(The FASEB Journal. 1999;13:S195-S200.)
© 1999 FASEB
Imaging biological matter across dimensions: from cells to molecules and atoms
DANIEL STOFFLER,
MICHEL O. STEINMETZ1 and
UELI AEBI2
Biozentrum, M.E. Müller Institute for Structural Biology, University of Basel, CH-4056 Basel, Switzerland
2Correspondence: Biozentrum, M. E. Müller Institute for Structural Biology, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. E-mail: aebi{at}ubaclu.unibas.ch
 |
INTRODUCTION
|
|---|
IN ADDITION TO X-ray diffraction and NMR spectroscopy,
microscopes (i.e., light, electron, and scanning probe microscopes)
definitely have become an integrated part of modern structure research.
The primary advantages of microscopes are threefold: 1) they
directly produce images rather than diffraction patterns or spectras;
2) they enable us to explore biological structure at all
levels from the macroscopic to the atomic scale; and 3) they
allow biological matter to be imaged in its functional environment.
Hence microscopes are complementary to–and not competing
with–diffraction methods and spectroscopies.
|
LIGHT MICROSCOPY: FROM OPTICAL SECTIONING TO TRACKING SINGLE PARTICLE EVENTS
|
|---|
At the light microscopic (LM) level, fluorescence microscopy (FM)
allows us to locate the relative distribution of distinct cellular
components (e.g., the different cytoskeletal filament systems) relative
to each other within their in vivo environment. The confocal
laser scanning microscope (CLSM) makes it possible to look inside cells
and tissues in a noninvasive manner by optical sectioning, thereby
dissecting the 3-dimensional cell architecture at the level of the
cytoskeleton and the various organelles. By using video-enhanced light
microscopy (VELM), cell structures with dimensions that are typically
10x below the diffraction-limited resolution of light can be
visualized; hence molecular motors can directly be watched at work,
and, in combination with optical tweezers, their mechano-chemical
performance can be quantified. For example, using a feedback-enhanced
double-laser trap system has allowed Finer et al. (1)
to
directly quantify the force and displacement that results from the
interaction of a single myosin motor molecule with a single suspended
fluorescent actin filament. By doing so, discrete stepwise movements
averaging 11 nm were depicted under conditions of low load, and
single-force transients averaging 3–4 pN were measured under isometric
conditions. Most exciting, Noji et al. (2)
watched single
F1-ATPase molecules acting as a rotary motor. To
achieve this remarkable result, these investigators attached a
fluorescent actin filament to the rotor (i.e., the 
-subunit) of the
motor as a marker, thereby enabling them to videotape the rotary motion
for >100 revolutions in a row in the presence of ATP.
|
TOWARD ATOMIC INTERPRETATION OF ELECTRON MICROSCOPIC DATA
|
|---|
The widest range of dimensions is covered by electron microscopes
(EMs). These microscopes are capable of imaging tissues and cells,
their organelles, and cytoskeleton and supramolecular assemblies, as
well as individual biomolecules, their 3-dimensional molecular
architecture, and, ultimately, single atoms (3)
.
Transmission EM (TEM) in combination with 3-dimensional image
reconstruction is now routinely used to determine the overall size and
shape of protein molecules and their supramolecular organization and
dynamic behavior at the 1–3 nm resolution level (4
5
6)
.
As an example, in the case of actin filaments, a major constituent of
muscle and the cytoskeleton of nonmuscle cells, we have been able to
interpret particular structural features at atomic detail (4
, 7
8)
. Moreover, we have prepared an undecagold-tagged
phalloidin derivative (Au11-PHD) to determine
this mushroom toxin’s binding site and orientation within the F-actin
filament by scanning transmission electron microscopy (STEM) and
3-dimensional helical reconstruction (9)
. Remarkably, when
stoichiometrically bound to F-actin, the Au11-PHD
moiety of the derivative could be directly visualized by STEM along the
two half-staggered long-pitch helical strands of single filaments
(Fig. 1C, D
), hence serving as a structural hallmark. The identified
phalloidin binding site within F-actin agreed well with the site
proposed by the atomic model of Lorenz et al. (10)
.
