Expansion microscopy reveals subdomains inC. elegansgerm granules

Abstract

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid–liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule inC. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine theC. elegansgerm granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT 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. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid–liquid phase separation.

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

“…Its utility was demonstrated in pan-ExM 2 and ester counterstain of membraneless organelles known as P-granules. 14…”

“…Several groups have used NHS esters with ExM to understand cellular structures, [3][4][5][6][7] with many others implementing esters alongside direct protein labels in order to understand the arrangement of specific molecular targets. [8][9][10][11][12][13][14][15] More recently, one nanometer ExM used NHS esters to enable unprecedented visualisations of the confor-mations of individual proteins. 16 The physical properties of the conjugated dye have been hypothesised as a major determinant of the labelling patterns, dictating which cellular structures are illuminated.…”