No subject

T-shaped junctions between the actin filaments; 12 of them are displayed 
in the gallery shown in Fig. 4. Among these examples, branching angles 
vary between 40° and 90°. Some branches show the arm linked to an actin 
filament out of plane, as illustrated in Fig. 4A (top). A special feature 
of some branches is visible in Fig. 4B: The side arms are sharply bent 
close to their site of attachment. This is presumably caused by an 
actin-binding protein that links one end of a filament to the side of 
another one. The putative linker protein sticks out of a filament at a 
wide angle. Cross-linking of actin filaments occurs often at a 90° angle 
(Fig. 4C).
Fig. 3. Visualization of actin network, membranes, and cytoplasmic 
macromolecular complexes. (A) A volume of 815 nm by 870 nm by 97 nm, 
corresponding to the area of Fig. 1A framed in black, was subjected to 
surface rendering. Colors were subjectively attributed to linear elements 
to mark the actin filaments (reddish); other macromolecular complexes, 
mostly ribosomes (green); and membranes (blue). (B) Stereo image of an 
actin layer taken from the upper left portion of (A) reveals a crowded 
network of branched and crosslinked filaments. [View Larger Version of 
this Image (85K GIF file)]

Fig. 4. Branches and crosslinks of actin filaments. These examples were 
chosen from the cortical regions of cells as in Fig. 3. The branching 
angles as displayed in 2D were extracted and maximized in 3D. (A) 
Compilation of branchings with different angles. (B) Examples 
characterized by a kink that changes the angle close to the branching 
site. (C) Cross-linkages between filaments in addition to branches. [View 
Larger Version of this Image (19K GIF file)]

Figure 5A shows a series of x-y slices from a tomogram of another cortical 
region with actin filaments predominantly arranged in parallel; for 
clarity, the sections were denoised. Although this tomogram is of inferior 
quality owing to the greater local thickness of the cell (~600 nm), the 
arrangement of actin filaments is clearly visible. Individual filaments 
can be traced over a length of 400 to 500 nm, which is twice the reported 
average length (36). Such parallel arrangements are typically generated 
and stabilized by bundling proteins forming serial bridges along the 
individual filaments. Indeed, at many locations such bridges are 
discernible in Fig. 5B; two of them are displayed in Fig. 5C.
Fig. 5. Bundles of actin filaments in the cell cortex. (A) Two denoised 
x-y slices of 10-nm thickness through the cortical region of a cell, 
sampled at 490 to 510 nm above the substrate. (B) A volume-rendered 
portion of the region depicted in (A) showing bridges between filaments 
and lateral connections of filaments to the membrane. (C) Representative 
bridges between actin filaments extracted from (B). [View Larger Version 
of this Image (83K GIF file)]

Because in cryo-ET the integrity of the cellular membranes is maintained, 
we were able to depict binding sites of actin filaments on the plasma 
membrane. Figure 6A shows a cortical actin-filament network locally 
connected to the plasma membrane, and in Fig. 6, B and C, surface-rendered 
actin filaments are presented that appear to be kinked close to the 
membrane surface. These images indicate that actin filaments are capable 
of end binding to the plasma membrane through a connecting element that 
forms the kink. This element might be an adaptor protein or the 
cytoplasmic domain of an actin-binding transmembrane protein.
Fig. 6. End binding of actin filaments to the plasma membrane. (A) 
Cortical region of 500 nm by 300 nm by 20 nm showing membrane-associated 
actin filaments presented as in Fig. 3. The arrow points to the site of 
actin-membrane interaction shown in (B) at higher magnification. (B and C) 
High-magnification image of cortical regions, depicting kinklike 
structures close to filament-membrane interaction sites. Kink angles were 
not maximized, and the membrane in (C) was not perpendicular to the actin 
filament. (B) and (C) were taken from different cortical regions. [View 
Larger Version of this Image (36K GIF file)]

In summary, cryo-ET of peripheral regions in Dictyostelium L cells 
depicted two different types of actin-filament arrays: almost isotropic 
networks (Fig. 3) and parallel arrangements of actin filaments that are 
tied together by bundling proteins (Fig. 5). Within the actin networks, we 
have found a broad distribution of branching angles and different types of 
cross-linkages (Fig. 4). These observations are consistent with the 
presence in eukaryotic cells of a large variety of actin-cross-linking and 
actin-branching proteins that connect actin filaments to each other at 
angles varying from 0° (parallel bundling) to 180° (antiparallel 
bundling). Groups of conserved branching and cross-linking proteins 
include the Arp2/3 complex, which is characterized by a 70° angle of 
branching (37, 38), and the filamin family (with filamin A as a 
prototype), which has a preference for orthogonal crosslinkage of actin 
filaments (23, 34). An advantage of cryo-ET is that branching angles can 
be determined precisely from the 3D maps. The quantitative analysis of 
network geometries will benefit from automated procedures, allowing the 
evaluation of large tomographic data sets.

