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 the rapidness of image acquisition and three-dimensional 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. Three-dimensional reconstruction of an entire volume of these cells provided details on the 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.

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…Some unicellular organisms are about as easy to access with expansion microscopy as cell culture. For example, the parasitic protist Trypanosoma brucei has been accessed using the U-ExM approach without any major adaptations. , Many unicellular organisms, however, possess unique features that make them difficult to access via expansion microscopy.…”

Unlocking Trypanosome Biology: A Comprehensive Protein-Tagging Toolkit for Localization and Functional Analysis

“…This new vector series encompasses over 20 different fluorescent proteins, three different split fluorescent proteins, three different tags for 'click' chemistry, two different proximity labelling tags, and several "fusion tags" allowing microscopy to be combined with another assay. In addition, we designed a set of vectors encoding ten tandem copies of an epitope tag (including FLAG, HA, Myc, Ty, and V5 [2]) to support the emerging technique of expansion microscopy [8][9][10]. A summary of the complete vector series is presented in Table 1, a comprehensive vector list is in Supplemental Table 1, and all vectors and their GenBank files are available via Addgene (https://www.addgene.org).…”

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research

“…Stimulated emission depletion (STED) ( Willig et al., 2006 ), stochastic optical reconstruction microscopy (STORM) ( Rust et al., 2006 ), and structured illumination microscopy (SIM) ( Vangindertael et al., 2018 ) have all been used in the context of Kinetoplastid and Apicomplexan research. Expansion microscopy (ExM) ( Chen et al., 2015b ) and ultrastructure expansion microscopy (U-ExM) ( Gambarotto et al., 2021 ) have also been incorporated with great success ( Amodeo et al., 2021 ; Bertiaux et al., 2021 ; Dos Santos Pacheco and Soldati-Favre, 2021 ; Gorilak et al., 2021 ; Kalichava and Ochsenreiter, 2021 ; Liffner and Absalon, 2021 ; Tomasina et al., 2021 ). Despite their value, important bottlenecks for using super-resolution in a high-throughput manner are the time required for image acquisition, and the volume of data produced for quantitation.…”