Cell Biol Educ 1(4): 111-114 2002
DOI: 10.1187/cbe.02-07-0018
© 2002 American Society for Cell Biology
Video Views and Reviews
Christopher Watters
Department of Biology, Middlebury College, Middlebury, Vermont
05753
Submitted July 3, 2002;
Revised July 17, 2002;
Accepted July 18, 2002
Some impressive video movies have appeared as supplemental material in
recent issues of the Journal of Cell Biology (JCB), and in
this article I review several of them. In general, the JCB format
provides each video clip with its own caption, in addition to any contextual
references in the article itself, and a separate descriptive
section—designated Online Supplemental Material (OSM)—at the end
of the article summarizes the caption detail. Thus, although the
"supplemental" videos are usually well-integrated research
records, many can be appreciated as stand-alone records, often with minimal
reference to the parent article. The JCB videos are also available
through a separate "Supplemental Material" list, which, unlike the
articles, may be viewed by nonsubscribers. The list may be accessed at
http://www.jcb.org/supplemental.
Unfortunately, this entry route provides no indication whether any given
supplement contains video records (and if so how many) or, instead, such other
ancillary material as still figures, additional tables, or protocol detail.
Thus, the casual browser must engage in a bit of optimal foraging through the
index linkages to find what he or she wants, which requires no great effort
for some unusually fine pickings.
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APOPTOSIS
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Cell death, as a genetically programmed event rather than an accidental
event, fascinates many undergraduates, irrespective of their career goals.
Possibly the most visually striking movie I have seen of apoptosis appeared in
an article by Morgan et al. in the June 19, 2002, issue (10.1083/jcb.
200204039; see Figure 1 for a
still image). The behavior of HeLa cells transfected with a plasmid containing
a chimeric gene for yellow fluorescent protein and the "death
domain" of the receptor for tumor necrosis factor (YFP-TRADD-DD) was
recorded during a 24-h period at a rate of one image every half-hour by using
fluorescence and phase-contrast microscopy. The fluorescent and phase-contrast
images at each time point were subsequently merged, and the published
QuickTime movie contains 36 frames and lasts about 12 s. Thus, the sequence of
portrayed events has been speeded up about 5400 times, and the results are
generally well resolved and contrasted and are cinematically spectacular. The
fluorescent chimera are seen localized in the HeLa nuclei, and, with time and
despite occasional changes in focus, the movie clearly documents their
aggregation as the nuclei of cells entering apoptosis become condensed.
Following such condensation, the cells relatively quickly enter the final
stages of apoptosis and begin to bleb along their peripheries. Curiously,
fluorescence disappears in some cells just before they enter the blebbing
stage, and with the benefit of hindsight I found myself wishing that images
had been collected more frequently following nuclear condensation.
The authors note the absence of nuclear localization and apoptosis in cells
transfected with the YFP gene alone, but no video record is provided of this
control. It is unclear how apoptosis resulting from the truncated form of
TRADD is related to that generated by the membrane-bound form of the receptor,
as the authors note. Nevertheless, the movie is a visually arresting document
and, in its brevity, a great lecture anthem on apoptosis for either an
introductory or a nonscience audience. It may be found at
http://www.jcb.org/cgi/content/full/jcb.200204039/DC1/1.
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MEMBRANE LIPID DIFFUSION
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For color overlays and technical refinement, my vote for the Best Video(s)
of Issue goes to those accompanying an article by Fujiwara et al.
(2002) describing the hop
diffusion of an unsaturated phospholipid analogue (DOPE) within the plane of
the plasma membranes of cultured normal rat kidney (NRK) fibroblasts
(10.1083/jcb.200202050). It has been clear for more than a decade that
integral membrane proteins (IMPs) are restricted in their diffusion within the
plane of a cell membrane by peripheral membrane "corrals" of
cytoskeletal elements or by transient "tethers" to these elements
(see, for example, earlier work from the same laboratory:
Tomishige and Kusumi, 1999;
PMID:10436005). Using high-speed video microscopy and both fluorescent and
gold-tagged DOPE, Fujiwara et al. present five temporally
well-resolved movies indicating that membrane phospholipids are similarly
restricted. Video 1 (see Figure
2A for a still image) depicts the diffusion of single,
fluorescent, and gold-tagged DOPE within an NRK membrane, in two separate
sequences, with 33-ms resolution. Video 2 (see
Figure 2B for a still image)
displays the more rapid behavior evident at higher temporal resolution (25
µs) in two sequences: first, that of a single, gold-tagged DOPE, which is
then repeated in a second sequence with an overlay of the particle
trajectories colored differentially to illustrate the transient restriction of
diffusion to 230-nm corrals or compartments. Video 3 illustrates the control
behavior of DOPE diffusing without restriction within the bilayer of a
phospholipid liposome, again with a colored trajectory overlay. Video 4
presents overlaid images that suggest that phospholipid diffusion is also
restricted by larger, 750-nm compartments, whereas Video 5 illustrates the
effects of latrunculin-A and jasplakinolide, which are, respectively, actin
destabilizing and stabilizing drugs, on the size of the 750-nm compartment and
on the phospholipid residency period. Video clips 1, 3, and 5 may be found at
http://www.jcb.org/cgi/content/full/jcb.200202050/DC1/3,
... /DC1/5, and ... /DC1/7, respectively.