However, atomic modeling of the Au11-PHD
derivative within the refined and averaged filament reconstruction
(Fig. 1E
) yielded an orientation of the toxin within its
binding site that was distinct from that obtained by Lorenz et al.
(10)
. In order for the Au11-tag,
which is linked to the phalloidin moiety by an ~17-Å spacer, not to
collide with an adjacent intersubunit contact, the phalloidin molecule
had to be rotated ~180° around an axis parallel to the filament
axis relative to the orientation proposed by Lorenz et al.
(10)
(Fig. 1F
). Moreover, we critically
evaluated two distinct atomic models of the F-actin filament [e.g.,
the Holmes-Lorenz model (10)
versus to the Schutt-Lindberg
model (11)
] by combining the structural data obtained by
various biochemical constraints (9)
. Whereas the
Holmes-Lorenz model of the F-actin filament produced a close match with
our EM-based Au11-PHD location and orientation
with the F-actin 3-dimensional reconstruction and crosslinking data
from Vandekerckhove et al. (12)
, both our EM and the
crosslinking data yielded a rather poor fit with the Schutt-Lindberg
model of F-actin.
View larger version (100K):
[in this window]
[in a new window]
|
Figure 1. Upper panel: Structural analysis of native, PHD- stabilized and
Au11-PHD stabilized rabbit muscle F-actin filaments and
orientation of PHD within its F-actin binding site (9
, 36)
. A) STEM dark-field (ADF) micrograph of a
negatively stained (i.e., with 0.75% uranyl formate, pH 4.25) PHD
stabilized F-actin filament stretch. B) As in panel
A, but freeze-dried and unstained. C)
Unstained and freeze-dried Au11-PHD stabilized F-actin
filament stretch. D) As in panel C, but
contrast adjusted so as to display the highest intensities only, which
correspond to single Au11-clusters (diameter ~1 nm)
spaced every ~5.5 nm along the two long-pitch helical strands that
are staggered by 2.75 nm relative to one another (see yellow lines).
E) An averaged and refined 3-dimensional helical
reconstruction computed from negatively stained Au11-PHD
stabilized F-actin filament stretches (see panel A) has
been surface-rendered to include 100% of its nominal molecular mass.
The location of the Au11-clusters has been determined from
a difference map (i.e., Au11-PHD stabilized F-actin
filament reconstruction minus PHD-stabilized F-actin filament
reconstruction) and visualized by 1 nm diameter gold spheres.
F) Alignment and overlay of an atomic PHD-stabilized
F-actin trimer (yellow ribbon; data from Lorenz et al.
(10)
with the Au11-PHD: F-actin 3-dimensional
reconstruction displayed in panel E. The PHD molecule
was rotated by ~180° roughly parallel to the filament axis and then
replaced by our Au11-PHD derivative (violet CPK with a
golden sphere). Bar, 25 nm (A–D). Lower
panel: Visualization of native (i.e., completely unfixed, never
dehydrated/rehydrated, and without any detergent treatment) NPCs by AFM
kept alive in near-physiological buffer medium (15)
.
