Bird's-eye view of the undercoat structure of the upper cell membrane
Glass coverslips precationized by a treatment with Alcian blue were placed on top of the cells cultured in 35-mm plastic dishes and were allowed to settle and attach to the upper cell membrane at 4°C for 15 min. The buffer containing 1% PFA and 0.25% glutaraldehyde was then added to the space between the coverslip and the plastic bottom of the culture dish. As the coverslip was floated apart, the cells were ruptured and the upper cell membrane was retained, still adhering to the overlaying Alcian blue–coated coverslip. The upper membrane was rapidly frozen by pressing its exposed cytoplasmic surface onto a pure copper block precooled by liquid helium. The frozen sample was deep etched, coated with platinum-carbon, and observed under an electron microscope. We have made extensive efforts to reproducibly prepare and observe large cell membrane fragments >10 μm in diameter.
is a typical electron micrograph providing a bird's-eye view of the cytoplasmic surface of a large area of the upper cell membrane of a cultured NRK cell. Many such EM images showing the cytoplasmic surfaces of large cell membrane fragments were obtained for NRK and FRSK cells, suggesting that the entire (upper) plasma membrane, except for the places where CCPs and caveolae exist, is coated with the filamentous netlike structure.
A bird's-eye view of the large cytoplasmic surface of the upper cell membrane (the membrane facing the buffer rather than the coverslip) of an NRK cell observed by rapid-freeze, deep-etch, freeze-replica EM. Bar, 2.5 μm.
(A and B), which was obtained for an NRK cell () and an FRSK cell (), shows the magnified images of the cytoplasmic surface of the plasma membrane, exhibiting extensive filamentous netlike structures, which are the MSK. The presence of clathrin-coated structures shows that this is indeed the cytoplasmic surface. The striped banding patterns with a 5.5-nm periodicity on individual filaments are characteristic of actin filaments and, thus, indicate that these are actin filaments (Heuser and Kirschner, 1980
; Heuser, 1983
; Katayama, 1998
; Schoenenberger et al., 1999
). Because almost all of these filaments contain this striped pattern, it is concluded that the MSK is predominantly composed of actin filaments. This was also confirmed by immunogold staining (see and related text).
Figure 2. Magnified MSK images of an NRK and FRSK cell on the cytoplasmic surface of the upper membrane. (A) NRK cell; (B) FRSK cell. Clathrin- coated structures (A and B) and a caveola (A) show the cytoplasmic surface. The striped banding patterns with the 5.5-nm (more ...)
Figure 3. Immunogold labeling also indicates that the major component of MSK is actin filaments (NRK cell). Actin filaments were indirectly immunolabeled with 5-nm colloidal gold particles coated with secondary antibodies. Each gold particle can be identified as (more ...)
The electron micrograph shown in the inset in indicates the spatial resolution: because each band in the striped pattern with a 5.5-nm periodicity is visibly separated, the effective resolution is thought to be ~2 nm (both the thickness of the platinum coating and the platinum granule size are ≤2 nm; Heuser and Kirschner, 1980
; Heuser, 1983
). The MSK structure observed here on the upper cell membrane is similar to that on the bottom cell membrane (the part of the cell membrane facing the coverslip) observed previously (Heuser and Anderson, 1989
These results suggest that the cytoplasmic surface of a portion of the upper cell membrane >10 μm in diameter was visualized with a spatial resolution of ~2 nm, which is much smaller than the width of a single actin filament or the repeat distance of the stripes. As shown in and (A and B), the MSK is likely to cover the entire cytoplasmic surface of the upper cell membrane except for the places where CCPs and caveolae are present in both NRK and FRSK cells. Such a notion of the complete coverage of the cytoplasmic surface of the plasma membrane by actin filaments might have existed for >30 yr in a part of the EM community (Byers and Porter, 1977
; for review see Sheetz et al., 2006
), but the data specifically indicating that the actin filaments of the MSK may cover the entire cell membrane has not been presented in the literature, as done here, nor shared in the cell biology community. The EM observations shown in this study are consistent with the MSK fence and anchored transmembrane protein picket models, in which the entire plasma membrane except for the specific membrane domains is partitioned into many small compartments with regard to lateral diffusion of the molecules incorporated in the plasma membrane.
