Calcium control of macrophage cytoplasmic gelation: evidence for the involvement of the 70,000 Mr actin-bundling protein

1987 ◽  
Vol 88 (1) ◽  
pp. 81-94
Author(s):  
M. Pacaud ◽  
M.C. Harricane
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Supernatant Fraction ◽  
Actin Networks ◽  
Dependent Inhibition ◽  
Section Electron ◽  
Filament Bundles ◽  
Gel Network

Under appropriate conditions macrophage cytosolic extracts can form a three-dimensional gel network of cross-linked actin filaments. These cytoplasmic gels are mainly composed of actin, filamin, alpha-actinin, and two new proteins of about 70,000 and 55,000 Mr (70 and 55 K). The behaviour of 70 K protein was found to be remarkably affected by Ca2+. Ca2+ treatment of isolated cytoplasmic gels led to the selective solubilization of the 70 K protein along with a 17 K polypeptide. Half-maximal recovery in the supernatant fraction was obtained from about 0.15 microM free Ca2+. The cytoplasmic gel constituents solubilized in high ionic strength buffer were able to re-assemble into an insoluble actin network when returned to near physiological ionic conditions. However, the inclusion of micromolar Ca2+ prevented the re-association of 70 K protein with actin in these complexes. As compared to the 70 K protein, alpha-actinin was fully resistant to any variations in Ca2+ concentrations. On the other hand, purified 70 K protein displayed the property of assembling actin filaments into bundles at low Ca2+ concentrations (less than 0.15 microM). However, the bundling activity decreased progressively at higher Ca2+, as detected by co-sedimentation and electron microscopy of the 70 K protein-actin mixtures. Half-maximal inhibition was observed at about 0.3 microM free Ca2+. Re-assembly of actin filaments into bundles occurred after chelation of Ca2+ by EGTA, indicating that the inhibitory effect of Ca2+ was reversible. Severing of actin filaments by 70 K protein was not observed in any of the solution conditions used. The Ca2+-dependent inhibition of the ability of 70 K protein to interact with actin networks resulted in a marked distortion of the overall organization of actin filaments, as revealed by thin-section electron microscopy of cytoplasmic gels formed in the presence and absence of Ca2+. Large zones of oriented bundles of filaments were replaced by a looser mesh. When the actin gel constituents were re-assembled in the presence of Ca2+ and exogenous gelsolin, it was also observed that the filament bundles (essentially generated by alpha-actinin) collapsed into dense aggregates. Furthermore, gelsolin did not significantly affect the ability of actin to re-combine with other proteins. The data presented here and previously led us to suspect that the Ca2+ control of the functional state of 70 K protein might be one of the prime factors in the triggering of rapid assembly and disassembly of microfilaments within macrophages.

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
Author(s):  
T.D. Pollard ◽  
P. Maupin
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Contractile Proteins ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Cytoplasmic Matrix ◽  
Computer Image Processing ◽  
Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

scholarly journals Centriolar remodeling underlies basal body maturation during ciliogenesis in Caenorhabditis elegans

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Inna V Nechipurenko ◽  
Cristina Berciu ◽  
Piali Sengupta ◽  
Daniela Nicastro
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Sensory Neurons ◽  
Basal Body ◽  
Three Dimensional ◽  
C Elegans ◽  
Mother Centriole ◽  
Section Electron ◽  
Serial Section Electron Microscopy ◽  
Section Electron Microscopy

The primary cilium is nucleated by the mother centriole-derived basal body (BB) via as yet poorly characterized mechanisms. BBs have been reported to degenerate following ciliogenesis in the C. elegans embryo, although neither BB architecture nor early ciliogenesis steps have been described in this organism. In a previous study (Doroquez et al., 2014), we described the three-dimensional morphologies of sensory neuron cilia in adult C. elegans hermaphrodites at high resolution. Here, we use serial section electron microscopy and tomography of staged C. elegans embryos to demonstrate that BBs remodel to support ciliogenesis in a subset of sensory neurons. We show that centriolar singlet microtubules are converted into BB doublets which subsequently grow asynchronously to template the ciliary axoneme, visualize degeneration of the centriole core, and define the developmental stage at which the transition zone is established. Our work provides a framework for future investigations into the mechanisms underlying BB remodeling.

scholarly journals Isolation and characterization of two forms of a cytoskeleton.