Corresponding AFM images revealed a distinct morphology for the
cytoplasmic (G) and the nuclear face (H)
of Xenopus oocyte NEs. The upper right inset in panel
G depicts a high-magnification view of the cytoplasmic
face so that the eightfold rotational symmetry of individual NPCs is
resolved. The lower right inset in panel G depicts a
pair of NPCs, one with a plug and one unplugged. H) On
the nuclear face, note the remnants of the nuclear lamina depicted in
areas devoid of NPCs (27)
. The right inset reveals a
high-magnification view of the nuclear face. Both the cytoplasmic
fibrils (G) and the nuclear basket structures
(H) were apparently too flexible to be resolved at
higher detail. Superimposed onto these two micrographs are schematic
representations of a consensus model of the ~125-MDa vertebrate NPC
(13
, 35)
. In this model, the ~55-MDa central framework
(in pink), made of eight multi-domain spokes embracing a central pore,
is sandwiched between a 32-MDa cytoplasmic ring (in blue) and a 21-MDa
nuclear ring (in red). From the cytoplasmic ring, eight short kinky
fibrils emanate, while the nuclear ring is topped with a basket
assembled from eight thin filaments joining distally into a ring. The
blue arrowheads indicate the AFM scanning tip relative to the NPC
periphery. I) Determination of possible structural
changes of the central channel region and possible induction of
plugging or unplugging occurring in response to adding calcium to
completely unfixed NEs kept in near-physiological buffer by time-lapse
AFM. For this purpose, the same specimen area, adsorbed with its
nuclear face, was imaged without calcium (upper micrograph) and after
addition of 100 µM calcium (lower micrograph). As documented, no
plugging or unplugging of individual NPCs was induced by addition of
calcium. Two corresponding pairs of NPCs, one unplugged and one
plugged, have been marked by blue arrowheads. For a more quantitative
comparison, the same 211 unplugged NPCs have been aligned and averaged,
and their average radial height profiles computed. In addition, 18
corresponding plugged NPCs have been selected, averaged and their
average radial height profiles computed. K)
Visualization of the reversible calcium-mediated opening (i.e., +100
µM Ca2+) and closing (i.e., -Ca2+) of the
nuclear baskets (i.e., via dilatation of the distal rings, which may
act as an iris-like aperture) by time-lapse AFM. The same specimen area
has been imaged in the two distinct conformational states with three
corresponding NPCs marked by blue arrowheads. For a more quantitative
comparison of the closed and open state, 30 corresponding NPCs have
been aligned and averaged, and their average radial height profiles
computed. Bars, 100 nm.
|
|
Nuclear pore complexes (NPCs) are large (~125 MDa) supramolecular
assemblies embedded in the double-membraned nuclear envelope (NE) of
interphase cells, representing the major gateways mediating
nucleocytoplasmic transport (13
14)
. To evaluate more
systematically the 3-dimensional molecular architecture of native NPCs,
energy-filtering transmission electron microscopy (EFTEM) of unfixed
and unstained Xenopus oocyte NEs embedded in thick (i.e.,
~250 nm) amorphous ice is now pursued and combined with tomographic
imaging and reconstruction (15)
. This approach, in
combination with specific labeling of distinct NPC constitutents, will
eventually extend the resolution of this massive organelle to ~2.5 nm
so that novel structural features may be located 3-dimensionally
andidentified molecularly. This information, in turn, will be a
prerequisite for a molecular understanding of NPC function (see below).
|
WATCHING MACROMOLECULAR MACHINES IN ACTION BY ATOMIC FORCE MICROSCOPY
|
|---|
Recently, spectacular advances have been achieved with the atomic
force microscope (AFM), the first imaging device allowing direct
correlation between structural and functional states of biomolecules in
their physiological environment (16
17
18
19
20
21)
. To directly
watch transport of cargo through individual NPCs, time-lapse AFM of
completely unfixed NE preparations kept in near-physiological buffer
environment has been performed.
To reproducibly visualize native NPCs under physiological conditions
(i.e., without detergent treatment, chemical fixation, or
dehydration/rehydration during preparation steps), novel preparation
techniques had to be explored (15
, 22)
. Typical AFM
specimen supports such as mica or glass could not be used for native
NEs because these failed to adsorb with stability on these supports.
Instead, the best image quality was achieved when carbon-coated EM
grids were used. In agreement with earlier EM data of chemically fixed
NEs (23
24
25)
, AFM images of native NEs revealed a distinct
morphology for the cytoplasmic and nuclear face of their NPCs. As the
highly flexible cytoplasmic fibrils and the filamentous architecture of
the fragile nuclear baskets could not be resolved by AFM, the
cytoplasmic face of the NPC exhibited donut-like rings protruding ~35
nm from the NE surface (Fig. 1G
), whereas its nuclear face
exhibited ~70-nm high dome-like structures (Fig. 1H
) that
were extremely susceptible to mechanical damage by the scanning tip.
These height measurements are comparable to values determined by EM of
thin sectioned/embedded NEs (26)
. In areas devoid of NPCs,
remnants of the nuclear lamina (27)
could readily be
depicted (Fig. 1H
). High-magnification views revealed
indications of the eightfold rotational symmetry of individual NPCs
(Fig. 1G
, upper right inset). Under near-physiological
preparation conditions, only a small fraction (i.e., 
10%) of the
NPCs harbored a plug within the central pore (Fig. 1G
).
