Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy

Photoactivated localization microscopy analysis of chemotaxis receptors in bacteria suggests that the non-random organization of these proteins results from random self-assembly of clusters without direct cytoskeletal involvement or active transport

Noise Contributions in an Inducible Genetic Switch: A Whole-Cell Simulation Study

Stochastic expression of genes produces heterogeneity in clonal populations of bacteria under identical conditions. We analyze and compare the behavior of the inducible lac genetic switch using well-stirred and spatially resolved simulations for Escherichia coli cells modeled under fast and slow-growth conditions. Our new kinetic model describing the switching of the lac operon from one phenotype to the other incorporates parameters obtained from recently published in vivo single-molecule fluorescence experiments along with in vitro rate constants. For the well-stirred system, investigation of the intrinsic noise in the circuit as a function of the inducer concentration and in the presence/absence of the feedback mechanism reveals that the noise peaks near the switching threshold. Applying maximum likelihood estimation, we show that the analytic two-state model of gene expression can be used to extract stochastic rates from the simulation data. The simulations also provide mRNA–protein probability landscapes, which demonstrate that switching is the result of crossing both mRNA and protein thresholds. Using cryoelectron tomography of an E. coli cell and data from proteomics studies, we construct spatial in vivo models of cells and quantify the noise contributions and effects on repressor rebinding due to cell structure and crowding in the cytoplasm. Compared to systems without spatial heterogeneity, the model for the fast-growth cells predicts a slight decrease in the overall noise and an increase in the repressors rebinding rate due to anomalous subdiffusion. The tomograms for E. coli grown under slow-growth conditions identify the positions of the ribosomes and the condensed nucleoid. The smaller slow-growth cells have increased mRNA localization and a larger internal inducer concentration, leading to a significant decrease in the lifetime of the repressor–operator complex and an increase in the frequency of transcriptional bursts

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200–300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring

Current understanding of the human microbiome

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Medicine 24 (2018): 392–400, doi:10.1038/nm.4517.Our understanding of the link between the human microbiome and disease, including obesity, inflammatory bowel disease, arthritis and autism, is rapidly expanding. Improvements in the throughput and accuracy of DNA sequencing of the genomes of microbial communities associated with human samples, complemented by analysis of transcriptomes, proteomes, metabolomes and immunomes, and mechanistic experiments in model systems, have vastly improved our ability to understand the structure and function of the microbiome in both diseased and healthy states. However, many challenges remain. In this Review, we focus on studies in humans to describe these challenges, and propose strategies that leverage existing knowledge to move rapidly from correlation to causation, and ultimately to translation.Many of the studies described here in our laboratories were supported by the NIH, NSF, DOE, and the Alfred P. Sloan Foundation.2018-10-1