The Fujiwara et al. video records are so clear and well described
in their captions that, with some background preparation, they could be used
for introductory courses in cell biology during an experimental session in a
computer laboratory. Alternatively, along with the article, they provide the
basis for an extended discussion of "membrane concepts: facts and
artifacts" in an advanced journal seminar. Using the records, students
could manually record residency times and estimate compartment sizes from the
color overlay sequences in Video clips 2
(http://www.jcb.org/cgi/content/full/jcb.200202050/DC1/4)
and 4
(http://www.jcb.org/cgi/content/full/jcb.200202050/DC1/6).
They could then compare these data with similar measurements of phospholipid
diffusion in a pure phospholipid bilayer (from Video 3). Following this
initial analysis, the possible effects of various cytoskeletal destabilizing
and stabilizing drugs could be discussed and hypotheses formulated. The latter
could then be tested by using the data in Video 5. (Alternatively, the effects
of similar drugs might be presented and discussed and then students might be
asked to determine from the data in Video 5 the functions of the two drugs
used for the experiment.) Observant students examining these records might
note in Video 4 the apparent stability of a larger compartment as the particle
being tracked first diffuses out of and then back into the same membrane
region. Moreover, a more thoughtful student might wonder whether the
"unrestricted movement" described for the singly colored
trajectory presented in the control video (Video 3) might also appear more
restricted and somewhat corralled if multiple colors had been used judiciously
in constructing the overlay. In considering these and other questions,
advanced students would necessarily want to consult the clearly written and
well-documented article and especially the statistical analyses. These are
excellent videos and a well-written article for undergraduate use at the
intermediate and advanced levels and for graduate students.
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MITOSIS AND EARLY DEVELOPMENT
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Looking for videos of mitosis or of early developmental events following
fertilization? Nice, clear, and well-resolved video movies of paired
differential interference contrast (DIC) and fluorescent images of dividing
Caenorhabditis elegans eggs were published by Hannak et al.
(2002; 10:1083/jcb.200202047),
accompanying their investigation of the importance of 
-tubulin in
centrosome formation. Chimeric proteins containing green fluorescent protein
(GFP) linked to -tubulin and to histone were expressed in eggs and
sperm and used to follow the behavior, respectively, of centrosomes and
chromosomes following fertilization. The paired images were all captured at
either 6- or 8-s intervals, and because the movies are shown at 10 frames per
second, the events depicted are speeded up 60- to 80-fold. Video 1
(http://www.jcb.org/cgi/content/full/jcb.200202047/DC1/1;
see Figure 3 for paired still
images) depicts early nematode development during the first 13 min or so. All
the crucial events are clearly evident in one or the other of the paired
movies: pronuclear migration and syngamy (fusion), establishment of the
mitotic poles, chromatin condensation into chromosomes, mitosis, and
cytokinesis and the formation of a two-celled embryo. Because -tubulin
is localized in centrosomes and centrosomes are contributed exclusively by
sperm in animal development, only one of the two pronuclei in the fertilized
egg is accompanied by a pair of brightly GFP-labeled centrosomes. Watching the
behavior of this organelle and the orderly progression of mitosis,
introductory students may wonder whether the centrosome exercises a
choreographic role in mitosis. They may also question how the chromosomes
become aligned during metaphase and separate during anaphase. To address these
questions, they can view Video 3
(http://www.jcb.org/cgi/content/full/jcb.200202047/DC1/3;
see Figure 4 for a still
image), which shows an identical sequence in an embryo expressing
GFP-
-tubulin and well-labeled astral and spindle microtubules, as well
as unorganized centrosomal tubulin. Observant students will also note the
periodic appearance of GFP-histone-labeled polar bodies at the egg periphery
in the various videos. Some quibbles, however: the Video 3 clip I was able to
view lacked a paired DIC movie. Also, because GFP was used to label both
proteins, instructors will need to identify for introductory students the
centrosome and chromatin as the loci of -tubulin and histone,
respectively. In sum, Videos 1 and 3 nicely illustrate the basic events of
mitosis and early animal development and are eminently suitable for an
introductory audience.