Intrigued by studies of Clapham and co-workers (28)
that
showed that manipulating the calcium stores residing in the lumen of
the NE caused reversible plugging or unplugging of the NPCs, we
performed time-lapse AFM experiments to monitor the cytoplasmic channel
topography in response to adding or removing micromolar amounts of
calcium to the buffer medium surrounding fully native NEs
(15)
. We started by scanning well-preserved NPC patches
kept in low-salt buffer (LSB) completely free of calcium (Fig. 1I
, top micrograph). Subsequently, micromolar amounts of
calcium were added to the buffer medium. After equilibration of the new
buffer condition, the same specimen area was rescanned (Fig. 1i
, lower micrograph) so that the same NPCs could be
compared with those of the scan before calcium addition. Under these
conditions, only ~10% of all NPCs harbored a central cytoplasmic
plug before addition of calcium. No plugging or unplugging of
corresponding native NPCs was induced by adding calcium; those NPCs
that were plugged before addition of calcium remained plugged on
addition and vice versa. For a more quantitative comparison,
the same 211 unplugged NPCs (Fig. 1I
, donut-shaped particle)
and 18 plugged NPCs (Fig. 1I
, donut-shaped particle with
clearly visible plug) before and after addition of calcium were aligned
and averaged, and their average radial height profiles were computed.
Consistent with earlier observations of negatively stained NEs imaged
by EM (23)
, the cytoplasmic face of the NPC did not appear
to undergo any significant change in response to calcium.
Next, we addressed the question of whether calcium could induce
structural changes of the NPC topography (15)
, given that
Jarnik and Aebi (23)
documented that the nuclear baskets
of NPCs are dynamic structures in the sense that they disassemble on
complete removal of divalent cations and reform on their addition. For
this purpose, the same specimen areas of completely unfixed NEs kept in
LSB medium were imaged by contact mode before and after adding or
removing calcium. By this scenario, topographic changes of the same
individual NPCs could be induced (Fig. 1K
) by adding
micromolar amounts of calcium to the buffer medium so that previously
closed baskets opened up at their tops. Conversely, it could be
demonstrated that NEs kept in near-physiological buffer containing
micromolar amounts of calcium harbored NPCs with open baskets that
could be closed to >90% by adding a calcium chelator (i.e., EGTA;
data not shown). To demonstrate the specificity of the calcium-mediated
opening and closing of the nuclear baskets, the same type of specimens
were imaged in response to adding or removing magnesium instead of
calcium. Accordingly, magnesium did not affect the basket structure
(data now shown). To analyze the calcium-induced structural changes of
the nuclear baskets more quantitatively, the same set of NPCs was
translationally aligned and averaged in the two distinct morphological
states. Average radial height profiles revealed openings appearing at
the distal end of the baskets with diameters of 20–30 nm after
addition of calcium (Fig. 1K
). This structural change may be
interpreted in terms of the distal ring of the basket acting as an
iris-like aperture, as suggested by Panté and Aebi
(14)
, which might act as a docking or even a gating site
for cargo transported in and out of the nucleus. Moreover, the overall
height of the baskets protruding out of the NEs did not significantly
change; it was 70 nm with or without calcium or magnesium.
|
OUTLOOK
|
|---|
Novel optical techniques have been developed recently that allow
mechanical and ligand-binding events of single myosin molecules to be
monitored simultaneously (29)
. Also, the axial rotation of
single actin filaments sliding over myosin molecules immobilized on a
glass surface could directly be visualized through fluorescence
polarization imaging of individual fluorophores sparsely bound to the
filaments (30)
. These examples effectively document that
LM, in addition to videotaping single particle events, can now also be
used to directly evaluate mechanical properties, chemical states, and
conformational changes of single macromolecular machines.
A milestone in high-resolution EM has been the elucidation of atomic
models of membrane and cytoskeletal proteins by electron
crystallography (31
32
33)
. Currently, EM at a resolution
sufficient to depict atomic detail (3.5 Å) requires a 2-dimensional
crystalline protein array analogous to the 3-dimensional crystal
required for X-ray crystallography. However, the potential exists to
bypass this limitation and collect the data from randomly oriented
single particles embedded in an amorphous ice film, thus rendering any
macromolecular assembly with a well-defined structure accessible to
high-resolution structural analysis.
