Particle-based simulations reveal two positive feedback loops allow relocation and stabilization of the polarity site during yeast mating.

Many cells adjust the direction of polarized growth or migration in response to external directional cues. The yeast Saccharomyces cerevisiae orient their cell fronts (also called polarity sites) up pheromone gradients in the course of mating. However, the initial polarity site is often not oriented towards the eventual mating partner, and cells relocate the polarity site in an indecisive manner before developing a stable orientation. During this reorientation phase, the polarity site displays erratic assembly-disassembly behavior and moves around the cell cortex. The mechanisms underlying this dynamic behavior remain poorly understood. Particle-based simulations of the core polarity circuit revealed that molecular-level fluctuations are unlikely to overcome the strong positive feedback required for polarization and generate relocating polarity sites. Surprisingly, inclusion of a second pathway that promotes polarity site orientation generated relocating polarity sites with properties similar to those observed experimentally. This pathway forms a second positive feedback loop involving the recruitment of receptors to the cell membrane and couples polarity establishment to gradient sensing. This second positive feedback loop also allows cells to stabilize their polarity site once the site is aligned with the pheromone gradient.

Mycobacterial Genetic Technologies for Probing the Host-Pathogen Microenvironment.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the oldest and most successful pathogens in the world. Diverse selective pressures encountered within host cells have directed the evolution of unique phenotypic traits, resulting in the remarkable evolutionary success of this largely obligate pathogen. Despite centuries of study, the genetic repertoire utilized by Mtb to drive virulence and host immune evasion remains to be fully understood. Various genetic approaches have been and continue to be developed to tackle the challenges of functional gene annotation and validation in an intractable organism such as Mtb. In vitro and ex vivo systems remain the primary approaches to generate and confirm hypotheses that drive a general understanding of mycobacteria biology. However, it remains of great importance to characterize genetic requirements for successful infection within a host system as in vitro and ex vivo studies fail to fully replicate the complex microenvironment experienced by Mtb. In this review, we evaluate the employment of the mycobacterial genetic toolkit to probe the host-pathogen interface by surveying the current state of mycobacterial genetic studies within host systems, with a major focus on the murine model. Specifically, we discuss the different ways that these tools have been utilized to examine various aspects of infection, including bacterial survival/virulence, bacterial evasion of host immunity, and development of novel antibacterial/vaccine strategies.

Profiling Myxococcus xanthus Swarming Phenotypes through Mutation and Environmental Variation.

Myxococcus xanthus is a bacterium that lives on surfaces as a predatory biofilm called a swarm. As a growing swarm feeds on prey and expands, it displays dynamic multicellular patterns such as traveling waves called ripples and branching protrusions called flares. The rate at which a swarm expands across a surface, and the emergence of the coexisting patterns, are all controlled through coordinated cell movement. M. xanthus cells move using two motility systems known as adventurous (A) and social (S). Both are involved in swarm expansion and pattern formation. In this study, we describe a set of M. xanthus swarming genotype-to-phenotype associations that include both genetic and environmental perturbations. We identified new features of the swarming phenotype, recorded and measured swarm expansion using time-lapse microscopy, and compared the impact of mutations on different surfaces. These observations and analyses have increased our ability to discriminate between swarming phenotypes and provided context that allows us to identify some phenotypes as improbable outliers within the M. xanthus swarming phenome. IMPORTANCE Myxococcus xanthus grows on surfaces as a predatory biofilm called a swarm. In nature, a feeding swarm expands by moving over and consuming prey bacteria. In the laboratory, a swarm is created by spotting cell suspension onto nutrient agar in lieu of prey. The suspended cells quickly settle on the surface as the liquid is absorbed into the agar, and the new swarm then expands radially. An assay that measures the expansion rate of a swarm of mutant cells is the first, and sometimes only, measurement used to decide whether a particular mutation impacts swarm motility. We have broadened the scope of this assay by increasing the accuracy of measurements and introducing prey, resulting in new identifiable and quantifiable features that can be used to improve genotype-to-phenotype associations.

Cellular Uptake of a Fluorescent Ligand Reveals Ghrelin O-Acyltransferase Interacts with Extracellular Peptides and Exhibits Unexpected Localization for a Secretory Pathway Enzyme.

Ghrelin O-acyltransferase (GOAT) plays a central role in the maturation and activation of the peptide hormone ghrelin, which performs a wide range of endocrinological signaling roles. Using a tight-binding fluorescent ghrelin-derived peptide designed for high selectivity for GOAT over the ghrelin receptor GHSR, we demonstrate that GOAT interacts with extracellular ghrelin and facilitates ligand cell internalization in both transfected cells and prostate cancer cells endogenously expressing GOAT. Coupled with enzyme mutagenesis, ligand uptake studies support the interaction of the putative histidine general base within GOAT with the ghrelin peptide acylation site. Our work provides a new understanding of GOAT's catalytic mechanism, establishes that GOAT can interact with ghrelin and other peptides located outside the cell, and raises the possibility that other peptide hormones may exhibit similar complexity in their intercellular and organismal-level signaling pathways.