Abstract
Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organization to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small molecule-induced, microtubule-based, cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in situ study of microtubule structure and contents.
Highlights
Contents of cytoplasmic microtubules are thought to be restricted to globular proteins such as tubulin-modifying enzymes (Coombes et al, 2016), higher order structures have been observed in sperm flagella microtubules and cilia (Ichikawa and Bui, 2018; Zabeo et al, 2018)
We describe the discovery of filamentous actin (F-actin) inside the microtubule lumen through in situ cryo-electron tomography analysis of these small molecule–induced projections
Formation and ultrastructure of small molecule–induced, microtubule-based projections To begin ultrastructural analysis of projections in their native state, HAP1 cells, incubated with a fluorescent membrane stain before kinesore treatment, were prepared for cryo–correlative light EM (CLEM). This allowed the unambiguous identification of projections using fluorescence microscopy that could be correlated with images from the electron microscope (Fig. 1 A)
Summary
Understanding of the lumenal contents of cytoplasmic microtubules has been classically driven by EM–based analysis in cells and tissues (Burton, 1984; Peters and Vaughn, 1967; Rodrıguez Echandıa et al, 1968), and most recently by high resolution cryoEM (Atherton et al, 2018; Bouchet-Marquis et al, 2007; Garvalov et al, 2006; Grange et al, 2017; Koning et al, 2008). Kinesore treatment results in dynamic looping and bundling of microtubules within the cytoplasm and their extrusion from the cell body as membrane-bound projections. We describe the discovery of filamentous actin (F-actin) inside the microtubule lumen through in situ cryo-electron tomography (cryo-ET) analysis of these small molecule–induced projections.
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