As outlined above, time-lapse AFM of native NPCs recorded in
near-physiological buffer environment has opened the possibility to
directly and reproducibly track functionally significant structural
changes such as the opening and closing of the nuclear baskets (i.e.,
the distal rings) at the level of individual NPCs (15)
.
Therefore, the NPC may become a role model for directly correlating
structural data with functional states and dynamic features of living
matter by time-lapse AFM at the level of individual macromolecular
machines. Another hallmark in single-molecule AFM has been the tracking
of the reversible unfolding of titin immunoglobulin domains by repeated
stretching of individual titin molecules (34)
. The
potential for such experiments to provide a more rational understanding
of single-molecule mechanics is evident, and the emergence of novel
findings along these lines is only limited by our imagination.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by the M. E. Müller Foundation
of Switzerland, the Swiss National Science Foundation Priority Program
NFP 36 (‘Nano-Sciences’), and a grant from the Chemical Industry
Foundation of Basel to D.S.
|
FOOTNOTES
|
|---|
1 Present address: Novartis Pharma Research, CH-4002 Basel, Switzerland. 
|
REFERENCES
|
|---|
-
Finer, J. T., Simmons, R. M., Spudich, J. A. (1994) Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature (London) 368,113-119[Medline]
-
Noji, H., Yasuda, R., Yoshida, M., Kinosita, K. (1997) Direct observation of the rotation of F-1-ATPase. Nature (London) 386,299-302[Medline]
-
Steinmetz, M., Stoffler, D., Hoenger, A., Bremer, A., Aebi, U. (1997) Actin filaments: from cell biology to atomic detail. J. Struct. Biol. 119,295-320[Medline]
-
Bremer, A., Henn, C., Engel, A., Baumeister, W., Aebi, U. (1992) Has negative staining still a place in biomacromolecular electron microscopy?. Ultramicroscopy 46,85-111[Medline]
-
Hoenger, A., Aebi, U. (1996) Three-dimensional reconstructions from ice-embedded and negatively stained macromolecular assemblies: a critical comparison. J. Struct. Biol. 117,99-116
-
Steinmetz, M., Goldie, K. N., Aebi, U. (1997) A correlative analysis of actin filament assembly, structure and dynamics. J. Cell Biol. 138,559-574[Abstract/Free Full Text]
-
Bremer, A., Henn, C., Goldie, K. N., Engel, A., Smith, P. R., Aebi, U. (1994) Towards atomic interpretation of 3-D reconstructions of F-actin filaments. J. Mol. Biol. 242,683-700[Medline]
-
Steinmetz, M. O., Hoenger, A., Tittmann, P., Gross, H., Aebi, U. (1998) An atomic model of crystalline actin tubes: combining electron microscopy with X-ray crystallography. J. Mol. Biol. 278,703-712[Medline]
-
Steinmetz, M. O., Stoffler, D., Müller, S. A., Jahn, W., Wolpensinger, B., Goldie, K. N., Engel, A., Faulstich, H., Aebi, U. (1998) Evaluating atomic models of F-actin with an undecagold-tagged phalloidin derivative. J. Mol. Biol. 276,1-6[Medline]
-
Lorenz, M., Popp, D., Holmes, K. C. (1993) Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. J. Mol. Biol. 234,826-836[Medline]
-
Schutt, C. E., Kreatsoulas, C., Page, R., Lindberg, U. (1997) Plugging into actin’s architectonic socket. Nature Struct. Biol. 4,169-172[Medline]
-
Vandekerckhove, J., Deboben, A., Nassal, M., Wieland, T. (1985) The phalloidin binding site of F-actin. EMBO J 4,2815-2818[Medline]
-
Panté, N., Aebi, U. (1996) Toward the molecular dissection of protein import into nuclei. Curr. Opin. Cell Biol. 8,397-406[Medline]
-
Panté, N., Aebi, U. (1996) Molecular dissection of the nuclear pore complex. Crit. Rev. Biochem. Mol. Biol. 31,153-199[Medline]
-
