Cytoskeletal organization in microtentacles

Abstract

Microtentacles are thin, flexible cell protrusions that have recently been described and whose presence enhances efficient attachment of circulating cells. They are found on circulating tumor cells and can be induced on a wide range of breast cancer cell lines, where they are promoted by factors that either stabilize microtubules or destabilize the actin cytoskeleton. Evidence suggests that they are relevant to the metastatic spread of cancer, so understanding their structure and formation may lead to useful therapies. Microtentacles are formed by microtubules and contain vimentin intermediate filaments, but beyond this, there is little information about their ultrastructure. We have used electron microscopy of high pressure frozen sections and tomography of cryo-prepared intact cells, along with super resolution fluorescence microscopy, to provide the first ultrastructural insights into microtubule and intermediate filament arrangement within microtentacles. By scanning electron microscopy it was seen that microtentacles form within minutes of addition of drugs that stabilize microtubules and destabilize actin filaments. Mature microtentacles were found to be well below one micrometer in diameter, tapering gradually to below 100 nanometers at the distal ends. They also contained frequent branches and bulges suggestive of heterogeneous internal structure. Super resolution fluorescence microscopy and examination of sectioned samples showed that the microtubules and intermediate filaments can occupy different areas within the microtentacles, rather than interacting intimately as had been expected. Cryo-electron tomography of thin regions of microtentacles revealed densely packed microtubules and absence of intermediate filaments. The number of microtubules ranged from several dozen in some areas to just a few in the thinnest regions, with none of the regular arrangement found in axonemes. Improved understanding of the mechanism of microtentacle formation, as well as the resultant structure, will be valuable in developing therapies against metastasis, if the hypothesized role of microtentacles in metastasis is confirmed. This work provides a significant step in this direction.

A mode of cell adhesion and migration facilitated by CD44-dependent microtentacles

“…STORM Imaging. The 3D-STORM imaging (71,72) was carried out on a homebuilt setup using a Nikon CFI Plan Apo λ 100× oil immersion objective (numerical aperture 1.45), as described previously (33,73). Briefly, the sample was mounted with an imaging buffer consisting of 5% (wt/vol) glucose, 100 mM cysteamine, 0.8 mg/mL glucose oxidase, and 40 μg/mL catalase in 1 M Tris•HCI (pH 7.5).…”

“…Furthermore, cells on HA generally lacked thick actin bundles and large lamellipodia typically observed on two-dimensional (2D), integrin-engaging substrates (27)(28)(29). The morphology of these protrusions, particularly their microtubule and actin architecture, seemed reminiscent of microtubule-positive McTNs that have been described in circulating breast cancer cells and implicated in endothelial docking and extravasation (30)(31)(32)(33). Thus, we refer to these protrusions as McTNs.…”

Vimentin Diversity in Health and Disease

“…These membrane extensions have been shown to help circulating tumour cells to reattach at the vasculature when leaving the blood stream, on their way to metastasis formation [ 231 ]. Microtentacles have been shown to contain vimentin [ 232 , 233 ], although the definition of the role of vimentin in these protrusions requires further studies.…”

LncRNAs regulate the cytoskeleton and related Rho/ROCK signaling in cancer metastasis

“…It plays an important role in the maintenance of cell shape, cell movement, transportation of substances in cells, and cytokinesis. Eukaryotic cells contain three main types of cytoskeletal filaments: microfilaments, microtubules, and intermediate filaments [ 12 ].…”