Preparation of Caenorhabditis elegans for Scoring of Muscle-derived Exophers

Abstract

Utilizingresources available from the mother's body to guarantee healthy offspring growth is the fundamental reproductive strategy. Recently, we showed that a class of the largest extracellular vesicles known as exophers, which are responsible for the removal of neurotoxic components from neurons ( Melentijevic et al., 2017 ) and damaged mitochondria from cardiomyocytes (Nicolás-Ávila et al., 2020), are released by the Caenorhabditis elegans hermaphrodite body wall muscles (BWM), to support embryonic growth ( Turek et al., 2021 ). Employing worms expressing fluorescent reporters in BWM cells, we found that exopher formation (exophergenesis) is sex-specific and fertility-dependent. Moreover, exophergenesis is regulated by the developing embryo in utero, and exophers serve as transporters for muscle-generated yolk proteins, which can be used to nourish the next generation. Given the specific regulation of muscular exophergenesis, and the fact that muscle-generated exophers are much larger than neuronal ones and have different targeting, their identification and quantification required a modified approach from that designed for neuronal-derived exophers ( Arnold et al., 2020 ). Here, we present a methodology for assessing and quantifying muscle-derived exophers that can be easily extended to determine their function and regulation in various biological contexts. Graphical abstract.

Keywords: Body wall muscle; Exopher; Extracellular vesicle; Fluorescent microscopy; Keywords: C. elegans.

Figure 1.. Fluorescently labelled (wrmScarlet) exophers extruded from the body wall muscles on day 2 of adulthood.

A. Characteristics of muscle-derived exophers in C. elegans . B–C. Cross-sections of ACH93 worms (with a focus on the midbody) visualized in the RFP and GFP channels of the confocal microscope (Zeiss LSM800). The square region on panel C marked with a dashed line is magnified 3.2× on its right side. The arrows point to exophers, and the asterisks indicate the position of the vulva. Structures of an irregular, non-round shape or inside the BWM are not classified as exophers. Scale bars are 20 μm in B and 50 and 20 μm in C. D. The wrmScarlet signal may also originate from scattered and smaller vesicles (< 2 μm) present in the body cavity (circled with a dotted line). In our readings, we do not count these structures as exophers, as these are coelomocytes that degrade fluorescent proteins taken up from a pseudocoelom. Scale bar is 10 µm. E. Zoom into the vulva region in separate channels: DIC, a merge of RFP and GFP, separate RFP and GFP. The square marked with a dashed line marks the boundaries of the enlarged region in the bottom left corner. The arrows indicate exophers with mitochondria. MOM—mitochondrial outer membrane. Scale bar is 20 µm.

Figure 2.. An example of an experiment subjected to quantitative, statistical analysis, and visualization in GraphPad Prism software.

The formation of muscle-derived exophers is affected by the depletion of EMB-8 ( emb-8 RNAi on strain ACH93). ACH93 worms fed on HT115 E. coli bacteria with the empty vector were used as control. **** P < 0.0001, Mann–Whitney test.