Impact of electron microscopy in cell biology: From past to present
A.B. Maunsbach”,
B.A. Afzelius#
*Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark.
#Department of Ultrastructure Research, The Arrhenius Laboratories E4, Stockholm University,
Stockholm, Sweden.
During half a century cell biologists have scrutinized all types of cells and tissues with the aid
of the electron microscope. It is today impossible to imagine what biology would be, if this instrument
had not been invented. By electron microscopy our conception of the cell is that of a compartment
furnished with distinct cell organelles in constant changes and interplay. What is less realized is that the
many advances in the exploration of the cell usually have come as the result of an improved specimen
preparation techniques rather than as the result of a straightforward exploration with standard methods
and have only very rarely been the result of an improved design of the electron microscope itself. The
resolving power of the electron microscope typically is a factor of ten better the actual resolution obtained
even in excellent micrographs of a biological material.
In this short presentation we will review some aspects of cell biology with emphasis on the role
that the specimen preparation techniques in electron microscopy have played in exploration of the cell and
hence in our present understanding of cell biology.
1. Mitochondria and bioenergetics
In 1953 the technique of fixation (with osmium ten-oxide), embedding (in methacrylates), and
sectioning (on newly constructed microtomes) had reached a stage at which some pioneers could study
the cell at high resolution by transmission electron microscopy. Thus Sjiistrand (1) and Palade (2) were
both able to make detailed electron micrographs of mitochondria. In Sjostrand’s high resolution
micrographs the wall of the mitochondrion was resolved as two separate membranes, now termed the
outer and the inner limiting membrane of the mitochondrion.
A few years later sectioning-staining had been developed and provided a simple means of
obtaining good image contrast. Nass and Nass (3) found that their section-stained mitochondria had
cristae-free areas of low electron density and that some fibrous structures resided in these areas,
reminescent of the bacterial genome. The fibers were shown to consist of DNA, as proven by DNAase
digestion; a mitochondrial genome had been discovered, where none had been expected. Its length about
5 micron was measured by using the Kleinschmidt-spreading
technique. Defects in the mitochondrial
genome lead to mitochondrial dysfunctions and deletions or point mutations in the mitochondrial DNA
have been observed to increase with age (4).
At about the same time, in the early 60s the negative staining procedure was tried on various
subcellular fragments, among them isolated mitochondria (5). Although the mitochondria
were
considerably distorted, a new structural element become visible: subunits projecting from the inner
membranes. These were originally interpreted as the compacted enzymatic components of the electron
transport chain but soon shown to contain a projecting
adding phosphate groups onto ADP when protons are
water mill. The working mechanism and the detailed
mainly by two of last year’s nobelprize-winners, Paul
portion of ATP-synthase, which is responsible for
flowing through the particles like water through a
structure or the ATP-synthase has been clarified
Boyer and John Walker (6,7).
2. Plasma membrane and transport proteins
Specific identification
of cell membrane proteins became possible with the aid of
immunocytochemical
methods at the electron microscopical level. These were initiated by Singer (8) and
expanded through the immunoperoxidase
procedure and not the least the immunogold method (9). A
wealth of information was obtained about the localization of membrane proteins within cells and among
cell types and have been particularly important for the understanding of epithelial physiology, e.g. the
transport pathways for salt and water (10).
The molecular structure of the cell membrane was gradually elucidated through the
development of methods for negative staining and freeze-fracture (11) but the seminal step in the analysis
of the molecular structure of membranes was the electron crystallographic studies by Unwin and
Henderson of the purple membrane in the mid 70s (12). Based upon the same principles an increasing
number of membranes have been analyzed over the last two decades to a high resolution, in some cases
to below 3 A, and electron crystallography has developed into an alternative method to x-ray
crystallography, in particular for membrane proteins that have not yet been crystallized in 3-dimensions.
3. Lysosomes and endocytosis
Lysosomes were discovered on the basis of biochemical studies by de Duve et al. (13), but the
understanding of their functions was greatly expanded through cytochemistry at the ultrastructure level.
This technique, initiated by Sheldon et al. (14), made it possible to characterize the structure and
distribution of lysosomes. The relationships between endocytosis and the lysosomal system were
determined at the ultrastructural level in the 60s by experimental cytochemical studies of cells absorbing
various protein tracers (15) and by combined autoradiographic and cytochemical analyses of the endocytic
pathway for physiologically occurring proteins (16).
4. Intracellular
membranes and secretion
In 1945 Porter et al. (17) examined by transmission electron microscopy thinly spread cultured
cells that had been grown on Formvar films and found certain structures in the cell interior that he later
named the endoplasmic reticulum. This network is not to be confused with another ‘endoplasmic reticular
apparatus’ (or ‘apparato reticular-e interno’ in Italian) that had been described much earlier by Camillo
Golgi from metal-impregnated
light microscopical preparations. The latter network, named the Golgi
complex after its discoverer, was first characterized at the ultrastructural level by Sjiistrand and Hanzon
(18) in 1954 when the thin-sectioning technique in electron microscopy had been elaborated.
