Cell Biol Educ 2(1): 9-13 2003
DOI: 10.1187/cbe.02-11-0057
© 2003 American Society for Cell Biology
Video Views and Reviews
Christopher D. Watters
Department of Biology, Middlebury College, Bicentennial Way, Middlebury,
Vermont 05753
Submitted November 6, 2002;
Revised November 14, 2002;
Accepted November 14, 2002
I could subtitle this feature "Kate's Cool Video Clips" because
the three Molecular Biology of the Cell (MBC) movies I
review here were suggested by Kate Durda, a biology major at Colby College in
Maine, who is compiling an index of MBC videos for ASCB.
On a technical note, many of the video records I review in these features
were obtained by "time-lapse photography": that is, the images
were captured at a slow rate (1 image s–1 or
min–1 or h–1, for example), not at the usual
rate of 16 or 24 images s–1. Most playback formats (such as
QuickTime), however, project images at a standard rate of 24
s–1, and consequently, videos are usually speeded up with
respect to the rate at which the events they record actually happened. The
effect is quite dramatic and arresting but also artifactual! Thus, a movie
containing 240 images may last 10 s, but if the events it portrays lasted 240
min (and were recorded at a rate of 1 image min–1), viewers
will observe those events as if they were speeded up "1440-fold"
or "1440 times." I use this terminology to indicate how much the
video records I review telescope the temporal events they record, but
unfortunately, it is not always possible to obtain this information from the
articles.
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NUCLEAR DYNAMICS AND NUCLEAR LOCALIZATION SIGNALS
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Videos that introductory-level students will likely find impressive
accompany a paper on nuclear dynamics in Arabidopsis by Chytilova
et al. (2000). Using
confocal fluorescence microscopy, the authors recorded the movements of root
hair nuclei labeled with a chimeric protein consisting of green fluorescent
protein (GFP), a nuclear localization signal (NLS) and
ß-glucuronidase (GUS) from Escherichia coli. As
demonstrated in a vivid time-lapse movie (their
Figure 3; speeded up
480-fold), nuclei are readily seen moving within root hairs, changing
their shapes, and even separating into small bodies (which, however, remain
connected by slender thread-like extensions, as illustrated in the paired
images in Figure 1). No student
seeing these dramatic movies will ever again think of nuclei as static
structures! Their Table 1 documents the effects of several inhibitors of
cytoskeletal organization, which indicate the nuclear movements are due to
microfilaments and not microtubules, but unfortunately these results are not
also presented as video records. Equally impressive, and possibly more
suitable for intermediate cell biology students, is the authors' video record
of the movement of the chimeric protein as a root hair nucleus passes through
mitosis (Chytilova et al.,
2000, Fig 6; speeded up about 235 times). Students must locate in
the video the nucleus encircled in the still image, which requires careful
observation. Tracking this nucleus during the video is complicated by the
microscopic field of view gradually shifting in a diagonal manner from the
upper right toward the lower left, which creates the somewhat disconcerting
illusion that all the nuclei are "drifting upward" toward the
upper-right corner. Even very attentive students may need to view this video
several times. This extra effort will prove worthwhile, however, when they
note the rapid dispersal of fluorescence (speeded up about 250-fold) as the
designated nucleus (Figure 2;
left-hand image) completes prophase of mitosis and its envelope breaks down
(middle image). Then, several minutes later, during telophase, the GFP chimera
may be seen slowly reaccumulating within the smaller daughter nuclei
(right-hand image). The sequence presents a striking demonstration of the
importance of the NLS, and it would be even more instructive were it paired
with a control sequence documenting the behavior of a GFP:GUS chimera lacking
the NLS.
View larger version (65K):
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Figure 3. Figures A and B are taken from videos that accompany Strome et al.
(2001;
http://www.molbiolcell.org/cgi/content/full/12/6/1751)
Figure 8, videos N2 GFPbetaB.mov and SevRNAi GFPbeta.mov respectively. Figures
C and D are taken from Strome et al., Figure 9, videos N2 GFPhist.mov
and SevRNAi GFPhist.mov. Reproduced from Molecular Biology of the
Cell, 2001, Vol. 12, pp. 1751–1764, by copyright permission of the
American Society for Cell Biology.
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SPINDLE AND CHROMOSOMAL MOVEMENTS DURING MITOSIS
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Those readers who missed the video review in the last issue of
CBE, concerning 
-tubulin, mitosis, and early
development in Caenorhabditis elegans
(Hannak et al., 2002),
may be interested in a similar paper published in MBC by Strome
et al. (2001). In the
latter paper, the authors present two sets of video fluorescence records
concerning, respectively, the wild-type behavior of GFP:
-tubulin and of GFP:ß-tubulin during early cleavage. These
records of control data are supplemented by additional videos that depict the
consequence of -tubulin depletion (by RNAi) on the spindle
organization of GFP:ß-tubulin and on the mitotic behavior of
chromosomes labeled with GFP:histone. Although the fluorescent images are not
paired with differential interference contrast images, as is the case in the
more recent study by Hannak et al.
