Expansion microscopy reveals subdomains in C. elegans germ granules

Abstract

Many membraneless organelles (MLOs) have been shown to form via liquid-liquid phase separation (LLPS). Light and electron microscopy techniques have been indispensable in the identification and characterization of LLPS MLOs. However, for complex MLOs such as the perinuclear germ granule in C. elegans, our understanding of how the organelle as a whole is regulated is hampered by 1) the technical limitations in confocal fluorescence imaging in which only a few granule protein markers can be examined at a time and 2) the inaccessibility of electron microscopy. In this study, we take advantage of the newly developed super-resolution method of expansion microscopy (ExM) and in-situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with wild type animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. The experimental workflow developed here will serve as an important tool in our understanding of germ granule biology as well as the biological role of LLPS.

Geometry-preserving Expansion Microscopy microplates enable high fidelity nanoscale distortion mapping

“…As a rapidly evolving discipline of innovation, ExM continues to improve in its versatility and compatibility with various sample types. A significant domain of ongoing research and development is in leveraging ExM for high-resolution medical/biopsy imaging [27] as well as imaging complex, multicellular organisms [19] and their organs or tissues [28]. Whilst the microplate approach can be viewed as a gateway to improving through-put, our experience is that the fragility of polyacrylamide gels requires a level of care and attention that may be prohibitive for large sample numbers.…”

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy

“…Fig 3B-i), or c.) demonstrating spatial relationships of an antibody epitope or highly localised organelle to neighbouring cellular compartments. Outside of these common experimental scenarios, we have also shown the capacity to visualise the full extent of membraneless compartments such as perinuclear germ granules in C. elegans that are formed through liquid-liquid phase separation (Suen et al, 2022).…”

“…NHS esters have recently been introduced in pan-ExM (M’Saad & Bewersdorf, 2020), capitalising on the decrowding effect from 16-fold expansion to observe the finest of cellular ultrastructures such as mitochondrial cristae, golgi cisternae and nucleolar sub-compartments. Several other groups have used NHS esters with ExM (Damstra et al, 2022; Lee et al, 2022; Louvel et al, 2022; Mao et al, 2020; Sim et al, 2022), with many others implementing NHS esters alongside specific protein labels in order to better understand the biology of a range of cell systems (Bertiaux et al, 2021; Chacko et al, 2023; Hinterndorfer et al, 2022; Laporte et al, 2022; M’Saad et al, 2022; Rashpa & Brochet, 2022; Suen et al, 2022; Yu et al, 2020). The same probes and method have enabled unprecedented visualisations of the conformations of individual proteins, as shown with one nanometer ExM (Shaib et al, 2022).…”

Unclearing Microscopy

“…Analogous to electron microscopy (EM), subcellular compartments can now be imaged in their entirety, without the need for specific labels and with a lateral spatial resolution of ~15 nm. Our pan-staining concept has been since adapted by many labs to examine contextual spatial information in a multitude of biological systems, and at different expansion levels, demonstrating the wide utility of this contrasting approach (Nijenhuis et al, 2021;Sim et al, 2021;Klimas et al, 2021;Damstra et al, 2022;Laporte et al, 2022;Shaib et al, 2022;Hinterndorfer et al, 2022;Rashpa and Brochet, 2022;Pacheco et al, 2021;Suen et al 2022).…”