Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites

Abstract

Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to rapidness of image acquisition and 3D reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic, and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. 3D reconstruction of an entire volume of these cells provided details on morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

3D FIB‐SEM structural insights into the architecture of sub‐pellicular microtubules of Trypanosoma cruzi epimastigotes

“…Some evidence indicates T. brucei SPMT has a minus‐end orientation toward the cell's anterior region and that the dynamic ends are located at the posterior region of the T. brucei (Sherwin and Gull, 1989b; Robinson et al., 1995, Wheeler et al., 2019). In Leishmania major , the end of individual corset microtubules is in the posterior part; the event was observed by Expansion microscopy (ExM), a powerful super‐resolution method in cell biology (Gorilak et al., 2021). Reconstruction of cellular volumes in three dimensions by ExM can be used in addition to FIB‐SEM analysis of T. cruzi to promote comparative data.…”

Kinetoplastid-specific X2-family kinesins interact with a kinesin-like pleckstrin homology domain protein that localizes to the trypanosomal microtubule quartet

“…ExM has recently been implemented in T. brucei (Gorilak et al ., 2021; Amodeo et al ., 2021), and accentuates the part of MtQ that wraps around the flagellar pocket upon immunolabeling with antibody recognizing MT-incorporated, acetylated α-tubulin (Fig. 5A) (Gorilak et al ., 2021). Additionally, this procedure allows the proximal MtQ to be distinguished from the nearby hook complex, which lacks MTs (Esson et al ., 2012).…”

“…To verify that TbKifX2 indeed binds the MtQ, we employed expansion microscopy (ExM), which enlarges cells by embedding them within a swellable polyacrylamide matrix prior to immunolabelling to allow examination of cellular structures in greater detail (Gambarotto et al, 2019). ExM has recently been implemented in T. brucei (Gorilak et al, 2021;Amodeo et al, 2021), and accentuates the part of MtQ that wraps around the flagellar pocket upon immunolabeling with antibody recognizing MT-incorporated, acetylated α-tubulin (Fig 6A) (Gorilak et al, 2021). Additionally, this procedure allows the proximal MtQ to be distinguished from the nearby hook complex, which lacks MTs (Esson et al, 2012).…”

The mitochondrial genome segregation system ofT. brucei: Single p197 molecules connect the basal body with the outer membrane

“…To more precisely analyze the intracellular localization of p197-HA we used U-ExM which was recently established for trypanosomes (33)(34)(35). U-ExM allows to increase the spatial resolution for microscopic imaging by physical expansion of the cell (36,37).…”