scholarly journals A new look for the growing microtubule end?

2018 ◽  
Vol 217 (8) ◽  
pp. 2609-2611 ◽  
Author(s):  
Luke M. Rice
Keyword(s):  
State Of The Art ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Fundamental Question ◽  
Cell Biol ◽  
Three Dimensional Images ◽  
In Cells ◽  
New Look

What does the end of a growing microtubule look like? In this issue, McIntosh et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201802138) use electron tomography to provide state-of-the-art three-dimensional images of microtubule ends in cells and in vitro, yielding an unexpected answer to this fundamental question.

scholarly journals Microtubule architecture in vitro and in cells revealed by cryo-electron tomography

2018 ◽  
Vol 74 (6) ◽  
pp. 572-584 ◽  
Author(s):  
Joseph Atherton ◽  
Melissa Stouffer ◽  
Fiona Francis ◽  
Carolyn A. Moores
Keyword(s):  
Molecular Motors ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
In Cells

The microtubule cytoskeleton is involved in many vital cellular processes. Microtubules act as tracks for molecular motors, and their polymerization and depolymerization can be harnessed to generate force. The structures of microtubules provide key information about the mechanisms by which their cellular roles are accomplished and the physiological context in which these roles are performed. Cryo-electron microscopy allows the visualization of in vitro-polymerized microtubules and has provided important insights into their overall morphology and the influence of a range of factors on their structure and dynamics. Cryo-electron tomography can be used to determine the unique three-dimensional structure of individual microtubules and their ends. Here, a previous cryo-electron tomography study of in vitro-polymerized GMPCPP-stabilized microtubules is revisited, the findings are compared with new tomograms of dynamic in vitro and cellular microtubules, and the information that can be extracted from such data is highlighted. The analysis shows the surprising structural heterogeneity of in vitro-polymerized microtubules. Lattice defects can be observed both in vitro and in cells. The shared ultrastructural properties in these different populations emphasize the relevance of three-dimensional structures of in vitro microtubules for understanding microtubule cellular functions.

scholarly journals Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments

2018 ◽  
Vol 217 (8) ◽  
pp. 2691-2708 ◽  
Author(s):  
J. Richard McIntosh ◽  
Eileen O’Toole ◽  
Garry Morgan ◽  
Jotham Austin ◽  
Evgeniy Ulyanov ◽  
...  
Keyword(s):  
High Pressure ◽  
Brownian Dynamics ◽  
Electron Tomography ◽  
Tubulin Polymerization ◽  
Guanosine Triphosphate ◽  
Simple Mechanism ◽  
In Cells

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate–tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.

scholarly journals Current data processing strategies for cryo-electron tomography and subtomogram averaging

2021 ◽  
Vol 478 (10) ◽  
pp. 1827-1845
Author(s):  
Euan Pyle ◽  
Giulia Zanetti
Keyword(s):  
Data Processing ◽  
Electron Tomography ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Current Data ◽  
Processing Strategies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
The Individual

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

scholarly journals Three-dimensional ultrastructure of the septin filament network inSaccharomyces cerevisiae

2012 ◽  
Vol 23 (3) ◽  
pp. 423-432 ◽  
Author(s):  
Aurélie Bertin ◽  
Michael A. McMurray ◽  
Jason Pierson ◽  
Luong Thai ◽  
Kent L. McDonald ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Yeast Cells ◽  
Lipid Monolayers ◽  
Morphological Description ◽  
Section Electron ◽  
Subunit Organization

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P2-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo–electron tomography. We found networks of filaments both perpendicular and parallel to the mother–bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

scholarly journals Modifying the Tumour Microenvironment: Challenges and Future Perspectives for Anticancer Plasma Treatments

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1920 ◽  
Author(s):  
Angela Privat-Maldonado ◽  
Charlotta Bengtson ◽  
Jamoliddin Razzokov ◽  
Evelien Smits ◽  
Annemie Bogaerts
Keyword(s):  
State Of The Art ◽  
Three Dimensional ◽  
Local Therapy ◽  
Tumour Microenvironment ◽  
In Vitro Models ◽  
Plasma Therapy ◽  
Secreted Factors ◽  
Physical Interactions ◽  
Cancerous Cells