(2002), the results are nonetheless quite impressive, as
illustrated in Figure 3. The
upper images (Figure 3, A and
B) illustrate the organization of ß-tubulin at
approximately metaphase during a normal mitosis (A) and at a similar stage in
an (RNAi) embryonic cell lacking -tubulin (B). Note, in
particular, the presence of a complete mitotic apparatus consisting of astral
and both interpolar and chromosomal microtubules in the control embryo and of
only astral microtubules surrounding two, poorly separated centrosomes in the
RNAi-treated embryo. The lower images
(Figure 3, C and D) illustrate
a similar effect of the same RNAi on chromosomal orientation during mitosis,
as inferred from the presence and condensed pattern of GFP:histone. The image
of the control preparation (C) shows two cells in different stages of mitosis:
the nearly invisible nucleus at the upper left is in metaphase, while the
chromosomes in the nucleus at the lower right are just beginning to condense.
In contrast, as illustrated in D, RNA interference with
-tubulin production allows chromosomal condensation but completely eliminates
the formation of a metaphase plate (or, indeed, any subsequent chromosomal
separation during anaphase). With appropriate guidance, introductory students
can follow the normal behavior of spindle and chromosomes in the control
videos and can synthesize the separate images into an accurate composite
picture of mitotic dynamics. More advanced students will likely enjoy
speculating with the authors concerning the action of
-tubulin on microtubule nucleation and subsequent function.
Moreover, the papers by Strome et al.
(2001) and Hannak et
al. (2002) provide
excellent examples of the use of RNAi, and a detailed comparison and analysis
of the two seem suitable for a graduate-level or an advanced undergraduate
seminar course or for a journal club discussion. In such a context, the RNAi
methodology could be examined in appropriate depth, and any difference in
results discussed with reference to differences in the RNAi employed in the
respective studies.
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FIBROBLAST MOTILITY AND SUBSTRATE ADHESIVENESS
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An actively moving cell continually attaches to the substratum along its
leading edge and then detaches at its trailing edge in a fairly regular
manner. If these changes in adhesiveness did not occur, a cell would exhibit
intracellular vesicular movement; additionally, it might seem to oscillate or
become elongated and greatly attenuated. But it wouldn't move! While focusing
on the gymnastics and mechanisms of myosin–actin interactions, students
(and their teachers) often overlook this crucial, anchoring aspect of cell
motility, and I was therefore pleased to see the recent MBC paper on
adhesiveness and fibroblast locomotion by Munevar et al.
(2001).
Despite Durda's recommendation, I almost passed over reviewing the paired
videos accompanying this article, because half of the images are quite complex
and were produced by a process—"traction force
microscopy"—likely to be unfamiliar to many CBE readers
and confusing to most students. Moreover, all video records are appended to
figures in the article without discussion or caption descriptions, leaving the
reader to infer what is happening in the movies from the more piecemeal
information accompanying the still images.
As I replayed the videos, however, I became fascinated by the manner in
which the paired videos depict locomotion: namely, as phase contrast images of
a moving fibroblast alongside "traction force" images of the
substrate distortion created by the moving cell. I was equally intrigued (and,
in my ignorance, uncritically so) by the use of fluorescent beads embedded in
a thin, distortable polyacrylamide substratum (containing Type I collagen) to
gauge the traction force exerted by moving fibroblasts. As a cell moves, it
distorts the substratum in proportion to its locomotion and to the strength of
its adhesions. An algorithm using the relative distance each bead moved from
its resting location estimated the tension of each distortion, and an image of
these distortions was pseudocolored to show a range from low (purple) to high
(red) intensity. Thus, for each of the four locomotive sequences recorded on
video, the viewer sees (on the left) a phase contrast image of a fibroblast
and, simultaneously (on the right), an image of the distorted substratum
beneath the fibroblast. While it is difficult to discern how intracellular
events generate the tensions that distort the substratum, or how locomotion
would appear on a substratum not as distortable as polyacrylamide, merely
watching a fibroblast move in concert with its pseudocolored doppelgänger
is impressive. And as the two still images in
Figure 4 suggest, the movies
seemed sufficiently interesting and pedagogically useful to warrant review and
recommendation to CBE readers.
I also think intermediate-level students will readily appreciate the use of
an artificial peptide containing a binding motif to inhibit competitively
integrin binding to collagen: Its effects are predictable, quite dramatic, and
very well documented in two of the movies. Unfortunately, the possible
relationship of intracellular actin-based events with the traction forces is
only cursorily discussed, and advanced students will likely want to explore
the interplay of actin assembly and treadmilling, vesicular transport and
endo- and exocytosis, and membrane anchoring by reference to
Bray's (2001) monograph on cell
motility or the more recent cell biology text by Pollard and Earnshaw
(2002).