1979 ◽  
Vol 83 (1) ◽  
pp. 109-115 ◽  
Author(s):  
K T Edds
Keyword(s):  
Sea Urchin ◽  
Electron Micrographs ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Strongylocentrotus Droebachiensis ◽  
Major Protein ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Filament Bundles

Isolated petaloid coelomocytes from the sea urchin Strongylocentrotus droebachiensis transform to a filopodial morphology in hypotonic media. Electron micrographs of negatively stained Triton-insoluble cytoskeletons show that the petaloid form consists of a loose net of microfilaments while the filopodial form consists of paracrystalline bundles of microfilaments. Actin is the major protein of both forms of the cytoskeleton. Additional polypeptides have molecular weights of approximately 220,000, 64,000, 57,000, and 27,000 daltons. Relative to actin the filopodial cytoskeletons have an average of 2.5 times as much 57k polypeptide as the petaloid cytoskeletons. Treatment with 0.25 M NaCl dissociates the filament bundles into individual actin filaments free of the actin-associated polypeptides. Thus, one or more of these actin-associated polypeptides may be responsible for crosslinking the actin filaments into bundles and maintaining the three-dimensional nature of the cytoskeletons.

Reducing Manual Operation Time to Obtain a Segmentation Learning Model for Volume Electron Microscopy Using Stepwise Deep Learning With Manual Correction

Microscopy ◽  
2021 ◽  
Author(s):  
Kohki Konishi ◽  
Takao Nonaka ◽  
Shunsuke Takei ◽  
Keisuke Ohta ◽  
Hideo Nishioka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Deep Learning ◽  
Three Dimensional ◽  
Operation Time ◽  
Training Data ◽  
Manual Operation ◽  
Annotation Method ◽  
Section Electron ◽  
Model Training ◽  
Manual Correction

Abstract Three-dimensional (3D) observation of a biological sample using serial-section electron microscopy is widely used. However, organelle segmentation requires a significant amount of manual time. Therefore, several studies have been conducted to improve their efficiency. One such promising method is 3D deep learning (DL), which is highly accurate. However, the creation of training data for 3D DL still requires manual time and effort. In this study, we developed a highly efficient integrated image segmentation tool that includes stepwise DL with manual correction. The tool has four functions: efficient tracers for annotation, model training/inference for organelle segmentation using a lightweight convolutional neural network, efficient proofreading, and model refinement. We applied this tool to increase the training data step by step (stepwise annotation method) to segment the mitochondria in the cells of the cerebral cortex. We found that the stepwise annotation method reduced the manual operation time by one-third compared with that of the fully manual method, where all the training data were created manually. Moreover, we demonstrated that the F1 score, the metric of segmentation accuracy, was 0.9 by training the 3D DL model with these training data. The stepwise annotation method using this tool and the 3D DL model improved the segmentation efficiency for various organelles.

scholarly journals Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1

2002 ◽  
Vol 365 (3) ◽  
pp. 707-719 ◽  
Author(s):  
Iram AFZAL ◽  
Philip CUNNINGHAM ◽  
Richard. J. NAFTALIN
Keyword(s):  
Binding Site ◽  
Oestrogen Receptor ◽  
Glucose Transporter ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Atp Binding ◽  
Binding Motif ◽  
Transmembrane Helices ◽  
Selective Oestrogen Receptor Modulators ◽  
Dependent Inhibition

17β-Oestradiol (ED when subscript to K) and the phytoestrogen isoflavone genistein (GEN) inhibit glucose transport in human erythrocytes and erythrocyte ghosts. The selective oestrogen receptor modulators or anti-oestrogens, faslodex (ICI 182780) (FAS) and tamoxifen (TAM), competitively antagonize oestradiol inhibition of glucose exit from erythrocytes (Ki(ED/FAS) = 2.84±0.16μM and Ki(ED/TAM) = 100±2nM). Faslodex has no significant inhibitory effect on glucose exit, but tamoxifen alone inhibits glucose exit (Ki(TAM) = 300±100nM). In ghosts, ATP (1–4mM) competitively antagonizes oestradiol, genistein and cytochalasin B (CB)-dependent inhibitions of glucose exit, (Ki(ATP/ED) = 2.5±0.23mM, Ki(ATP/GEN) = 0.99±0.17mM and Ki(ATP/CB) = 0.76±0.08mM). Tamoxifen and faslodex reverse oestradiol-dependent inhibition of glucose exit with ATP>1mM (Ki(ED/TAM) = 130±5nM and Ki(ED/FAS) = 2.7±0.9μM). The cytoplasmic surface of the glucose transporter (GLUT)1 contains four sequences with close homologies to sequences in the ligand-binding domain of human oestrogen receptor β (hesr-2). One homology is adjacent to the Walker ATP-binding motif II (GLUT1, residues 225–229) in the large cytoplasmic segment linking transmembrane helices 6 and 7; another GLUT (residues 421–423) contains the Walker ATP-binding motif III. Mapping of these regions on to a three-dimensional template of GLUT indicates that a possible oestrogen-binding site lies between His337, Arg349 and Glu249 at the cytoplasmic entrance to the hydrophilic pore spanning GLUT, which have a similar topology to His475, Glu305 and Arg346 in hesr-2 that anchor the head and tail hydroxy groups of oestradiol and genistein, and thus are suitably placed to provide an ATP-sensitive oestrogen binding site that could modulate glucose export.

