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Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA
1Correspondence: Shinya Inoué, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543-1015.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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In the late 1940s and early 1950s, amid the discovery of a series of
respiratory enzymes and other wonderful proteins, many physiologists
and biochemists considered a living cell to be a sackful of enzymes.
Despite the careful earlier work by E. B. Wilson, Karl
B
l
r, and other cytologists, the presence of a structural
framework in the cell was seriously debated, and I recall the real heat
with which Dr. L. Victor Heilbrunn greeted the observations of Eric Kao
(who was working with Dr. Robert Chambers). Kao showed that
microinjected oil drops took on an elongated, nonspherical shape,
implying the presence of microscopically invisible fibrils in neurons
and muscle cells (1)
. Said Heilbrunn: "I determined the
viscosity of the living cytoplasm using the standard method by
measuring the sedimentation rate of particles in the living cell,
including neurons, and it is virtually the same as water. There cannot
be any invisible filaments." As a brash graduate student, I myself
was harshly reprimanded for asking: "But Dr. Heilbrunn, how do you
explain the shape of those oil drops?"
Around the same time, I was able to demonstrate the reality of spindle
fibers and fibrils and their dynamic nature in active living cells by
using polarized light microscopy, whose sensitivity and resolution I
had been improving (2)
. Because with polarized light
microscopy contrast is generated noninvasively by the anisotropic
alignment of molecules, I argued that we were observing the actual
formation and changes in the fine structure inside living cells.
Neither those fine structural filaments, nor their bundle, spindle
fibers, had been visible in living cells earlier with the microscope
unless the cells were fixed and stained, or the cells had been made
acidic.
The notion that spindle fibers and fibrils seen after fixation and staining are fixation artifacts was so strong that, after seeing my time-lapse movies showing the birefringence of those structures and their changes in actively dividing pollen mother cells of Easter lilies, Dr. Ethyl Brown Harvey asked: "Were those cells alive?"
A film clip from those days shows mitosis in living endosperm cells of Hemanthus captured by Andrew and Wishia Bajer with phase contrast microscopy. The chromosomes, which show clearly because of their higher refractive index, move mysteriously to the spindle poles in anaphase (video)2 . A second film clip shows mitosis and cell plate formation in Lily pollen mother cells, which I recorded with a sensitive polarizing microscope (video)2 . In conjunction with these observations, I have done experiments using antimitotic agents such as colchicine and cold, and demonstrated that what we were seeing as birefringent spindle fibers were a bundle of protein filaments that could be reversibly depolymerized by the anti-mitotic agents.
Thus, in the living cell, we had a pool of material that could be
transiently polymerized around orienting centers as needed by the cell,
only to be taken apart again (5–7). I was able to say polymerization
states, using the LM despite its limited resolving power, because I was
using the polarizing microscope to deduce the reversible dynamic
changes of fine structure by measuring the birefringence and changes in
dimensions of the aligned fibers in the spindle (8
, 9
).
That was in 1950–1951. But for 15 years after that, Dr. Keith Porter,
whose many contributions we celebrate today, kept telling me that what
I was observing in the spindle was not protein filaments but membranes.
And my explanation that the sign of birefringence would be wrong for
membranes fell on deaf ears. I pointed out that if these were
membranes, the slow axis would be perpendicular to what is observed for
the spindle fibers. Our friendly dispute lasted until the spindle fiber
region that had appeared empty in the electron microscope (EM), except
for a few membrane fragments, was found to be full of microtubules once
Sabatani came out with an improved fixative using glutaraldehyde. Then
in the mid-1960s, Dr. Porter gave the famous Ciba Foundation Symposium
talk (10)
, where he demonstrated the wide presence of
microtubules, including in spindle fibers. And to show that the
electron micrographs were not fixation artifacts, he came to borrow my
slides that showed their birefringence in living cells
(11)
!
So microtubules are in spindle fibers, but, people argued, they
couldn’t possibly polymerize and depolymerize the way I said they
would because protein fibers just don’t do such things. So it took
another several years before Dick Weisenberg figured out how to obtain
LABILE microtubules from cells (by sequestering the
calcium ions that would otherwise disassemble, for example, the spindle
microtubules) (12)
. This was immediately followed by
Joanna Olmsted and Gary Borisy’s classical studies that showed how the
isolated microtubules could be made to reversibly disassemble and
assemble by cold and other agents (13)
. So now it was
acceptable because these changes could be made to happen outside the
cell, rather than be based on observations inside the cell.
In the past few years, model experiments performed by some ingenious
investigators have also confirmed that depolymerizing microtubules can
remain attached, and by the very act of depolymerizing and shortening
pull organelles, as I had postulated on the basis of polarization
optical studies of metaphase-arrested spindles treated with colchicine
(14
, 15
). Of course, I am happy to learn that the
conclusions that we had drawn from polarization microscopy of living
cells could finally be accepted (although after decades) by being
verified also in isolated, cell-free systems.