The development of cytochemical methods at the electron microscopical level led to the
detection of glycosylation enzymes in the Golgi complex (19). It was seen that different enzymes are
located in the cis- and in the trans-cistema. With immunoelectron microscopy it has recently been possible
to demonstrate
the detailed distribution of several other enzymes (20). Electron microscope
autoradiography was also performed to investigate what role, if any, the Golgi complex has in the
synthesis and secretion of proteins in exocrine pancreas (21). From autoradiographic and cytochemical
data, combined with biochemical information, it could be concluded that secretory proteins’ are
synthesized at the endoplasmic reticulum, transferred to the cis-side of the Golgi complex, and pass from
the cis-cistema to the next one and on to the trans-side cisterna and that they then are released at a portion
of the Golgi complex named the trans-Golgi network.
5. Tubulin and cytoskeleton
The concept of a cytoskeleton, with microtubules as one of its major element, is of a relatively
young date. The triggering event was the introduction of glutaraldehyde as a fixative in 1963 (22). In that
year Ledbetter and Porter (23) wrote: ‘Finally, the image after glutaraldehyde + 0~0, fixation differs from
others in showing large numbers of thin but uniform filaments which we shall refer to as “tubules”,
“microtubules” or “cytotubules”.’ Whereas all the kinds of eukaryotic cells were shown to possess
microtubules the structural organization of the microtubules themselves could not be further clarified until
another fixative had been introduced: tannic acid as an additive to the glutaraldehyde fixation (24). The
microtubules were then shown to have a wall that in most cases consists of 13 protofilaments. An even
more detailed three-dimensional view came with computer-aided spectral analysis using Fourier methods.
In one such study (25) the microtubules had been fixed with a modified glutaraldehyde-tannic acid fixative
(no osmium postfixation) and showed the tubulin heterodimers, and in another study they were seen in
their native state after plunge freezing and examination by cryo-electron microscopy (26).
The cover of the second issue this year of Nature shows a further step in the exploration of the
microtubules; namely the secondary structure of tubulin. This achievement is characterized in an editorial
note as “a veritable tour de force.” A crystallographic study had been used on tubulin sheets that had been
polymerized in the presence of zinc, immersed in tannic acid and glucose and examined at liquid-nitrogen
temperature (27). By using electron diffraction Nogales et al. were able to resolve the tubulin molecules
to a resolution of 3.7 A, one of the highest resolutions recorded from a biological specimen. Thus over
a time span of 35 years from their discovery the structure of microtubules has been elucidatd to near
atomic level through stepwise improvements of the analytical methods.
6. Dynein and cell motility
Dynein arms were first described from sections of the sperm tail using a new fixative (28) .
Some years later, dynein arms were isolated and deposited onto filmed specimen grids that were rotary
shadowed with platinum and carbon (29). The dynein arms were then shown to have either two or three
dynein heads. A rather similar morphology was seen also in other motor proteins, such as the kinesins.
These findings together with video-enhanced microscopy of living systems led to a better understanding
of the mechanisms behind a.o. mitotic division and of cytoplasmic organization.
The complex three-dimensional structure of dynein arms in cilia and flagella was elucidated
by Goodenough and Heuser (30), who invented a sophisticated technique in which quick-frozen deepetched replicas of cilia and flagella are prepared. They found that each dynein arm is composed of five
discrete components among which there is a very slender linker that extends from the dynein arm of one
microtubule to its neighboring microtubule. The spatial relationship between the various components of
the dynein arms were shown to be different in a ‘rigor’and a ‘relaxed position.
7. Nucleic acids and splicing
The nucleus, in contrast to the cytoplasm, has no membrane-linked
compartments and its
ultrastructural appearance in sections is rather uninformative. Preparation methods other than fixation,
embedding and sectioning had to be invented in order to elucidate some of the functions of the nuclear
components. One of the successful alternative techniques was the Kleinschmidt-technique,
so named after
its inventor (3 1). Strands of nucleic acids are spread on a water surface from where they are picked up
onto filmed specimen grids and rotary shadowed. In a study of the transcription of the information in a
DNA strand into messenger RNA, it was found that the RNA strand was not a strictly linear copy of that
in the DNA strand but that there were gaps not replicated; these places were loops made by DNA not
accompanied by the RNA (32). This phenomenon was against the generally held belief that the messenger
RNA is a faithful replica of all the DNA. The finding gave rise to such concepts as introns and exons and
of the splicing phenomenon. The nuclear chromatin was similarly examined and found to be a pearl-string
of small beads, now named nucleosomes (33). The core of the nucleosomes consists of histone octamers;
these were crystallized and examined by Klug et al. (34) who were able to resolve the 3-dimensional
structure of the nucleosomes: Nucleic acid strands are wound nearly two turns around the histone core,
which arrangement serves to pack the long nucleic acids effectively in the nucleus.
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