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ANGIOGENESIS (THE DEVELOPMENT OF BLOOD VESSELS)
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Often we are so caught up studying subcellular mechanisms that we lose
sight of the fact that most eukaryotic cells are specialized components of
tissues, organs, and organisms. As such, much cellular behavior is
understandable only in histological or larger contexts. Students, however,
usually come at cellular biology from these more familiar, macroscopic
vantages, and it is always useful when teaching undergraduates to be able to
situate cellular events in their broader settings. I was delighted, therefore,
to read a recent article in the Tube Morphogenesis Series of Trends in
Cell Biology that contains a brief, but interesting video clip of the
growth and differentiation of a blood vessel, during the process called
angiogenesis (Weinstein,
2002). The phenomenon was recorded from the forebrain of a
transparent zebrafish embryo whose vasculature had been made fluorescent
through the expression of a transfected plasmid containing a GFP gene fused
with a promoter specific for vascular endothelium. The URL for the clip is
http://archive.bmn.com/supp/tcb/movie3.html.
View larger version (25K):
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Figure 5. Still image from Weinstein
(2002). Reproduced from
Trends in Cell Biology, 2002, Vol. 12, pp. 439–445, by
copyright permission of Elsevier Science.
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In Figure 5, taken from the
video, an endothelial cell is shown arching its way across the top of the
image and down toward a larger cerebral artery, with which it will form a
communicating vessel (in a process that takes about 8 h). In the video,
numerous filopodial extensions are readily evident extending (and retracting)
both from the leading edge of the growing cell and from cells of the target
artery. Interestingly, when contact is made between endothelial cell and
target artery, filopodial activity ceases. Students with some knowledge of
embryonic development will immediately recognize the similarity of these
"searching"filopods to the pathfinding behavior of archenteron
cells during sea urchin gastrulation or of axonal growth cones during
vertebrate neurogenesis, and they might well speculate about common mechanisms
involving attractive and repulsive, diffusible and substratum cues. These
discussions might lead, for example, to consideration of such plasma membrane
proteins as Ephs, which are tyrosine kinase receptors, and ephrins, which are
lipid-linked proteins that are Eph ligands. Questions might also arise as to
how possible Eph and ephrin involvement in pathfinding could be tested and to
the consequences within target and pathfinding cells of this
ligand–receptor interaction. Students might also want to investigate
what is known of the GFP promoter (for Fli1, a transcription factor)
and why it is also expressed during embryogenesis in neural crest and certain
myeloid derivatives. Moreover, instructors interested in producing fluorescent
embryos for other vascular investigations, or simply for general demonstration
purposes, will be interested in the well-illustrated and well-organized
article and in the description of the more advanced "molecular
epistasis" experiments. All in all, this article is a good read.
Corresponding author. E-mail address:
watters{at}middlebury.edu.
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REFERENCES
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Bray, D. (2001). Cell Movements from Molecules
to Motility, 2nd ed. New York: Garland.Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S.M., Lambert,
G.M., and Galbraith, D.W. (2000). Nuclear dynamics in Arabidopsis
thaliana. Mol. Biol. Cell. 11, 2733–2741. (Available at
http://www.molbiolcell.org/cgi/content/full/11/8/2733)
Hannak, E., Oegema, K., Kirkham, M., Gonczy, P., Habermann, B., and
Hyman, A.A. (2002). The kinetically dominant assembly pathway for centrosomal
asters in Caenorhabditis elegans is -tubulin
dependent. J. Cell Biol. 157, 591–602. (Available at
http://www.jcb.org/cgi/doi/10.1083/jcb200202047)
Munevar, S., Wang, Y., and Dembo, M. (2001). Distinct roles of
frontal and rear cell-substrate adhesions in fibroblast migration. Mol. Biol.
Cell. 12, 3947–3954. (Available at
http://www.molbiolcell.org/cgi/content/full/12/12/3947)
Pollard, T.D., and Earnshaw, W.C. (2002). Cell
Biology. New York: Saunders.
Strome, S., Powers, J., Dunn, M., Reese, K., Malone, C.J., White,
J., Seydoux, G., and Saxton, W. (2001). Spindle dynamics and the role of
g-tubulin in early Caenorhabditis elegans embryos. Mol. Biol. Cell
12, 1751–1764. (Available at
http://www.molbiolcell.org/cgi/content/full/12/6/1751)
Weinstein, B.M. (2002). Vascular cell biology in
vivo: A new piscine paradigm. Trends Cell Biol
12,439
–445.[CrossRef][Medline]
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