Automated segmentation of electron tomograms for a quantitative description of actin filament networks

Rigort, Alexander; Günther, David; Hegerl, R.; Baum, Daniel; Weber, Britta; Prohaska, Steffen; Medalia, Ohad; Baumeister, Wolfgang; Hege, Hans-Christian

doi:10.1016/j.jsb.2011.08.012

Cited by 193 publications

(211 citation statements)

References 37 publications

Supporting

Mentioning

202

Contrasting

Unclassified

Order By: Relevance

“…The increase in cell tomogram contrast using the Zernike phase plate technology in cryo-ET will likely ease the recognition and quantification of the observed subcellular features (32). Software algorithms are being actively developed for automatic feature extraction of filamentous features (33). Therefore, the ability of cryo-ET to measure fine ultrastructure aberrations opens the possibility for this technology to be used for large-scale biomarker development in this and other disease states.…”

Section: Discussionmentioning

confidence: 99%

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Wang

Stone

Kaelber

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.electron | cryotomography | platelet | microtubule | cancer

show abstract

Section: Discussionmentioning

confidence: 99%

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Wang

Stone

Kaelber

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Actin centerlines were computationally extracted using a template matching/tracing algorithm implemented within the Amira image segmentation software (10). Before filament extraction, tomograms were denoised using Nonlinear Anisotropic Diffusion filtering in IMOD.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…4. The plasma membranes were segmented by hand, while the central line of each actin filament was segmented computationally using an actin segmentation algorithm within the Amira software package (10). Using these 3D segmentations, we counted the number of filaments in each tomogram.…”

mentioning

confidence: 99%

Structure of the fission yeast actomyosin ring during constriction

Swulius

Nguyen

Ladinsky

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that "saddles" the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane.C ytokinesis, the final step of cell division in eukaryotic cells, is typically driven by a contractile actomyosin ring (AMR) primarily composed of actin (1) and myosin (2). Our understanding of the molecular mechanisms of cytokinesis is most detailed in the rodshaped unicellular eukaryote Schizosaccharomyces pombe (otherwise known as fission yeast), which shares a remarkably conserved set of cytokinesis genes with metazoans (3). In S. pombe, the AMR undergoes multiple phases known as assembly, maturation, constriction, and disassembly (4), with open questions in each of these four stages. Due to a lack of information about the precise arrangement of filamentous actin (F-actin) within the force-generating network of the AMR, we chose to focus on imaging the AMR during constriction.In S. pombe, glancing sections through plastic-embedded, dividing cells gave the first glimpse of actin filaments running parallel to the division plane at the front of the septum (5). Unfortunately, the study yielded limited examples and lacked 3D information for a full analysis. In an ambitious pioneering effort, Kamasaki et al. (6) produced 3D reconstructions of entire S. pombe AMRs by imaging serial sections through permeabilized cells decorated with myosin S1 fragments. The amount of F-actin and the size of the rings appeared significantly altered by the procedure used for preserving them (details in Discussion), but the continuous bundles that were reconstructed were composed of mixed polarity filaments running circumferentially around the cell.Here we sought to visualize the precise arrangement of F-actin within the AMR and its interface with the membrane by imaging intact cells in a cryopreserved state using electron cryotomography (ECT) (7). Because whole S. pombe cells are too thick for ECT, which is limited to specimens thinner than a few hundred nanometers, we overcame this obstacle by first rapid freezing dividing cells and then either cryosectioning them or using the recently developed method cryofocused ion beam (cryo-FIB) milling to ...

show abstract

“…Notably, the limited available EM data suggests that a considerably higher number of actin fibers might be found in the terminal sarcomere (Patla et al, 2010) compared with those in the central stress fiber sarcomeres (Cramer et al, 1997;Rigort et al, 2012). High-resolution cryo-EM tomography of focal adhesions reveals an actin inter-filament spacing of∼8 nm (Patla et al, 2010), which is probably due to bundling by fascin (Jansen et al, 2011).…”

Section: Stress Fiber Diversity and Interdependence With Focal Adhesionsmentioning

confidence: 99%

The inner workings of stress fibers − from contractile machinery to focal adhesions and back

Livne

Geiger

2016

Journal of Cell Science

161

153

View full text Add to dashboard Cite

Ventral stress fibers and focal adhesions are physically coupled structures that play key roles in cellular mechanics and force sensing. The tight functional interdependence between the two is manifested not only by their apparent proximity but also by the fact that ventral stress fibers and focal adhesions are simultaneously diminished upon actomyosin relaxation, and grow when subjected to external stretching. However, whereas the apparent co-regulation of the two structures is well-documented, the underlying mechanisms remains poorly understood. In this Commentary, we discuss some of the fundamental, yet still open questions regarding ventral stress fiber structure, its force-dependent assembly, as well as its capacity to generate force. We also challenge the common approach -i.e. ventral stress fibers are variants of the well-studied striated or smooth muscle machinery -by presenting and critically discussing alternative venues. By highlighting some of the less-explored aspects of the interplay between stress fibers and focal adhesions, we hope that this Commentary will encourage further investigation in this field.

show abstract

Automated segmentation of electron tomograms for a quantitative description of actin filament networks

Cited by 193 publications

References 37 publications

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Structure of the fission yeast actomyosin ring during constriction

The inner workings of stress fibers − from contractile machinery to focal adhesions and back

Contact Info

Product

Resources

About