The MSK predominantly consists of actin filaments: immunogold labeling of actin and actin-binding proteins
To further examine whether the MSK is predominantly composed of actin filaments (and partly because the 5.5-nm periodicity of the banding pattern is somewhat difficult to discern in some of the filaments), we examined it using an indirect immunolabeling method with 5-nm-diameter colloidal gold particles (see Materials and methods; ). On the filaments with striped patterns, the enlarged images () show the presence of many colloidal gold actin probes, which appear as distinct white spots surrounded by somewhat blurred white halos, reflecting the platinum shadow over the antibody molecules attached to the gold particle. The electron micrographs in revealed that almost all of the colloidal gold probes are bound to the filaments located on the cytoplasmic surface (yellow dots). Therefore, it is concluded that actin is the main constituent molecule of the MSK.
Electron tomography of the undercoat structure on the cytoplasmic surface of the plasma membrane
The 3D structure of the undercoat within 100–134 nm from the cytoplasmic surface of the plasma membrane, which includes CCPs, caveolae, and the actin-based MSK, was reconstructed using electron tomography for the platinum-replicated samples. Based on the 97–141 tilt images acquired in the range of ±48–70° every 1° step for a single EM view field, 100–121 sliced images of every 0.85–1.34 nm perpendicular to the z axis (parallel to the image obtained at 0° of the tilt angle) were calculated by a computer (long wavelength [≥~500 nm] undulations of the cell membrane were corrected by the 3D reconstruction software IMOD). The 3D image was reconstructed based on these serial thin slices. Representative images obtained for an EM view field are shown in Video 1 (131 tilt images; an anaglyph produced from images taken at ±12° is shown in ) and Video 2 (showing the 3D image by rotating the 3D reconstructed undercoat structure; a typical view is shown in ; videos are available at http://www.jcb.org/cgi/content/full/jcb.200606007/DC1
). Throughout the present research, this protocol was used to obtain 3D images.
Figure 4. Stereo electron micrographs and 3D reconstructed images of the undercoat structure, CCPs, and caveolae in NRK cells. (A) An EM anaglyph of the undercoat structure generated at ±12° of the tilt angle among the 131 tilt images (acquired (more ...)
In these images, because of their 3D representation, it is especially clear that the MSK, which is mostly composed of actin filaments, generally spreads along the membrane, covering almost the entire cytoplasmic surface of the upper membrane except for the places with caveolae and CCPs. In addition, CCPs and caveolae are very closely associated with the actin filaments in the MSK, as seen in these images and also in (A and B) and 3. These results are consistent with Rothberg et al. (1992)
, Fujimoto et al. (2000)
, and Parton (2003)
, but in NRK cells studied here, many more actin filaments were found to be associated with each CCP or caveola. Furthermore, 92 and 93% of CCPs and caveolae (n
= 200) were bound by the actin filaments. These results are consistent with the requirement of filamentous actin for CCP internalization (Qualmann et al., 2000
; Merrifield et al., 2002
Many short, thin filaments protrude toward the cytoplasm, mostly perpendicularly, from the membrane surface (they were short probably because they were broken at the time of the membrane rip off; , arrows). Note that these perpendicular filaments are almost always connected to the MSK network lying on the cytoplasmic surface (see the tips of the arrows; ). Thus, the part of the MSK that is located on the cytoplasmic surface is connected three dimensionally to the cytoskeleton. Together, they will provide mechanical support for the membrane and the force for deforming the membrane.
