Real-time monitoring of replication errors' fate reveals the origin and dynamics of spontaneous mutations.

The efficiency of replication error repair is a critical factor governing the emergence of mutations. However, it has so far been impossible to study this efficiency at the level of individual cells and to investigate if it varies within isogenic cell populations. In addition, why some errors escape repair remains unknown.

High-throughput detection and tracking of cells and intracellular spots in mother machine experiments.

The analysis of bacteria at the single-cell level is essential to characterization of processes in which cellular heterogeneity plays an important role. BACMMAN (bacteria mother machine analysis) is a software allowing fast and reliable automated image analysis of high-throughput 2D or 3D time-series images from experiments using the 'mother machine', a very popular microfluidic device allowing biological processes in bacteria to be investigated at the single-cell level. Here, we describe how to use some of the BACMMAN features, including (i) segmentation and tracking of bacteria and intracellular fluorescent spots, (ii) visualization and editing of the results, (iii) configuration of the image-processing pipeline for different datasets and (iv) BACMMAN coupling to data analysis software for visualization and analysis of data subsets with specific properties.

Real-time visualization of mutations and their fitness effects in single bacteria.

Mutations are the driving force of evolution and the source of important pathologies. The characterization of the dynamics and effects of mutations on fitness is therefore central to our understanding of evolution and to human health. This protocol describes how to implement two methods that we recently developed: mutation visualization (MV) and microfluidic mutation accumulation (µMA), which allow the occurrence of mutations created by DNA replication errors (MV) and the evolution of cell fitness during MA (µMA) to be followed directly in individual cells of Escherichia coli.

Mutation dynamics and fitness effects followed in single cells.

Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in , we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations.

Proliferation-dependent positioning of individual centromeres in the interphase nucleus of human lymphoblastoid cell lines.

The cell nucleus is a highly organized structure and plays an important role in gene regulation. Understanding the mechanisms that sustain this organization is therefore essential for understanding genome function. Centromeric regions (CRs) of chromosomes have been known for years to adopt specific nuclear positioning patterns, but the significance of this observation is not yet completely understood.

Analysis of nuclear organization with TANGO, software for high-throughput quantitative analysis of 3D fluorescence microscopy images.

The cell nucleus is a highly organized cellular organelle that contains the genome. An important step to understand the relationships between genome positioning and genome functions is to extract quantitative data from three-dimensional (3D) fluorescence imaging. However, such approaches are limited by the requirement for processing and analyzing large sets of images.

TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization.

Motivation: The cell nucleus is a highly organized cellular organelle that contains the genetic material. The study of nuclear architecture has become an important field of cellular biology. Extracting quantitative data from 3D fluorescence imaging helps understand the functions of different nuclear compartments.