In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen
Posted on: 2 December 2019
Preprint posted on 18 November 2019
Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.201911154
Letting the cat out of the bag: Cytoplasmic microtubules contain actin filaments
Selected by Sina Knapp
Categories: biophysics, cell biology
Background
The interaction between actin filaments and microtubules have been extensively studied during the last decades. This interplay between the two cytoskeletal systems is crucial during migration, polarization and division of the cell. Depending on the cellular context, the interaction of the two main cytoskeletal components occurs through either physical crosslinkers or passive interference. Although much is known about the structures of actin filaments and microtubules, a key question remains unresolved: What does the microtubule lumen consist of? In cytoplasmic microtubules, tubulin modifying enzymes have been identified and in sperm flagella microtubules, higher structures have been described. However, the content of cytoplasmic microtubules has thus far not been thoroughly revealed, as Cryo-EM analysis requires a very thin sample and super-resolution fluorescence microscopy is problematic in microtubules due to impaired antibody epitope accessibility.
Recently, the authors of this study identified a small-molecule activator (kinesore) of the microtubule motor kinesin-1, by which cytoplasmic microtubules bundle and reorganize in a nonradial network spread throughout the cytoplasm and expand in membrane-bound projections. Here the authors, by taking advantage of kinesore’s ability of inducing thin protrusive structures, set up a Cryo Electron Tomography (Cryo-ET) imaging protocol for analysis of the lumen of microtubules residing in the kinesore-induced cellular projections. Remarkably, the approach of Paul et al. revealed that filamentous actin can be found within the microtubule’s lumen.
Key findings
Kinesore treatment induces bundling of microtubules in cell projections
Cryo Correlative Light Electron Microsopy (Cryo-CLEM) after fluorescent membrane stain and kinesore treatment revealed microtubule bundles in cell projections in HAP1 cells. Live cell imaging using siR-tubulin labelled microtubules confirmed that these microtubule extensions are caused by the progressive addition of microtubules by loop extrusion events, following kinesore treatment. The cryo-EM analysis of these cell projections identified mainly aligned bundles of microtubules, containing 4-30 single ones (Fig. 1A).
The lumen of microtubules in cell projections contain actin filaments
Within the lumen of kinesore induced microtubules, cryo-ET analysis identified structures with filamentous density and helical appearance (Fig. 1B). To understand if the 5-9 nm wide filaments could be actin, kinesore treated cells were stained for tubulin and F-actin, revealing patches of F-actin linking gaps in ß-tubulin staining.
The morphology of the lumenal filaments could be grouped in two classes, differing in thickness. The filament thickness correlates with the microtubule thickness, as thinner filaments (class I filaments) were found in microtubules with smaller outer and lumenal diameters. Further, on average 27% of the total luminal length of the microtubule was filled with actin-like filaments. Thicker actin-like filaments (class II filaments) were shorter compared to thinner filaments, but the frequency of the two did not differ.
To further validate that the luminal filaments are actin fibers and to understand the difference between the two groups of filaments, layer line images after Fourier transforms of 2D projection images from 3D subvolumes were analyzed. This analysis showed that the class I filaments can be identified as F-actin by reflecting an actin layer line pattern. The class II filaments also resemble an actin layer pattern with a distinctive meridional reflection indicating affiliated proteins at the lumenal actin. This meridional reflection is speculated to resemble a formin-like encircled actin backbone.
Why I like this preprint
By additional accumulation of cytoplasmic microtubules in cell projections, the approach of Paul et al., allows the analysis of the microtubule lumen via Cryo-ET, overcoming previous limitations in sample thickness. The identification of filamentous actin within the lumen of microtubules sheds a new light on the crosstalk of actin with microtubules. Previously, actin filaments and microtubules were thought to merely interact, but the idea of one incorporating the other gives rise to new questions in the field.
Questions
- Actin filament occupancy does highly vary within microtubules (ranging from 4% to 76%), what distinguishes the highly occupied from less occupied microtubules?
- Is filamentous actin within the lumen specific to microtubules of the cell projections? How about microtubules during mitosis or at the leading edge of a migrating cell?
- Do you suggest with the hypothesis of class II filaments reflecting formin-like encircling of the actin backbone a possibility for actin filament assembly and elongation within the microtubules? Why are the class II filaments shorter compared to class I filaments?
doi: https://doi.org/10.1242/prelights.15513
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