3D reconstruction of the MSK structure
The part of the actin-based MSK that is in contact with the cytoplasmic surface of the cell membrane has been proposed to partition the cell membrane into 30–230-nm compartments by the fence and picket effect (Edidin et al., 1991
; Kusumi and Sako, 1996
; Kusumi et al., 2005
). If these fence and picket models are correct, the distribution of the mesh size of the MSK on the cytoplasmic surface of the plasma membrane would be practically the same as that of the compartment size determined by diffusion measurements of membrane molecules. To carry out this examination, the 3D reconstruction of MSK by electron tomography provides a unique opportunity because the obtained images provide quantitative data on how far the individual filaments are located from the membrane surface.
In , a typical MSK structure quantitatively analyzed in this study is shown in an anaglyph, and its 8.5-nm–thick sections (created by superimposing 10 0.85-nm sections) of the MSK of an NRK cell, starting from the cytoplasmic side toward the membrane, are shown (; a series of the original tilt images is shown in Video 3, and a series of sliced images of every 0.85 nm is shown in Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200606007/DC1
). The actin-based MSK is visible on image sections 81–110. Individual actin filaments, forming a network as well as bundles, can be identified. Given the high density of the actin filament meshwork, which is much smaller than the optical resolution, conventional fluorescence microscopy will be unable to visualize the MSK meshwork and can visualize only the bundles of actin filaments.
Figure 5. A series of sliced images of the actin MSK on the plasma membrane cytoplasmic surface of an NRK cell. (A) A typical actin MSK structure used for analysis of the mesh size on the cytoplasmic surface of the plasma membrane using computed tomography. An (more ...)
Interface structure of MSK on the cytoplasmic surface of the plasma membrane
The filaments of the MSK that are directly associated with the cytoplasmic surface of the plasma membrane and may be involved in partitioning the plasma membrane were systematically determined. Out of the stack of 121 image slices taken every 0.85 nm from the cytoplasmic surface (~100-nm thick altogether), 16 consecutive image slices from the membrane surface (~13.6-nm thick altogether) were used for this analysis ().
Figure 6. The method for determining the MSK mesh on the cytoplasmic surface of the plasma membrane, which possibly delimits the compartments of the plasma membrane, using the 3D reconstructed images of the MSK (an NRK cell). (A and B) The images on the far left (more ...)
In (four images on the right) and (the second to fourth images), the boxed areas in the left-most images were expanded, and the sections of every 1.7 nm (superposition of two 0.85-nm–thick slices; 330 × 330 nm) are displayed between 0 and 11.9 nm. Using these sections, the filaments that are closely associated with the cytoplasmic surface of the cell membrane were determined. Because the thickness (width in the image) of the actin filament after platinum shadowing is between 9 and 11 nm (consistent with Heuser, 1983
) and the thickness of the platinum replica is ≤2 nm (consistent with Heuser, 1983
and Moritz et al., 2000
), the height of the actin filament that is associated with the membrane will be 7–9 nm (because the height is given by the actin thickness and one replica thickness, whereas the width in the image is determined by the actin thickness plus two replica thicknesses), with 8 nm being a reasonable estimate. In the series of electron tomography sections shown in (A and B), the existence of three major classes of filaments with regard to the distance from the membrane surface can be discerned (for details of this analysis, see Materials and methods).
The first class of filaments is distinct in computer-reconstructed sections close to the cytoplasmic surface of the plasma membrane, even in the first ~0–1.7-nm section (because the contrast is reversed in these micrographs, they look more lucent or white), but fade out of the reconstructions 8–10 nm away from the membrane surface (for details, see Materials and methods). These filaments are drawn in green in . We interpret that these filaments are in true contact with the plasma membrane (the gap between the filament and the inner membrane surface is <0.85 nm) because they can be seen clearly even in the first 0.85-nm section. These filaments are likely to be the significant ones for generating membrane corrals.
