Cytoskeletal architecture of neuromuscular junction: Localization of vinculin

Cytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick-freeze deep-etched method and immunoelectron microscopy of ultrathin frozen sections. In a quick-freeze deep-etched replica of fresh, unfixed muscles, 8.9 ± 1.5-nm particles were present on the true postsynaptic membrane surface. Underneath this receptor-rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized—12 nm, 9 nm, and 7 nm in diameter. The 12-nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty-six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.     

I5M: 3D widefield light microscopy with better than 100 nm axial resolution

Sevenfold improved axial resolution has been achieved in three-dimensional widefield fluorescence microscopy, using a novel interferometric technique in which the sample is observed and/or illuminated from both sides simultaneously using two opposing objective lenses. Separate interference effects in the excitation light and the emitted light give access to higher resolution axial information about the sample than can be reached by conventional widefield or confocal microscopes. Here we report the experimental verification of this resolution performance on complex biological samples.     

Quick-freeze,deep-etch studies of endothelial components,with special reference to cytoskeletons and vesicle structures

A three-dimensional study of the ultrastructure of endothelial cells is helpful in understanding important endothelial functions such as vascular transport and cell permeability. For this purpose, in addition to serial sectioning electron microscopy and high-voltage electron microscopy, the quick-freeze, deep-etching technique also enables us to analyze structures at the molecular level by its high resolution and is useful for three-dimensional morphological studies. Some modifications on the conventional deep-etching method were made in this study to reduce the undesirable aggregation of proteins and salts during etching. Using this technique, we examined the rat aortic endothelium, particularly the membrane structures and cytoskeletons. The luminal surface of the endothelium was covered with a fine filamentous coat, which was anchored to the plasma membrane. In the cytoplasm, actin filaments were prominent and were oriented randomly or in a parallel fashion near the plasma membrane. Of the vesicles seen in the endothelium, some had basket coats of clathrin, and others had striped coats on the cytoplasmic membrane surface. These surface structures of the vesicles suggest the transport mechanism of the vesicles in association with the fine filaments attached to the vesicles.     

Organization of microtubules in cochlear hair cells

The organization of microtubules in hair cells of the guinea-pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin-associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells. In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo-vesicular track between the apex and base. In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells. The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.     

Polymer physics of the cytoskeleton

The cytoskeleton is generally visualized by light or electron microscopy as a meshwork of protein filaments that spans the space between the nuclear envelope and the plasma membrane. In most cell types, this meshwork is formed by a three dimensional composite network of actin filaments, microtubules (MT), and intermediate filaments (IF) together with the host of proteins that bind to the sides or ends of these linear polymers. Cytoskeletal binding proteins regulate filament length, crosslink filaments to each other, and apply forces to the filaments. One approach to modeling the mechanical properties of the cytoskeleton and of cell in general is to consider the elements of the cytoskeleton as polymers, using experimental methods and theoretical models developed for traditional polymers but modified for the much larger, stiffer, and fragile biopolymers comprising the cytoskeleton. The presence of motor proteins that move actin filaments and microtubules also creates a new class of active materials that are out of thermodynamic equilibrium, and unconstrained by limitations of the fluctuation–dissipation theorem. These active materials create rich opportunities for experimental design and theoretical developments. The degree to which the mechanics of live cells can usefully be modeled as highly complex polymer networks is by no means certain, and this article will discuss recent progress in quantitatively measuring cytoskeletal polymer systems and relating them to the properties of the cell.     

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.