Skipping to the present and future, we have seen ample, remarkable examples of how microscopy is shedding ever more light on dynamic cell fine structure, including at the level of individual molecules. And we may ask: Does polarized light microscopy still offer attractive windows for studying dynamic fine structures in living cells? My belief is a resounding yes. While, unfortunately, very few biologists still have a good intuitive grasp of the interaction of polarized light with molecular and atomic order, and perhaps even less in terms of how in practice one takes advantage of such interactions, my personal conviction is that we are still at the foothills of a grand mountain of treasures that are awaiting to be explored using polarized light microscopy, by those who are prepared. Fortunately, more individuals are better prepared in physics and chemistry today, so I hope these treasurers will be explored.
Naturally, the specificity and sensitivity of fluorescent markers have great appeal, as does the much higher image resolution given by electron microscopy. Fluorescence microscopy is now yielding spectacular images signaling the activity of single protein motors and of unexpected molecular fluxes in membranes and mitotic microtubules. Combined with video enhancement and ingenious analyses, differential interference is shedding light even on transcription rates along single strands of DNA. And the resolution by cryo-EM now competes with X-ray analyses of dynamic atomic arrangements, using much smaller, 2-dimensional samples than the 3-dimensional crystals that are needed for X-ray diffraction analyses.
Following are some of the images obtained with the new Pol-Scope
developed by Rudolf Oldenbourg in our Architectural Dynamics in Living
Cells Program at the MBL. With the new Pol-Scope, unlike the images
formed with classical polarization microscopy, image contrast is
strictly proportional to the local specimen birefringence and
independent of slow axis orientation (Fig. 1
). This shows the distribution of microtubules in an isolated sea-urchin
spindle. It typifies the sensitivity and striking image quality of the
new system.
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The videotape shows the dynamic behavior of the microtubule bundles and organelles in a dividing newt lung epithelial cell taken by Ted Salmon, Phong Tran, and Rudolf Oldenbourg. The degree of resolution here is truly remarkable, and the measurement of birefringence changes along the length of each fiber should tell us much about their local concentration kinetics.
The idea behind this new Pol-Scope is that, instead of using an
ordinary compensator, one uses 2 liquid-crystal plates to which voltage
is applied to generate a half-wave plate and a quarter-wave plate
plus-minus delta phase differences. That effectively changes the
crossed polarizer images into those with a regular Brace-Koehler
compensator turned clockwise, then counterclockwise, then parallel to
the initial polarizer axes, and finally in crossed circularly polarized
light. A computer grabs and calculates from all of these 4 images
(which together are acquired in 0.25 s without involving any
mechanical movements) and displays the retardance image
(16)
. The images seen in the video were grabbed as a
time-lapsed series every few seconds. Unlike polarized light images in
the past, all the microtubules in the plane of focus show their
birefringence regardless of their orientation, and the intensity that
you observe in the image is strictly proportional to the retardance at
the image point. So you can count, for example, the number of
microtubules or actin filaments that are making up a particular part of
a fiber by measuring the image brightness. Also, one can obtain an
image that maps the slow axis orientation and one can follow the fine
structures change dynamically.
Another example of dynamic fine structures that Kaoru Katoh and Rudolf
have captured with the new Pol-Scope is the growth cone of
Aplysia neurons (in collaboration with Peter Smith’s
Biocurrents Research Center group at the MBL). In a low-power video, we
saw the active movements and changes in these filaments. At higher
power, the video shows the generation, treadmilling, and dynamic fusion
of actin filament bundles (17)
. As mentioned above, one
can count the number of the unresolvable actin filaments that make up
each part of the filament bundle and follow their changes.
Another video sequence was taken with a centrifuge polarizing microscope (CPM), an instrument that I had dreamed of being able to use since I first observed the spindle fibers and fibrils in Chaetopterus and Lilium nearly half a century ago. Because if we could apply centrifugal force, we could stratify and visualize the structure that had been obscured by the light-scattering cellular contents. We could also follow how molecules and fine structures become aligned or stretched by centrifugal forces inside the living cells and perhaps even discover new dynamic fine structures. By collaborating with Hamamatsu Photonics (Hamamatsu City, Japan) and Olympus Optical (Hachioji, Japan), we were finally able to develop a prototype CPM that allows us to gain contrast from fine structures that are stratified and ordered in living cells under up to 10,000 times gravity.
Looking at cells in a centrifuge microscope as such is not at all new
(18). In fact, here at the MBL in the 1930s and 1940s, Ethyl Brown
Harvey, using a centrifuge microscope that her husband E. Newton Harvey
and Bill Loomis had designed, was able to observe
Arbacia eggs under centrifugation (Fig. 2
). The eggs were made to float on a gradient between seawater and
isotonic sucrose solution. She was able to show the stratification,
from the light side, of oil drop layer, nucleus, clear cytoplasm,
mitochondrial layer, yolk, pigment, and so on. And as the
centrifugation continued, she showed that the eggs turn into a dumbbell
shape and eventually are pinched into a white half and a red half, then
into 4 quarters. The real surprise came when she removed from the
centrifuge clear quarters, which presumably did not have much
mitochondria, they could be fertilized and some would develop into
pluteus larvae. Even more surprising was that she could artificially
activate fragments without using any sperm–and it’s not too
surprising that the clear quarter develops since it has an egg
nucleus–but even the heavy quarter that had no egg or sperm nucleus
could be activated and would form asters and spindles
(20)
! One should seriously think about this experience of
Harvey’s when today one is considering what organizes the cytoplasm.
