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In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Akahoshi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Compartmentalized protein recruitment is a fundamental feature of signal transduction. Accordingly, the cell cortex is a primary site of signaling supported by the recruitment of protein regulators to the plasma membrane. Recent emergence of optogenetic strategies designed to control localized protein recruitment has offered valuable toolsets for investigating spatiotemporal dynamics of associated signaling mechanisms. However, determining proper recruitment parameters is important for optimizing synthetic control. In this chapter, we describe a stepwise process for building linear differential equation models that characterize the kinetics and spatial distribution of optogenetic protein recruitment to the plasma membrane. Specifically, we outline how to construct (1) ordinary differential equations that capture the kinetics, efficiency, and magnitude of recruitment and (2) partial differential equations that model spatial recruitment dynamics and diffusion. Additionally, we explore how these models can be used to evaluate the overall system performance and determine how component parameters can be tuned to optimize synthetic recruitment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E. coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E. coli. In contrast, E. coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Optogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long time scales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, efficient recruitment over wider gene expression ranges, and improved spatial control over signaling outputs. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications.

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.