Stoffler, D., Goldie, K. N., Feja, B., Aebi, U. (1999) Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy. J. Mol. Biol. 287,741-752[Medline]
-
Müller, D. J., Schabert, F. A., Büldt, G., Engel, A. (1995) Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy. Biophys. J. 68,1681-1686[Abstract/Free Full Text]
-
Müller, D. J., Büldt, G., Engel, A. (1995) Force-induced conformational change of bacteriorhodopsin. J. Mol. Biol. 249,239-243[Medline]
-
Müller, D. J., Baumeister, W., Engel, A. (1996) Conformational change of the hexagonally packed intermediate layer imaged by atomic force microscopy. J. Bacteriol. 178,3025-3030[Abstract/Free Full Text]
-
Müller, D. J., Schoenenberger, C.-A., Schabert, F., Engel, A. (1997a) Structural changes in native membrane proteins monitored at sub-nm resolution with the atomic force microscope. J. Struct. Biol. 119,149-157[Medline]
-
Müller, D. J., Engel, A., Amrein, M. (1997b) Preparation techniques for the observation of native biological specimens with the atomic force microscope. Biosens. Bioelectronics 12,867-877
-
Ludwig, M., Dettmann, W., Gaub, H. E. (1997) Atomic force microscope imaging contrast based on molecular recognition. Biophys. J. 72,445-448[Abstract/Free Full Text]
-
Aebi, U., Stoffler, D., Feja, B., Goldie, K. (1998) Nuclear pore transport: Insight in situ. Microsc. Microanal. 4((Suppl. 2)),956-957
-
Jarnik, M., Aebi, U. (1991) Towards a 3D model of the nuclear pore complex. J. Struct. Biol. 107,291-308[Medline]
-
Panté, N., Aebi, U. (1993) The nuclear pore complex. J. Cell Biol. 122,977-984[Free Full Text]
-
Goldie, K. N., Panté, N., Engel, A., Aebi, U. (1994) Exploring native nuclear pore complex structure and conformation by scanning force microscopy in physiological buffers. J. Vac. Sci. Technol. B 12,1482-1485
-
Fahrenkrog, B., Hurt, E. C., Aebi, U., Panté, N. (1998) Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes. J. Cell Biol. 143,577-588[Abstract/Free Full Text]
-
Aebi, U., Cohn, J., Buhle, E. L., Gerace, L. (1986) The nuclear lamina is a meshwork of intermediate-type filaments. Nature (London) 323,560-564[Medline]
-
Perez-Terzic, C., Pyle, J., Jaconi, M., Stehno-Bittel, L., Clapham, D. E. (1996) Conformational sates of the nuclear pore complex induced by depletion of nuclear Ca2+ stores. Science 273,1875-1877[Abstract/Free Full Text]
-
Ishijima, A., Kojima, H., Funatsu, T., Tokunaga, M., Higuchi, H., Tanaka, H., Yanagida, T. (1998) Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92,161-171[Medline]
-
Sase, I., Miyata, H., Ishiwata, S., Kinosita, K. (1997) Axial rotation of sliding actin filaments revealed by single-fluorophore imaging. Proc. Natl. Acad. Sci. USA 94,5656-5650[Abstract/Free Full Text]
-
Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M., Henderson, R. (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259,393-421[Medline]
-
Kuehlbrandt, W., Wang, D. N., Fujiyoshi, Y. (1994) Atomic model of plant light-harvesting complex by electron crystallography. Nature (London) 367,614-621[Medline]
-
Nogales, E., Wolf, S. G., Downing, K. H. (1998) Structure of the alpha/beta-tubulin dimer by electron crystallography. Nature (London) 391,199-203[Medline]
-
Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., Gaub, H. E. (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276,1109-1112[Abstract/Free Full Text]
-
Nigg, E. (1997) Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature (London) 386,779-787[Medline]
-
Steinmetz, M. O., Stoffler, D., Aebi, U. (1998) Actin: Dissecting the structural basis of its oligomerization, polymerization, and polymorphism. Biol. Bull. 194,337-341[Medline]
TOP
INTRODUCTION
LIGHT MICROSCOPY: FROM OPTICAL...
