scholarly journals Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

2006 ◽  
Vol 174 (6) ◽  
pp. 851-862 ◽  
Author(s):  
Nobuhiro Morone ◽  
Takahiro Fujiwara ◽  
Kotono Murase ◽  
Rinshi S. Kasai ◽  
Hiroshi Ike ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
High Speed ◽  
Plasma Membranes ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Single Particle Tracking ◽  
Coated Pits ◽  
Cytoplasmic Surface

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

scholarly journals Identification of a membrane skeleton in platelets.

1988 ◽  
Vol 106 (5) ◽  
pp. 1525-1538 ◽  
Author(s):  
J E Fox ◽  
J K Boyles ◽  
M C Berndt ◽  
P K Steffen ◽  
L K Anderson
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Protein ◽  
Amorphous Material ◽  
Three Dimensional ◽  
Amorphous Layer ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Filamentous Form ◽  
Actin Binding Protein

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

scholarly journals Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

2016 ◽  
Vol 27 (7) ◽  
pp. 1101-1119 ◽  
Author(s):  
Takahiro K. Fujiwara ◽  
Kokoro Iwasawa ◽  
Ziya Kalay ◽  
Taka A. Tsunoyama ◽  
Yusuke Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Diffusion ◽  
Electron Tomography ◽  
Transmembrane Protein ◽  
Mesh Size ◽  
Domain Size ◽  
Membrane Skeleton ◽  
Reaction Rates ◽  
Single Particle Tracking ◽  
Major Mechanism

The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed “hop diffusion”) for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

scholarly journals Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

1982 ◽  
Vol 94 (3) ◽  
pp. 613-623 ◽  
Author(s):  
J Aggeler ◽  
Z Werb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
High Resolution Electron Microscopy ◽  
Peritoneal Macrophages ◽  
Coated Pits ◽  
Thin Sections ◽  
Particle Uptake ◽  
Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

scholarly journals Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus.

1979 ◽  
Vol 83 (2) ◽  
pp. 338-347 ◽  
Author(s):  
M Büechi ◽  
T Bächi
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Sendai Virus ◽  
Light And Electron Microscopy ◽  
Distal Portion ◽  
Double Labeling ◽  
Virus Particles ◽  
Viral Antigens ◽  
Cytoplasmic Surface

A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double-labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.

scholarly journals High-Density Single Particle Tracking on the Plasma Membrane using High-Speed Hyperspectral Imaging

2012 ◽  
Vol 102 (3) ◽  
pp. 581a
Author(s):  
Patrick J. Cutler ◽  
Michael D. Malik ◽  
Sheng Liu ◽  
Jason M. Byars ◽  
Diane S. Lidke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hyperspectral Imaging ◽  
Particle Tracking ◽  
Single Particle ◽  
High Speed ◽  
Single Particle Tracking ◽  
High Density

Automation of Tilt Series Acquisition in Transmission Electron Microscopy for Applications in Biology and Materials Science

2001 ◽  
Vol 7 (S2) ◽  
pp. 88-89
Author(s):  
Ingo Daberkow ◽  
Bernhard Feja ◽  
Peter Sparlinek ◽  
Hans R. Tietz
Keyword(s):  
3D Reconstruction ◽  
Data Acquisition ◽  
San Francisco ◽  
High Speed ◽  
Materials Science ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Max Planck ◽  
Automatic Data ◽  
Tilt Series

During the last decade, computation of a three-dimensional image from a tilt series (3D reconstruction) has become a well established method, of which a variety of implementations are available. The term “electron tomography” is now generally used for this type of data acquisition and 3D reconstruction. An overview over the techniques involved is given in.With the introduction of micro-processor-controlled TEMs and cooled slow-scan CCD cameras and with the progress in performance of high-speed computers, automation of complex imaging procedures became mainly a task of developing appropriate software, using the control facilities of the microscope. in this way, automated electron tomography was realized in 1990 at the Max- Planck-Institute for Biochemistry in Martinsried, and at about the same time at the University of California in San Francisco (UCSF). New techniques for automatic focusing and alignment, developed somewhat earlier , have been integrated in these automated tomography procedures. in the following we discuss the requirements of automatic data acquisition and the present implementation for several TEMs.

scholarly journals Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

2004 ◽  
Vol 24 (20) ◽  
pp. 9102-9123 ◽  
Author(s):  
Shaohui Huang ◽  
Larry Lifshitz ◽  
Varsha Patki-Kamath ◽  
Richard Tuft ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Ph Domain ◽  
Membrane Ruffling ◽  
Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

scholarly journals F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes.

1987 ◽  
Vol 105 (4) ◽  
pp. 1741-1751 ◽  
Author(s):  
L J Wuestehube ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Binding Site ◽  
Dictyostelium Discoideum ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Binding Activity ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Cytoplasmic Surface ◽  
Actin Binding Site

F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.

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