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Another figure shows the arrangement of the CPM (Fig. 3
). Unlike our system, the Harvey centrifuge microscope had the
microscope optics spinning with the specimen. We can’t do that when we
use polarized light because all the lens elements would be strained and
become birefringent. So in the CPM, we placed the whole microscope
outside of the centrifuge. The specimen is illuminated by a short flash
from a laser each time the spinning specimen lines up exactly under the
microscope objective. The speed needed for the synchronized laser flash
can be imagined by considering Edgerton’s famous flash pictures that
froze the image of a bullet flying at supersonic speed that had just
shattered a light bulb or penetrated an apple (22)
. Those
pictures were taken with cleverly triggered flashes that lasted on the
order of one-millionth of a second. Since a microscope magnifies speed
of movements by the same factor as it magnifies the image, for the CPM
with a radius of 7.5 cm to give an image resolution of 0.5 µM at
10,000 RPM, we need laser flashes that last <6 ns (100 times shorter
than Edgerton’s Xenon flashes). These flashes need to be bright enough
to be used for high-extinction polarization microscopy and also have to
be precisely synchronized to the centrifuge rotation so that the image
doesn’t jump all over the place with the succeeding flashes. Our
collaborators, Hamamatsu Photonics and Olympus Optical, solved these
difficult technical problems.
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As seen in Fig. 4
, the image of a test target including the 1.0-µM scale is dead steady
in the CPM spinning at 10,000 RPM. We are thus resolving the image to
better than 1 µM. The image of a thin section of frog striated muscle
shows that we are also detecting birefringence retardance to better
than 1 nm while the CPM rotor is spinning at 10,000 RPM. Therefore, we
should be able to make measurements on a rather few molecules that are
lined up in the CPM.
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Figure 5a and b
show in the CPM a
Chaetopterus oocyte that had been shed in
Ca2+-free seawater and thus whose nuclear envelope had not
broken down. In such centrifuged and stratified oocytes, we see a
large, relatively clear zone above the heavier organelles, yolk, and
other cell inclusions. Near the top of this zone, abutting the oil cap,
we see the large germinal vesicle (oocyte nucleus) with its nucleolus
protruding below. Surrounding and just below the nucleolus, we see a
strange curtain of negatively birefringent material (which appears to
be membrane material) that has become organized by the centrifugation
in the clear zone.
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After activation of the oocyte in the CPM with calcium ions, the
nucleolus, nuclear envelope, and the negatively birefringent material
all disappear quite rapidly, while we also see a shower of particles
released from the cortex above the germinal vesicle raining down. And
in place of the negatively birefringent material (that eventually
reappears before the next division cycle), 2 small, positively
birefringent asters emerge. The astral microtubules continue to grow
and eventually form the meiosis-I metaphase spindle (Fig. 5c, d
).
When Chaetopterus oocytes are shed into normal
(Ca2+-containing) seawater, they proceed through germinal
vesicle breakdown and develop the 1st meiotic metaphase spindle that is
arrested for many hours unless the oocyte is activated. Observed in the
CPM, the pattern of stratification and shape of the spindle in such a
metaphase-arrested oocyte is quite different from those that were shed
in Ca2+-free seawater and activated with Ca2+
in the CPM. We see 3–4 layers of (negatively) birefringent material on
the lighter, centripetal pole. And the spindle, lying in the narrow
neck of the elongated stratified cell, is now stretched, strongly
birefringent, and its poles are pointed (Fig. 5e
,
f). Clearly, the organization of the basic
cytoplasm and interaction of its fine structural components have
changed dramatically by maturation of the egg.
The CPM thus gives us an opportunity to study the fine structures that become stratified, to collect them in mini-cells that have pinched apart from the rest of the cell, as well as to follow the dynamic physiological changes that take place under centrifugal stratification in the living cell. The CPM is also expected to open up new windows for capturing surprising transient orders in other quasi-fluid systems such as solutions of liquid crystals and emulsions whose contents are being stratified or aligned by centrifugation.
In closing, I would like to point out how much I have been struck by the undreamed of pace by which microscopic revelation of dynamic molecular events, especially aided by electronic imaging and enhancement, has advanced.
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FOOTNOTES |
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. Uncompressed versions
of the first two scenes, together with many others showing experimental
modifications of the spindle microtubule assembly, and chromosome
movement, can be seen in Video Supplement II of Cell Motility,
and the Cytoskeleton (4)
. (The FASEB
Journal cannot guarantee the availability of references to the
Web.) 
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REFERENCES |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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