More advanced undergraduates and their instructors may want to explore the
function of -tubulin in greater depth. If so, they will want to grapple
with the figures and data of the Hannak et al. article and their use
of RNAi as an experimental tool. Video 2
(http://www.jcb.org/cgi/content/full/jcb.200202047/DC1/2)
provides an excellent entry for such an exploration because it depicts the
striking effect of an RNAi (against gip-1) that prevents the expression of a
protein (CeGrip-1), which, in turn, is crucial for the centrosomal
localization of -tubulin. Video 4
(http://www.jcb.org/cgi/content/full/jcb.200202047/DC1/4)
would also prove useful in this regard because it illustrates the effect of an
RNAi for tbg-1 (the C. elegans -tubulin gene) on the
organization of -tubulin during early development.
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ERRATUM
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Finally, I want to revise some comments in my last review
(Watters, 2002) about the
arrangement of videos in the Molecular Biology of the Cell
(MBC) paper by Sharpless and Harris
(2002; 10.1091/mbc01.07.0356).
Certain of the criticisms I voiced then concerning the confusing manner in
which video clips were associated with unrelated figures have since been
addressed. The clips are now linked with the appropriate figures and, in
certain instances, renumbered as well. Movie 1 is now linked with Figure 5 and
illustrates the localization of an actin-scaffolding protein called SEPA in a
cytokinetic ring. Movie 3, which illustrates the location of SEPA at the tip
of a growing hypha, has inexplicably been renumbered "2," but its
identification has not been changed in either the caption or the text. Four
other movies—5 and 7 and 6 and 8—have been linked appropriately
with Figures 7A and 7B, respectively, and they show the effects of
cytochalasin A on SEPA dynamics in the contractile ring and the control data
(Figure 7A) and in the hyphal tip also with control data (Figure 7B). Clip 2
seems to have been deleted and Clip 4 can now be located, without caption,
through the link (MBC Video) that appears with the article title in
the Table of Contents. These changes greatly improve the ease with which the
videos can be accessed and, in turn, understood in context.
Again, I invite your comments on these reviews and your suggestions of
other peer-reviewed videos for possible review as educational material.
Corresponding author. E-mail address:
watters{at}middlebury.edu.
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REFERENCES
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Fujiwara, T., Ritchie, K., Murakoshi, H., Jacobson, K., and Kusumi,
A. (2000). Phospholipids undergo hop diffusion in
compartmentalized cell membrane. J. Cell Biol.
157(6), 1071-1082/10.1083
.
http://www.jcb.org/cgi/doi/10.1083/jcb.200202050[CrossRef]Hannak, E., Oegema, K., Kirkham, M., Gönczy, 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(4), 591-602.
http://www.jcb.org/cgi/doi/10.1083/jcb.200202047[Abstract/Free Full Text]
Morgan, M., Thorburn, J., Pandolfi, P.P., and Thorburn, A.
(2002). Nuclear and cytoplasmic shuttling of TRADD induces
apoptosis via different mechanisms. J. Cell Biol.
157(6), 975-984.
http://www.jcb.org/cgi/doi/10.1083/jcb.200204039[Abstract/Free Full Text]
Sharpless, K.E., and Harris, S.D. (2002). Functional
characterization and localization of the Aspergillus nidulans formin
SEPA. Mol. Biol. Cell 13,469
-479/10.1091/mbc.01-07-0356
.[Abstract/Free Full Text]
Tomishige, M., and Kusumi, A. (1999).
Compartmentalization of the Erythrocyte Plasma Membrane by the membrane
skeleton: intercompartmental hop diffusion of Band 3. Mol. Biol.
Cell 10,2475
-2479.[Free Full Text]
Watters, C. (2002). Video views and reviews.Cell Biology Education
1(1-2),9
-10.
TOP
APOPTOSIS
MEMBRANE LIPID DIFFUSION