The second class of filaments also looks clear in sections very close to the membrane surface but does not fade out until ~14 nm away from it. We interpret that these may be the actin filaments that had platinum coatings all around their surfaces because they stood off the surface somewhat, which slightly exaggerated their thickness and made them appear as though they were in contact with the plasma membrane when in fact they probably were not quite in direct contact. We did not consider these filaments to be close enough to generate membrane corrals.
The third class of filaments is not apparent in sections closest to the plasma membrane but becomes clear some distance away from it (>2–4 nm) and also does not fade out until ~14 nm. We interpret these as being filaments that definitely do not contact the plasma membrane directly and, thus, should not contribute to forming corrals. The second and third classes of filaments are drawn in red in .
Therefore, we considered that only the first class of filaments (those drawn in green in ) forms the MSK fences and pickets, and the area surrounded by these filaments is colored green in the 0–6.8-nm section shown in . Note that areas are excluded from this analysis in which bundles of actin filaments are present (e.g., the structure crossing diagonally from the bottom left to the top right in ), actin filaments are too crowded to be individually discerned, an actin filament is terminated in the middle of a domain (domains that contain a loose end of an actin filament), or CCPs, caveolae, and the smooth surface membrane invaginations are present (the white regions in ).
Figure 7. The MSK meshwork directly on the cytoplasmic surface of the plasma membrane. The central parts of the figures in the top row (bar, 300 nm) are magnified by a factor of three and are shown in the bottom row (bar, 100 nm). (A) Typical stereo views of the (more ...)
Comparison of the MSK mesh size on the plasma membrane determined by electron tomography with the compartment size for the diffusion of membrane molecules
Similar determination of the MSK meshwork was performed for FRSK cells. Representative meshes of the MSK are shown in (for an FRSK cell, colored to aid in visualization). We performed such analyses for 10 representative stacks of image sections (1,290 × 1,290-nm plane) each for NRK cells and FRSK cells (eight different cell membrane sheets for each cell type) and identified 76 and 1,300 areas bounded by the MSK meshwork, respectively (excluding the regions occupied by stress fibers and other membrane undercoat structures such as CCPs and caveolae; about the same total membrane areas were examined for each cell type, and, thus, the difference in the number of identified areas represents the difference in the area size between these two cell lines). The 2D area size for each domain was measured by Amira software. The distributions of the square root of the area size (the side length, assuming a square shape for the area) for NRK (, pink open bars) and FRSK (blue open bars) cells are shown in . The median values of the area and its square root are 3.9 × 104 nm2 and 200 nm, respectively, for NRK cells and 2.7 × 103 nm2 and 52 nm, respectively, for FRSK cells.
Figure 8. Comparison of the distributions of the MSK mesh size on the cytoplasmic surface of the plasma membrane estimated by electron tomography with that of the compartment size determined from the phospholipid diffusion data for NRK and FRSK cells. Electron (more ...)
The size distributions of the compartments for the diffusion of membrane molecules were obtained for an unsaturated phospholipid, l
-α-dioleoylphosphatidylethanolamine, by Fujiwara et al. (2002)
and Murase et al. (2004)
for NRK and FRSK cells, respectively. The distributions of the side lengths for NRK (, pink closed bars) and FRSK (blue closed bars) cells are shown in the histograms in . The median values of the compartment area and the side length are 4.3 × 104
and 230 nm, respectively, for NRK cells and 2.1 × 103
and 41 nm, respectively, for FRSK cells (Murase et al., 2004
These results indicate that in the same cell line (for both the NRK and FRSK cases), the MSK mesh size determined by electron tomography and the diffusion compartment size determined by the high speed single-particle tracking of a phospholipid are similar to each other. However, between these two cell lines, both the MSK mesh and the diffusion compartment sizes differ greatly. The similarities between the MSK mesh sizes and the diffusion compartment sizes in cell lines that exhibit quite different distributions strongly support the MSK fence and picket models.