Investigating the cytoskeleton and microtubule-based transport with in situ cryo-electron tomography

Abstract

The cytoskeleton is composed of microtubules, actin, and intermediate filaments, which function both as a cellular scaffold and as tracks for motor proteins that drive intracellular transport. Cytoplasmic dynein, in combination with dynactin and activating adaptors, is a microtubule-based motor that drives long-range transport of many cargo types towards microtubule minus ends. Structural and cellular work to date has revealed how dynein-dynactin complexes form and arrange on microtubules. However, due to limitations of in vitro reconstitution, the molecular details of cargo interaction are not well described. Cryo-electron tomography (cryo-ET) can be applied to cellular samples to resolve macromolecules spatially at nanometer resolution. When many copies of a target are present, sub-tomogram averaging (STA) can be applied to improve resolution. I aimed to use cryo-ET to visualise cargo-bound dynein-dynactin complexes, directly in cells. I also aimed to visualise the specialised cytoskeletal environments in different cellular compartments in which dynein functions. I initially aimed to use cryo-ET to visualise the cellular projections of SH-SY5Y cells, a human neuroblastoma line. Cryo-ET of these projections revealed microtubule bundles and organelles. Immunofluorescence and STA analysis revealed uniform polarity microtubule bundles in MAP2-expressing projections, suggesting that differentiated SH-SY5Y projections showed a mixed neurite phenotype. The signal:noise ratio (SNR) of these tomograms was too low to visualise most macromolecules in the cytoplasm. This made this approach sub-optimal to visualise dynein-dynactin complexes. To address the SNR issues, I next employed cryo-focused ion beam (cryo-FIB) milling to thick rodent hippocampal neurites. This was to 1) target cellular regions with more motor-driven transport and 2) to remove unnecessary cellular noise in attempt to improve tomogram SNR. I found that, even with sample optimisation, cryo-FIB milling of neurites was low-throughput and produced poor quality lamellae. This undermined the aim of visualising dynein-dynactin complexes in situ. I also used a cryo-correlative light and electron microscopy (cryo-CLEM) approach to target the axon initial segment (AIS), providing insights into its microtubule and neurofilament networks. Due to the poor lamella quality, I next pursued more conventional cryo-FIB milling cellular targets. I next investigated cryo-FIB milling of cancer cell lines with dynein-driven cargo clustering systems. This was to increase the number of dynein-dynactin complexes on recognisable cargo, for a targeted cryo-ET approach. I first trialled a dynein and peroxisome clustering system that harnessed the inducible FKBP-FRB binding system. Cryo-CLEM allowed the targeting and identification of possible clusters in lamellae; however, tomograms lacked microtubules, which is an essential pre-requisite for visualising motile dynein-dynactin complexes. I next trialled a sodium arsenite (AS)-induced dynein-driven organelle clustering system. While many instances of cargo in the vicinity of microtubules were observed, no putative dynein-dynactin complexes were observed, likely as a result of the thickness of the lamellae in this dataset which were on average >200nm. However, the tomograms did reveal surprising ribosome localisations; membranes of vesicles of many forms were decorated with ribosomes in AS-treated HeLa cells. STA revealed a subset of these ribosomes that appeared to be non-active, that bound via a novel binding mode. Finally, I trialled a dynein-driven mitochondrial clustering system. This system yielded a large tomogram dataset with many microtubules and mitochondrial cargo, in which I could manually identify possible dynein-dynactin complex densities. Going forward, a computational particle picking and STA approach would confirm if these densities are in fact dynein-dynactin complexes. This work also showed that in situ cryo-ET is promising for identifying dynein-dynactin complex on cargo.