Prospects of cryo-ET applied to cell biology. Cryo-ET has the potential to 
bridge the gap between cellular and molecular structural studies (39). By 
combining the power of 3D imaging with the close-to-life preservation 
achieved by vitrification, we have visualized macromolecular complexes and 
supramolecular structures inside intact eukaryotic cells. The tomograms of 
Dictyostelium cells contain an imposing amount of information, even at the 
present resolution of 5 to 6 nm. Essentially, they are 3D images of the 
cell's entire proteome, and they represent the network of macromolecular 
interactions that provides the basis for coordinated cellular activities. 
Exploitation of this information is confronted with a number of problems. 
Despite advanced image-acquisition procedures and the application of 
denoising techniques, cryoelectron tomograms still suffer from substantial 
residual noise and distortions because of missing data. Moreover, the 
cytoplasm and organelles are crowded with macromolecules, which often 
makes it impossible to perform segmentation and feature extraction on the 
basis of visual inspection of the tomograms, except for large and 
continuous structures such as membranes and filaments.

The less-crowded cytoplasm of eukaryotic relative to prokaryotic cells 
(40) made it possible to visualize 26S proteasomes within their cellular 
environment. For the detection of smaller complexes and less idiosyncratic 
structures, it will be necessary to rely on pattern recognition 
techniques, such as template matching (41). To make the search for 
specific complexes objective and reproducible, it should be automated and 
machine based, not requiring manual intervention. Such methods are under 
development (42). One current limitation of cryo-ET is the lack of methods 
that allow the electron microscopic data to be correlated with light 
microscopic images. Thus, we cannot yet relate the different patterns of 
actin arrangement shown in Figs. 3 and 5 to specific activities in the 
cell cortex. Future developments will aim at combining video recording and 
fluorescence microscopy of green fluorescent protein-tagged proteins with 
cryo-ET to characterize the dynamic state and protein inventory of the 
structures under investigation.

