Revealing the polarity of actin filaments by cryo-electron tomography

Abstract

Supplementary Figure 1 | Missing wedge and projection of subtomograms. (a) Structure of an actin filament (EMD-6179 [1]), that was oriented parallel to the tilt-axis (y-axis), and then distorted by the missing wedge. (b) However, if the filament was oriented parallel to the x-axis (orthogonal to the tilt-axis), the anisotropic distortion caused by the missing wedge in z-direction is substantially more pronounced. (c) Left to right: projection of filament (a) in z-direction, projection of filament (b) in z-direction, and difference image between the two projections. The difference image is featureless, which indicates that the missing wedge induced anisotropy vanishes in the projection images. (d) However, if a mask in z-direction is applied before projection (in this case the height of the mask was 11 nm), the difference image is not featureless anymore. The influence of this mask on the precision of APT is part of the validation of the method. Scale bars 10 nm.

Peering into tunneling nanotubes—The path forward

“…It is therefore essential researchers increase the resolution of cryo‐TEM and ‐ET technologies (e.g., TITAN Krios cryo‐TEM) in order to correctly assess the arrangement and polarity of actin filament bundles within TNTs. The application of novel computational approaches elegantly developed by Medalia and colleagues to study actin filament polarity in cryo‐EM datasets will assist in this endeavor (Martins et al , 2021).…”

The role of mode switching in a population of actin polymers with constraints

“…Note however that experimental approaches to measure the length of actin filaments, based on electron micrographs (e.g., [29]) or more recently on cry-electron tomography (e.g. [18]), typically yield estimates that vary from a few hundreds nanometers [3,9] to a few micrometers [26,30], covering at least one order of magnitude.…”

Talin-activated vinculin interacts with branched actin networks to initiate bundles

“…More importantly, we reconstructed actin filaments to a resolution of 14 Angstrom and unambiguously determined filaments' polarity within these in vitro bundles. Next, we determined the polarity of the original filaments ( Figure 3 figure supplement 4A-D and Materials and Methods section, (Martins et al, 2020)). Evaluating the polarity of each actin filament with respect to its neighboring filaments in 3D revealed a mixed-polarity organization, wherein neighboring filaments could exhibit both uniform-and mixed-polarity orientation ( Figure 3figure supplement 4E).…”

“…Actin filaments were automatically segmented, as we described in (Martins et al, 2020), from 16 tomograms using a convolutional neural network algorithm (Chen et al, 2017). Based on the segmentations, 3D coordinates of actin segments were chosen along the filaments with a spacing of 16.5 nm.…”

“…the black filaments in Figure 3 figure supplement 4D. In order to measure the distance between the filaments and to quantify the distribution of uniform polarity and mixed polarity regions within the bundles, we performed a 3D neighborhood analysis that described in details in Martins et al (Martins et al, 2020).…”