Expansion of metabolically labelled endocytic organelles and cytoskeletal cell structures in Giardia lamblia using optimised U-ExM protocols

Abstract

Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.

Keywords: Giardia lamblia; cytoskeleton; endocytosis; endoplasmic reticulum; expansion microscopy; metabolic labelling; subcellular compartment.

Figure 1. Protocol of a post-and a substantially shortenedpre-expansion staining method. (A) An overview of the post-expansion staining protocol. Briefly, trophozoites of G. lamblia are left to settle on a cover slip (1). After addition of formaldehyde (FA) and acrylamide (AA), cells are embedded into a polymer and proteins are denatured (2). After a first round of expansion (3), the gels are shrunk in PBS (4). Compared to regular fluorescent assays, antibody incubations are longer for both the primary (5) and secondary antibodies requiring up to three hours per incubation (6). During secondary antibody incubation, DNA is stained with DAPI (6). Following the final expansion (7) gels are imaged (8). (B) Cells are harvested and fixed (1) prior to permeabilization and blocking (2). Epitopes are stained with primary (3) and secondary (4) antibodies before settling cells on a cover slip. Stained cells are then anchored during FA and AA treatment. Subsequently, gelation and denaturation are performed (6). After briefly washing the cells in PBS, the DNA is stained with DAPI (7) and after a final expansion step (8), the sample is imaged (9). Created with BioRender.com.

Figure 2. Comparison of a post- and a substantially shortened pre-expansion staining method used to visualise different cell compartments in G. lamblia. (A) Representative widefield light microscopy images of antibody labelled HA epitope tagged proteins expressed in G. lamblia trophozoites. Displayed are overview images of unexpanded cells in the first column. These cells were split off from the samples that were then further treated in the pre-expansion staining ExM method displayed in this figure. Representative cells of the pre- and post-expansion staining methods are depicted in columns 2-5 with the composite images including DNA stain in cyan. On the left the expected localisation and below the tagged protein is indicated. Signal was enhanced for better visibility. Scale bars: 20µm. (B) Expansion factors measured for unexpanded, pre- and post-expansion stained nuclei and ventral discs (n=20 cells). (C) Maximal Z-projection of widefield images of pre- and post-expanded cells treated with NHS-Ester. Different compartments are colour coded. Scale bars: 20 µm.

Figure 3. Cholera toxin-dependent metabolic labelling of peripheral endocytic compartments inExM. The workflow of metabolic cholera toxin labelling with subsequent antibody staining is depicted in (A). Briefly, harvested cells (1) are incubated with cholera toxin (CTX) which binds to glycans on cell membranes (2). Cells are allowed to settle on a cover slip (3) before performing the anchoring with formaldehyde (FA) and acrylamide (AA). After gelation and denaturation (4), the gels are briefly washed in PBS (5) before performing tubulin staining with primary (6) and secondary antibodies together with DAPI (7). An example image showing the CTX staining in magenta, tubulin in yellow and a composite including DAPI staining is depicted in part (B). Signal was enhanced for better visibility and colours are displayed in cmyk rather than original fluorophore colour. Scale bar 5 µm. Created with BioRender.com.