in BWM cells. Boxed region is displayed in higher magnification. BWM (M), intestine (I) and coelomocytes (CC) are outlined. (F) Single-plane confocal micrographs showing intestinally expressed R2E2i::RFP in vesicles located in intestinal and BWM cells. Intestine (I) and BWM cell (M) are outlined. Note the parallel aligned myofilaments (arrowheads) in the brightfield image as a muscle-specific marker. Scale bars: 10 Āµm.</p
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