scholarly journals A Rapid Self-Assembly Peptide Hydrogel for Recruitment and Activation of Immune Cells

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 419
Author(s):  
Ruyue Luo ◽  
Yuan Wan ◽  
Xinyi Luo ◽  
Guicen Liu ◽  
Zhaoxu Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dendritic Cells ◽  
Self Assembly ◽  
Immune Cell ◽  
Three Dimensional ◽  
Hydrophobic Surface ◽  
Inhibitory Effect ◽  
Blue Staining ◽  
Peptide Hydrogel

Self-assembly peptide nanotechnology has attracted much attention due to its regular and orderly structure and diverse functions. Most of the existing self-assembly peptides can form aggregates with specific structures only under specific conditions and their assembly time is relatively long. They have good biocompatibility but no immunogenicity. To optimize it, a self-assembly peptide named DRF3 was designed. It contains a hydrophilic and hydrophobic surface, using two N-terminal arginines, leucine, and two c-terminal aspartate and glutamic acid. Meanwhile, the c-terminal of the peptide was amidated, so that peptide segments were interconnected to increase diversity. Its characterization, biocompatibility, controlled release effect on antigen, immune cell recruitment ability, and antitumor properties were examined here. Congo red/aniline blue staining revealed that peptide hydrogel DRF3 could be immediately gelled in PBS. The stable β-sheet secondary structure of DRF3 was confirmed by circular dichroism spectrum and IR spectra. The observation results of cryo-scanning electron microscopy, transmission electron microscopy, and atomic force microscopy demonstrated that DRF3 formed nanotubule-like and vesicular structures in PBS, and these structures interlaced with each other to form ordered three-dimensional nanofiber structures. Meanwhile, DRF3 showed excellent biocompatibility, could sustainably and slowly release antigens, recruit dendritic cells and promote the maturation of dendritic cells (DCs) in vitro. In addition, DRF3 has a strong inhibitory effect on clear renal cell carcinoma (786-0). These results provide a reliable basis for the application of peptide hydrogels in biomedical and preclinical trials.

scholarly journals Three-dimensional arrangement of F-actin in the contractile ring of fission yeast

2007 ◽  
Vol 178 (5) ◽  
pp. 765-771 ◽  
Author(s):  
Tomoko Kamasaki ◽  
Masako Osumi ◽  
Issei Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Single Site ◽  
Yeast Cells ◽  
Contractile Ring ◽  
Division Plane ◽  
Early Stages ◽  
Myosin S1

The contractile ring, which is required for cytokinesis in animal and yeast cells, consists mainly of actin filaments. Here, we investigate the directionality of the filaments in fission yeast using myosin S1 decoration and electron microscopy. The contractile ring is composed of around 1,000 to 2,000 filaments each around 0.6 μm in length. During the early stages of cytokinesis, the ring consists of two semicircular populations of parallel filaments of opposite directionality. At later stages, before contraction, the ring filaments show mixed directionality. We consider that the ring is initially assembled from a single site in the division plane and that filaments subsequently rearrange before contraction initiates.

scholarly journals The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy.

1986 ◽  
Vol 102 (5) ◽  
pp. 1654-1665 ◽  
Author(s):  
E G Fey ◽  
G Krochmalnic ◽  
S Penman
Keyword(s):  
Electron Microscopy ◽  
Nuclear Matrix ◽  
Intact Cell ◽  
Three Dimensional ◽  
Soluble Proteins ◽  
Sequential Fractionation ◽  
Nonhistone Proteins ◽  
Nuclear Rna ◽  
Section Electron ◽  
Section Electron Microscopy

The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged. Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E. G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles.(ABSTRACT TRUNCATED AT 400 WORDS)

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