Folding of prestin's anion-binding site and the mechanism of outer hair cell electromotility.

Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl- anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl- binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin's unique voltage-sensing mechanism and electromotility.

Folding of Prestin's Anion-Binding Site and the Mechanism of Outer Hair Cell Electromotility.

Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl - anion at a conserved binding site formed by amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl - binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion-binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and helps define prestin's unique voltage-sensing mechanism and electromotility.

Conserved NDR/LATS kinase controls RAS GTPase activity to regulate cell growth and chronological lifespan.

Adaptation to the nutritional environment is critical for all cells. RAS GTPase is a highly conserved GTP-binding protein with crucial functions for cell growth and differentiation in response to environmental conditions. Here, we describe a novel mechanism connecting RAS GTPase to nutrient availability in fission yeast. We report that the conserved NDR/LATS kinase Orb6 responds to nutritional cues and regulates Ras1 GTPase activity. Orb6 increases the protein levels of an Ras1 GTPase activator, the guanine nucleotide exchange factor Efc25, by phosphorylating Sts5, a protein bound to efc25 mRNA. By manipulating the extent of Orb6-mediated Sts5 assembly into RNP granules, we can modulate Efc25 protein levels, Ras1 GTPase activity, and, as a result, cell growth and cell survival. Thus, we conclude that the Orb6-Sts5-Ras1 regulatory axis plays a crucial role in promoting cell adaptation, balancing the opposing demands of promoting cell growth and extending chronological lifespan.

Synthesis of polymer nanoparticles via vapor phase deposition onto liquid substrates.

In this article, the growth of polymer nanoparticles formed at the liquid-vapor interface via vapor phase polymerization is studied. The particles grow by polymer aggregation, which is driven by the surface tension interaction between the liquid and polymer. It is demonstrated that the mechanism of particle growth is determined by whether polymer particles remain at the liquid-vapor interface or submerge into the liquid. The position of the particles depends on the interaction between the polymer and the liquid. For example, the deposition of poly(n-butyl acrylate) onto poly(dimethyl siloxane) and Krytox liquids leads to the formation of nanoparticles that remain at the liquid-vapor interface. The size of these particles increases as a function of deposition time. The deposition of poly(4-vinylpyridine) onto poly(dimethyl siloxane) and Krytox leads to the formation of nanoparticles that submerge into the liquid. The size of these particles does not significantly change with deposition time. Our study offers a new rapid, one-step synthetic approach for fabricating functional polymer nanoparticles for applications in catalysis, photonics, and drug delivery.

Effect of surface tension, viscosity, and process conditions on polymer morphology deposited at the liquid-vapor interface.

We have observed that the vapor-phase deposition of polymers onto liquid substrates can result in the formation of polymer films or particles at the liquid-vapor interface. In this study, we demonstrate the relationship between the polymer morphology at the liquid-vapor interface and the surface tension interaction between the liquid and polymer, the liquid viscosity, the deposition rate, and the deposition time. We show that the thermodynamically stable morphology is determined by the surface tension interaction between the liquid and the polymer. Stable polymer films form when it is energetically favorable for the polymer to spread over the surface of the liquid, whereas polymer particles form when it is energetically favorable for the polymer to aggregate. For systems that do not strongly favor spreading or aggregation, we observe that the initial morphology depends on the deposition rate. Particles form at low deposition rates, whereas unstable films form at high deposition rates. We also observe a transition from particle formation to unstable film formation when we increase the viscosity of the liquid or increase the deposition time. Our results provide a fundamental understanding about polymer growth at the liquid-vapor interface and can offer insight into the growth of other materials on liquid surfaces. The ability to systematically tune morphology can enable the production of particles for applications in photonics, electronics, and drug delivery and films for applications in sensing and separations.