Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii

ABSTRACT Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKG I is plasma membrane associated whereas PKG II is cytosolic in Toxoplasma gondii . To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii . By combining AID regulation with genome editing strategies, we determined that PKG I is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKG II is functionally insufficient and dispensable in the presence of PKG I . The difference in functionality mapped to the first 15 residues of PKG I , containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in T. gondii . IMPORTANCE Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii . We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii . </jats:p

Green investment strategies: a positive force in cities

Deterioration of urban neighborhoods is known to induce out migration, but how well do public investments to reverse decline actually work? To evaluate Philadelphia’s greening investment, researchers measured property buyers’ willingness to pay more—and found that greening works.Community development

Multi-heme Cytochromes in Shewanella oneidensis MR-1:Structures, functions and opportunities

Multi-heme cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometers. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-heme cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-heme cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward there are opportunities to engage multi-heme cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-heme cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-heme cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies

High Spatial Resolution Kelvin Probe Force Microscopy With Coaxial Probes

Kelvin probe force microscopy (KPFM) is a widely used technique to measure the local contact potential difference (CPD) between an AFM probe and the sample surface via the electrostatic force. The spatial resolution of KPFM is intrinsically limited by the long range of the electrostatic interaction, which includes contributions from the macroscopic cantilever and the conical tip. Here, we present coaxial AFM probes in which the cantilever and cone are shielded by a conducting shell, confining the tip-sample electrostatic interaction to a small region near the end of the tip. We have developed a technique to measure the true CPD despite the presence of the shell electrode. We find the behavior of these probes agrees with an electrostatic model of the force, and we observe a factor of 5 improvement in spatial resolution relative to unshielded probes. Our discussion centers on KPFM, but the field confinement offered by these probes may improve any variant of electrostatic force microscopy.Engineering and Applied Science

The Importance of Cantilever Dynamics in the Interpretation of Kelvin Probe Force Microscopy

A realistic interpretation of the measured contact potential difference (CPD) in Kelvin probe force microscopy (KPFM) is crucial in order to extract meaningful information about the sample. Central to this interpretation is a method to include contributions from the macroscopic cantilever arm, as well as the cone and sharp tip of a KPFM probe. Here, three models of the electrostatic interaction between a KPFM probe and a sample are tested through an electrostatic simulation and compared with experiment. In contrast with previous studies that treat the KPFM cantilever as a rigid object, we allow the cantilever to bend and rotate; accounting for cantilever bending provides the closest agreement between theory and experiment. We demonstrate that cantilever dynamics play a major role in CPD measurements and provide a simulation technique to explore this phenomenon.Physic