In applying cryo-ET to dynamic cytoskeletal arrangements, a major goal 
will be to assign the types of branches and cross-linkages to specific 
linker proteins. This might be accomplished by different routes. The 
structural analysis of mutants that miss specific actin-cross-linking 
proteins or that lack combinations of these proteins will help to identify 
the individual contributions of major players in the dynamic organization 
of actin networks. Another possibility is the combination of cryo-ET with 
the labeling of specific proteins by microinjected gold-conjugated 
antibodies. Finally, at higher resolution it will be possible to detect 
and identify cross-linking proteins by a template-matching approach, as 
currently explored for larger cytoplasmic protein complexes (42). Because 
prospects for a significant improvement in resolution to 1.5 to 2.0 nm are 
good (43), it will ultimately be possible to generate pseudoatomic maps of 
large supramolecular assemblies or even whole organelles by docking 
high-resolution structures of molecular components into cellular 
tomograms.
REFERENCES AND NOTES
1. W. Baumeister, R. Grimm, J. Walz, Trends Cell Biol. 9, 81 (1999) 
[CrossRef][ISI][Medline].
2. B. F. McEwen and J. Frank, Curr. Opin. Neurobiol. 11, 594 (2001) 
[CrossRef][ISI][Medline].
3. R. G. Hart, Science 159, 1464 (1968) [ISI][Medline].
4. Reviewed in A. J. Koster, et al., J. Struct. Biol. 120, 276 (1997) 
[CrossRef][ISI][Medline].
5. Reviewed in B. F. McEwen and M. Marko, J. Histochem. Cytochem. 49, 553 
(2001) [Abstract/Free Full Text].
6. K. Dierksen, D. Typke, R. Hegerl, A. J. Koster, W. Baumeister, 
Ultramicroscopy 40, 71 (1992) [CrossRef][ISI].
7. K. Dierksen, D. Typke, R. Hegerl, W. Baumeister, Ultramicroscopy 49, 
109 (1993) [CrossRef][ISI].
8. R. Hegerl and W. Hoppe, Z. Naturforsch. Sect. A Phys. Sci. 31, 1717 
(1976) [ISI].
9. B. F. McEwen, K. H. Downing, R. M. Glaeser, Ultramicroscopy 60, 357 
(1995) [CrossRef][ISI][Medline].
10. R. Grimm, et al., Biophys. J. 72, 482 (1997) [Abstract].
11. R. Grimm, et al., Biophys. J. 74, 1031 (1998) [Abstract/Free Full 
Text].
12. D. Nicastro, A. S. Frangakis, D. Typke, W. Baumeister, J. Struct. 
Biol. 129, 48 (2000) [CrossRef][ISI][Medline].
13. R. Grimm, A. J. Koster, U. Ziese, D. Typke, W. Baumeister, J. Microsc. 
183, 60 (1996) [ISI].
14. Materials and methods are available as supporting material on Science 
Online.
15. A. Muller-Taubenberger, et al., EMBO J. 20, 6772 (2001) [Abstract/Free 
Full Text].
16. D. Voges, P. Zwickl, W. Baumeister, Annu. Rev. Biochem. 68, 1015 
(1999) [Abstract/Free Full Text].
17. J. M. Peters, Z. Cejka, J. R. Harris, J. A. Kleinschmidt, W. 
Baumeister, J. Mol. Biol. 234, 932 (1993) [CrossRef][ISI][Medline].
18. M. Rechsteiner, L. Hoffman, W. Dubiel, J. Biol. Chem. 268, 6065 (1993) 
[Free Full Text].
19. H. Holzl, et al., J. Cell Biol. 150, 119 (2000) [Abstract/Free Full 
Text].
20. M. H. Glickman, et al., Cell 94, 615 (1998) [ISI][Medline].
21. J. E. Heuser and M. W. Kirschner, J. Cell Biol. 86, 212 (1980) 
[Abstract].
22. T. M. Svitkina, A. B. Verkhovsky, K. M. McQuade, G. G. Borisy, J. Cell 
Biol. 139, 397 (1997) [Abstract/Free Full Text].
23. L. A. Flanagan, et al., J. Cell Biol. 155, 511 (2001) [Abstract/Free 
Full Text].
24. G. P. Resch, K. N. Goldie, A. Krebs, A. Hoenger, J. V. Small, J. Cell 
Sci. 115, 1877 (2002) [Abstract/Free Full Text].
25. Reviewed in J. E. Bear, M. Krause, F. B. Gertler, Curr. Opin. Cell 
Biol. 13, 158 (2001) [CrossRef][ISI][Medline].
26. Reviewed in D. Pantaloni, C. Le Clainche, M. F. Carlier, Science 292, 
1502 (2001) [Abstract/Free Full Text].
27. Reviewed in J. V. Small, T. Stradal, E. Vignal, K. Rottner, Trends 
Cell Biol. 12, 112 (2002) [CrossRef][ISI][Medline].
28. C. Hug, et al., Cell 81, 591 (1995) [ISI][Medline].
29. M. Haugwitz, A. A. Noegel, J. Karakesisoglou, M. Schleicher, Cell 79, 
303 (1994) [ISI][Medline].
30. J. Niewöhner, I. Weber, M. Maniak, A. Muller-Taubenberger, G. Gerisch, 
J. Cell Biol. 138, 349 (1997) [Abstract/Free Full Text].
31. M. Tsujioka, L. M. Machesky, S. L. Cole, K. Yahata, K. Inouye, Curr. 
Biol. 9, 389 (1999) [CrossRef][ISI][Medline].
32. E. L. de Hostos, Trends Cell Biol. 9, 345 (1999) 
[CrossRef][ISI][Medline].
33. R. Insall, et al., Cell Motil. Cytoskel. 50, 115 (2001) 
[CrossRef][ISI][Medline].
34. T. P. Stossel, et al., Nature Rev. Mol. Cell. Biol. 2, 138 (2001) 
[CrossRef][ISI][Medline].
35. E. Lee, K. Pang, D. A. Knecht, Biochim. Biophys. Acta 1525, 217 (2001) 
[ISI][Medline].
36. J. L. Podolski and T. L. Steck, J. Biol. Chem. 265, 1312 (1990) 
[Abstract/Free Full Text].
37. R. D. Mullins, J. A. Heuser, T. D. Pollard, Proc. Natl. Acad. Sci. 
U.S.A. 95, 6181 (1998) [Abstract/Free Full Text].
38. H. N. Higgs and T. D. Pollard, Annu. Rev. Biochem. 70, 649 (2001) 
[Abstract/Free Full Text].
39. M. Schliwa, Nature Rev. Mol. Cell. Biol. 3, 291 (2002) 
[CrossRef][ISI][Medline].
40. Reviewed in R. Grünewald, O. Medalia, A. Gross, A. C. Steven, W. 
Baumeister, Biophys. Chem., in press.
41. J. Böhm, et al., Proc. Natl. Acad. Sci. U.S.A. 97, 14245 (2000) 
[Abstract/Free Full Text].
42. A. S. Frangakis, et al., Proc. Natl. Acad. Sci. U.S.A. 99, 14153 
(2002) [Abstract/Free Full Text].
43. J. M. Plitzko, et al., Trends Biotechnol. 20, S40 (2002) 
[CrossRef][Medline].
44. J. Walz, et al., J. Struct. Biol. 121, 19 (1998) 
[CrossRef][ISI][Medline].
45. O.M. thanks the European Commission for a Marie Curie fellowship. I.W. 
was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 266 
to G.G.). We thank J. Köhler for the movie and A. C. Steven for critical 
reading of the manuscript.
16 July 2002; accepted 9 September 2002
10.1126/science.1076184
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