Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.

Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.

Experiments and Lagrangian simulations on the formation of droplets in continuous mode

Experimental and computational studies on the dynamics of millimeter-scale cylindrical liquid jets are presented. The influences of the modulation amplitude and the nozzle geometry on jet behavior have been considered. Laser Doppler anemometry (LDA) was used in order to extract the velocity field of a jet along its length, and to determine the velocity modulation amplitude. Jet shapes and breakup dynamics were observed via shadowgraph imaging. Aqueous solutions of glycerol were used for these experiments. Results were compared with Lagrangian finite-element simulations with good quantitative agreement. © 2011 The American Physical Society.

Experiments and Lagrangian simulations on the formation of droplets in drop-on-demand mode

The creation and evolution of millimeter-sized droplets of a Newtonian liquid generated on demand by the action of pressure pulses were studied experimentally and simulated numerically. The velocity response within a model, large-scale printhead was recorded by laser Doppler anemometry, and the waveform was used in Lagrangian finite-element simulations as an input. Droplet shapes and positions were observed by shadowgraphy and compared with their numerically obtained analogues. © 2011 American Physical Society.