Tumours are complex systems formed by cellular (malignant, immune, and endothelial cells, fibroblasts) and acellular components (extracellular matrix (ECM) constituents and secreted factors). A close interplay between these factors, collectively called the tumour microenvironment, is required to respond appropriately to external cues and to determine the treatment outcome. Cold plasma (here referred as ‘plasma’) is an emerging anticancer technology that generates a unique cocktail of reactive oxygen and nitrogen species to eliminate cancerous cells via multiple mechanisms of action. While plasma is currently regarded as a local therapy, it can also modulate the mechanisms of cell-to-cell and cell-to-ECM communication, which could facilitate the propagation of its effect in tissue and distant sites. However, it is still largely unknown how the physical interactions occurring between cells and/or the ECM in the tumour microenvironment affect the plasma therapy outcome. In this review, we discuss the effect of plasma on cell-to-cell and cell-to-ECM communication in the context of the tumour microenvironment and suggest new avenues of research to advance our knowledge in the field. Furthermore, we revise the relevant state-of-the-art in three-dimensional in vitro models that could be used to analyse cell-to-cell and cell-to-ECM communication and further strengthen our understanding of the effect of plasma in solid tumours.

scholarly journals Digital Twins for Tissue Culture Techniques—Concepts, Expectations, and State of the Art

Processes ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 447
Author(s):  
Johannes Möller ◽  
Ralf Pörtner
Keyword(s):  
Tissue Culture ◽  
Mathematical Models ◽  
Product Life Cycle ◽  
State Of The Art ◽  
Three Dimensional ◽  
Culture Techniques ◽  
Digital Twins ◽  
On Chip ◽  
Tissue Culture Techniques

Techniques to provide in vitro tissue culture have undergone significant changes during the last decades, and current applications involve interactions of cells and organoids, three-dimensional cell co-cultures, and organ/body-on-chip tools. Efficient computer-aided and mathematical model-based methods are required for efficient and knowledge-driven characterization, optimization, and routine manufacturing of tissue culture systems. As an alternative to purely experimental-driven research, the usage of comprehensive mathematical models as a virtual in silico representation of the tissue culture, namely a digital twin, can be advantageous. Digital twins include the mechanistic of the biological system in the form of diverse mathematical models, which describe the interaction between tissue culture techniques and cell growth, metabolism, and the quality of the tissue. In this review, current concepts, expectations, and the state of the art of digital twins for tissue culture concepts will be highlighted. In general, DT’s can be applied along the full process chain and along the product life cycle. Due to the complexity, the focus of this review will be especially on the design, characterization, and operation of the tissue culture techniques.

scholarly journals Novel cryo-electron tomography structure of Arp2/3 complex in cells reveals mechanisms of branch formation

2020 ◽  
Author(s):  
Florian Fäßler ◽  
Georgi Dimchev ◽  
Victor-Valentin Hodirnau ◽  
William Wan ◽  
Florian KM Schur
Keyword(s):  
Actin Filament ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Branch Formation ◽  
Complex Structural ◽  
Filament Networks ◽  
Subtomogram Averaging ◽  
Resolution Structure ◽  
In Cells

AbstractThe actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Our structure indicates a central role for the ArpC3 subunit in stabilizing the active conformation and suggests that in the branch junction relocation of the ArpC5 N-terminus and the C-terminal tail of Arp3 is important to fix Arp2 and Arp3 in an actin dimer-like conformation. Notably, our model of the branch junction in cells significantly differs from the previous in vitro branch junction model.

scholarly journals The Supramolecular Organization of Fibrillin-Rich Microfibrils

2001 ◽  
Vol 152 (5) ◽  
pp. 1045-1056 ◽  
Author(s):  
Clair Baldock ◽  
Abraham J. Koster ◽  
Ulrike Ziese ◽  
Matthew J. Rock ◽  
Michael J. Sherratt ◽  
...  
Keyword(s):  
Binding Sites ◽  
Colloidal Gold ◽  
Symmetry Axis ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Supramolecular Organization ◽  
Staggered Arrangement ◽  
Cell Layers ◽  
Proline Rich Region

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-Å resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of ∼100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

scholarly journals Conserved and Variable Features of Gag Structure and Arrangement in Immature Retrovirus Particles

2010 ◽  
Vol 84 (22) ◽  
pp. 11729-11736 ◽  
Author(s):  
Alex de Marco ◽  
Norman E. Davey ◽  
Pavel Ulbrich ◽  
Judith M. Phillips ◽  
Vanda Lux ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Supramolecular Organization ◽  
Gag Proteins ◽  
Terminal Domain ◽  
Rous Sarcoma ◽  
Subtomogram Averaging

ABSTRACT The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

Superresolution three-dimensional images of fluorescence in cells with minimal light exposure

Science ◽  
1995 ◽  
Vol 268 (5216) ◽  
pp. 1483-1487 ◽  
Author(s):  
W. Carrington ◽  
R. Lynch ◽  
E. Moore ◽  
G Isenberg ◽  
K. Fogarty ◽  
...  
Keyword(s):  
Light Exposure ◽  
Three Dimensional ◽  
Three Dimensional Images ◽  
In Cells
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