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Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a recently established and powerful model system for mycobacterial infection. In this chapter, two simple protocols for live imaging of Dictyostelium discoideum infection are described. The first method is used to monitor the dynamics of recruitment of GFP-tagged Dictyostelium discoideum proteins at single time-points corresponding to the main stages of the infection (1.5-72 h post infection). The second method focuses at the early stages of the establishment of an infection (0-3 h post infection). In addition, several procedures to improve the imaging of the bacterium-containing compartment are described. Basic bacterial parameters such as bacterial growth and the recruitment of host proteins to the bacterium-containing compartment can be easily and precisely quantified using macros for ImageJ. These methods can be adapted to monitoring mycobacteria infection in other systems using mammalian cells.

Phagocytosis and phagosome maturation lead to killing and digestion of bacteria by protozoans and innate immune phagocytes. Phagocytosis of particles expressing or coupled to various fluorescent reporters and sensors can be used to monitor quantitatively various parameters of this central biological process. In this chapter we detail different labeling techniques of bacteria and latex beads used to measure adhesion and uptake by FACS analysis. We also describe methods to use fluorescent reporter dyes (FITC or DQgreen) coupled to silica beads to measure the kinetics of acidification and proteolysis. Measurements can be performed either at the single-cell level, using live microscopy, or for a whole cell population, with a fluorescence microplate reader.

Mycobacterium marinum is the causative agent of fish and amphibian tuberculosis in the wild. It is a genetically close cousin of Mycobacterium tuberculosis, and thereby the infection process remarkably shares many of the hallmarks of M. tuberculosis infection in human, at both the cellular and organism levels. Therefore, M. marinum is used as a model for the study of mycobacterial infection in various host organisms. Recently, the Dictyostelium-M. marinum system has been shown to be a valuable model that recapitulates the main features of the intracellular fate of M. marinum including phagosome maturation arrest, as well as its particular cell-to-cell dissemination mode. We present here a "starter kit" of detailed methods that allows to establish an infection of Dictyostelium with M. marinum and to monitor quantitatively the intracellular bacterial growth.

The amoeba Dictyostelium discoideum is an established model to study phagocytosis. The sequence of events leading to the internalization and degradation of a particle is conserved in D. discoideum compared to metazoan cells. As its small haploid genome has been sequenced, it is now amenable to genome-wide analysis including organelle proteomics. Therefore, we adapted to Dictyostelium the classical protocol to purify phagosomes formed by ingestion of latex beads particles. The pulse-chase protocol detailed here gives easy access to pure, intact, and synchronized phagosomes from representative stages of the entire process of phagosome maturation. Recently, this protocol was used to generate individual temporal profiles of proteins and lipids during phagosome maturation generating a proteomic fingerprint of six maturation stages (1). In addition, immunolabeling of phagosomes on a coverslip was developed to visualize and quantitate antigen distribution at the level of individual phagosomes.

Recent years have seen a powerful revival of fluorescence microscopy techniques, both to observe live cells and fixed objects. The limits of sensitivity, simultaneous detection of multiple chromophores, and spatial resolution have all been pushed to the extreme. Therefore, it is essential to improve in parallel the quality of the structural and antigenic preservation during fixation and immunostaining. Chemical fixations are broadly used but often lead to antigenicity loss and severe membrane damages, such as organelle vesiculation. They also must be followed by membrane permeabilization by detergents or solvents, which can lead to extensive extraction and cytosol leakage. Fixation with solvents bypasses the need for permeabilization, but when carried out at "high" temperatures, leads to severe extraction of soluble proteins and lipids and cytosol wash-out, and has therefore been used routinely to visualize the cytoskeleton. Here, we describe a few modifications to the common aldehyde fixation protocol that help decrease the usual artifacts induced by chemical fixation. Alternatively, new techniques have now been established that are based on rapid freezing using a variety of coolants followed by fixation in solvents at low temperature. We present detailed protocols and notes that allow the achievement of optimal preservation and permeabilization for both light and electron microscopy.

Phagocytosis plays a fundamental role in the immune system for the defense against invading microorganisms and the clearing of apoptotic and cancerous cells. The common amoeba Dictyostelium discoideum is a recognized model for professional immune phagocytes and is now commonly used to study host-pathogen interactions. Dictyostelium is genetically and biochemically tractable and is a most versatile experimental system. The classical protocol for purifying phagosomes formed by ingestion of latex beads particles has been adapted to Dictyostelium. It was improved in yield, purity, and synchronicity, allowing isolation of milligram amounts of phagosomal proteins and lipids. This method has been used successfully to highlight membrane trafficking and phagosome maturation. Here, we present a step-by-step protocol including detailed notes necessary for ensuring access to a large number of highly synchronized phagosomes of high purity and integrity.