scholarly journals The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis

2017 ◽  
Vol 28 (5) ◽  
pp. 613-623 ◽  
Author(s):  
John H. Henson ◽  
Casey E. Ditzler ◽  
Aphnie Germain ◽  
Patrick M. Irwin ◽  
Eric T. Vogt ◽  
...  
Keyword(s):  
Animal Cell ◽  
Myosin Ii ◽  
Three Dimensional ◽  
String Model ◽  
Ultrastructural Organization ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Contractile Ring ◽  
Transmission Electron ◽  
Ring Constriction

Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical “purse-string” model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.

scholarly journals CYK-4 regulates Rac, but not Rho, during cytokinesis

2017 ◽  
Vol 28 (9) ◽  
pp. 1258-1270 ◽  
Author(s):  
Yelena Zhuravlev ◽  
Sophia M. Hirsch ◽  
Shawn N. Jordan ◽  
Julien Dumont ◽  
Mimi Shirasu-Hiza ◽  
...  
Keyword(s):  
Alternative Model ◽  
Single Cell Analysis ◽  
Myosin Ii ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Filamentous Actin ◽  
Contractile Ring ◽  
Division Plane ◽  
Ring Constriction

Cytokinesis is driven by constriction of an actomyosin contractile ring that is controlled by Rho-family small GTPases. Rho, activated by the guanine-nucleotide exchange factor ECT-2, is upstream of both myosin-II activation and diaphanous formin-mediated filamentous actin (f-actin) assembly, which drive ring constriction. The role for Rac and its regulators is more controversial, but, based on the finding that Rac inactivation can rescue cytokinesis failure when the GTPase-activating protein (GAP) CYK-4 is disrupted, Rac activity was proposed to be inhibitory to contractile ring constriction and thus specifically inactivated by CYK-4 at the division plane. An alternative model proposes that Rac inactivation generally rescues cytokinesis failure by reducing cortical tension, thus making it easier for the cell to divide when ring constriction is compromised. In this alternative model, CYK-4 was instead proposed to activate Rho by binding ECT-2. Using a combination of time-lapse in vivo single-cell analysis and Caenorhabditis elegans genetics, our evidence does not support this alternative model. First, we found that Rac disruption does not generally rescue cytokinesis failure: inhibition of Rac specifically rescues cytokinesis failure due to disruption of CYK-4 or ECT-2 but does not rescue cytokinesis failure due to disruption of two other contractile ring components, the Rho effectors diaphanous formin and myosin-II. Second, if CYK-4 regulates cytokinesis through Rho rather than Rac, then CYK-4 inhibition should decrease levels of downstream targets of Rho. Inconsistent with this, we found no change in the levels of f-actin or myosin-II at the division plane when CYK-4 GAP activity was reduced, suggesting that CYK-4 is not upstream of ECT-2/Rho activation. Instead, we found that the rescue of cytokinesis in CYK-4 mutants by Rac inactivation was Cdc42 dependent. Together our data suggest that CYK-4 GAP activity opposes Rac (and perhaps Cdc42) during cytokinesis.

scholarly journals Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

2021 ◽  
Author(s):  
Anna Loeschberger ◽  
Yauheni Novikau ◽  
Ralf Netz ◽  
Marie-Christin Spindler ◽  
Ricardo Benavente ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Brain Slices ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution ◽  
Coated Pits ◽  
Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

scholarly journals Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

2020 ◽  
Author(s):  
Jiji Chen ◽  
Hideki Sasaki ◽  
Hoyin Lai ◽  
Yijun Su ◽  
Jiamin Liu ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Network Performance ◽  
Resolution Enhancement ◽  
Three Dimensional ◽  
Ground Truth ◽  
Time Lapse ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Attention Networks

Abstract We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ~1.4-fold laterally and ~3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

scholarly journals A novel workflow for three-dimensional analysis of tumour cell migration

2020 ◽  
Vol 10 (2) ◽  
pp. 20190070 ◽  
Author(s):  
Sophie Ketchen ◽  
Arndt Rohwedder ◽  
Sabine Knipp ◽  
Filomena Esteves ◽  
Nina Struve ◽  
...  
Keyword(s):  
Dimensional Analysis ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Tumour Spheroid ◽  
3D Data ◽  
Biological Studies ◽  
Research Findings ◽  
Imagej Plugin

The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal–amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

scholarly journals High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism

Nanophotonics ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 143-148
Author(s):  
Adrien Descloux ◽  
Marcel Müller ◽  
Vytautas Navikas ◽  
Andreas Markwirth ◽  
Robin van den Eynde ◽  
...  
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
Optical Element ◽  
Spatial Light Modulators ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Image Stack ◽  
Light Modulators ◽  
3D Information

AbstractSuper-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live-cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with high-speed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS-7 cells.

scholarly journals Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse

2016 ◽  
Vol 215 (3) ◽  
pp. 383-399 ◽  
Author(s):  
Sricharan Murugesan ◽  
Jinsung Hong ◽  
Jason Yi ◽  
Dong Li ◽  
Jordan R. Beach ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Immunological Synapse ◽  
Three Dimensional ◽  
Cell Receptor ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Tcr Signaling ◽  
Immune Synapse ◽  
Distal Edge

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T–B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

scholarly journals CEP19 cooperates with FOP and CEP350 to drive early steps in the ciliogenesis programme

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170114 ◽  
Author(s):  
Bahareh A. Mojarad ◽  
Gagan D. Gupta ◽  
Monica Hasegan ◽  
Oumou Goudiam ◽  
Renata Basto ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Three Dimensional ◽  
Structured Illumination ◽  
Loss Of Function ◽  
Structured Illumination Microscopy ◽  
C Terminus ◽  
Interaction Partners ◽  
Cilia Formation ◽  
Mother Centriole ◽  
Section Electron

Primary cilia are microtubule-based sensory organelles necessary for efficient transduction of extracellular cues. To initiate cilia formation, ciliary vesicles (CVs) are transported to the vicinity of the centrosome where they dock to the distal end of the mother centriole and fuse to initiate cilium assembly. However, to this date, the early steps in cilia formation remain incompletely understood. Here, we demonstrate functional interplay between CEP19, FOP and CEP350 in ciliogenesis. Using three-dimensional structured-illumination microscopy (3D-SIM) imaging, we mapped the relative spatial distribution of these proteins at the distal end of the mother centriole and show that CEP350/FOP act upstream of CEP19 in their recruitment hierarchy. We demonstrate that CEP19 CRISPR KO cells are severely impaired in their ability to form cilia, analogous to the loss of function of CEP19 binding partners FOP and CEP350. Notably, in the absence of CEP19 microtubule anchoring at centromes is similar in manner to its interaction partners FOP and CEP350. Using GFP-tagged deletion constructs of CEP19, we show that the C-terminus of CEP19 is required for both its localization to centrioles and for its function in ciliogenesis. Critically, this region also mediates the interaction between CEP19 and FOP/CEP350. Interestingly, a morbid-obesity-associated R82* truncated mutant of CEP19 cannot ciliate nor interact with FOP and CEP350, indicative of a putative role for CEP19 in ciliopathies. Finally, analysis of CEP19 KO cells using thin-section electron microscopy revealed marked defects in the docking of CVs to the distal end of the mother centrioles. Together, these data demonstrate a role for the CEP19, FOP and CEP350 module in ciliogenesis and the possible effect of disrupting their functions in ciliopathies.

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