Cell Biol Educ 2(3): 137-140 2003
DOI: 10.1187/cbe.03-04-0021
© 2003 American Society for Cell Biology
Video Views and Reviews
Christopher Watters
Department of Biology, Middlebury College, Bicentennial Way, Middlebury,
Vermont 05753
Submitted April 30, 2003;
Accepted May 1, 2003
For this column I return to the Supplemental Material list of the
Journal of Cell Biology (JCB), which I last examined for
Winter 2002 and which is readily accessible by subscribers and nonsubscribers
alike at
http://www.jcb.org/supplemental.
Generally speaking, the research videos published in JCB can be
appreciated as stand-alone records, which is fortunate because the articles
themselves can be accessed on-line only by subscribers. The journal format
provides each video with its own caption, in addition to any contextual
references. A separate descriptive section—designated Online
Supplemental Material (OSM)—is located at the end of each article, which
summarizes the caption detail. Unfortunately, however, the "Supplemental
Material" entry provides no indication whether any given supplement
contains video records (and, if so, how many) or such ancillary material as
still figures, additional tables, or protocol detail. Moreover, the citations
are arranged more or less by year (in reverse chronological order), but they
are not delineated further within any given year, by volume, issue, or first
author.
Thus, if you enjoy browsing in used book stores as I do, you will enjoy
scanning the Supplemental Material listing for interesting material, and as in
any good used book store you are sure to be rewarded with a find or two! I
provide URLs for the videos that accompany each of the articles I review
below, for the more focused reader and viewer.
Again, I invite your comments on these reviews and your suggestions of
other peer-reviewed videos for possible review as educational material
[watters{at}middlebury.edu].
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CALCIUM SIGNALING AND PHAGOCYTOSIS IN NEUTROPHILS
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Neutrophils are white blood cells specialized for scavenging cellular
debris and consuming infectious microorganisms in a process called
phagocytosis. What seems to make neutrophils especially voracious is
the presence of antibodies (or opsonins) coating the surface of foreign cells.
Recently, Dewitt and Hallett
(2002) examined the role of
cytoplasmic calcium in mediating neutrophil phagocytosis of opsonin-coated
particles, using neutrophils that had been loaded with a compound (Fura-2)
that changes its fluorescence properties when it binds with calcium. Their
observations are accompanied by two videos, the first recording changes in
calcium concentrations as an opsonized particle is phagocytosed
(Figure 1) and the second
exhibiting similar changes when a particle is touched to the surface of a
neutrofil and then withdrawn (Figure
2).
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Figure 1. Phagocytosis of an opsonized particle (white sphere) by a neutrophil loaded
with the calcium fluorochrome, Fura-2. Changes in the fluorescence ratio of
Fura excited at two wavelengths is due to increasing cytoplasmic calcium
concentrations and is reflected by changes in pseudocoloring from blue
(ca. 100 nM) to green (ca. 600 nM).
http://www.jcb.org/cgi/content/full/jcb.200206089/DC1/1.
Reproduced from The Journal of Cell Biology, 2002, vol. 159, pp.
181–189, by copyright permission of The Rockefeller University
Press.
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Figure 2. Tryptich images of the time course of the rise in cytoplasmic calcium
concentration (a) in the fluorescent image of a neutrophil (b) being touched
with an opsonized particle tethered to a micropipette (c).
http://www.jcb.org/cgi/content/full/jcb.200206089/DC1/2.
Reproduced from The Journal of Cell Biology, 2002, vol. 159, pp.
181–189, by copyright permission of The Rockefeller University
Press.
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The changes in Fura fluorescence are quite dramatic, as
Figure 1 illustrates,
especially the wave of calcium that spreads throughout the neutrofil following
phagocytosis. The video documents this wave for an introductory audience.
Unfortunately, the initiation of the wave and the early stages of phagocytosis
are not temporally well resolved, however, due to the time-lapse nature of the
process required to obtain reliable Fura emission ratio measurements and to
produce pseudocolored images that accurately reflect cytoplasmic calcium
levels. It is therefore difficult to distinguish the rapid sequence of events
associated with particle binding, and many observant students will likely
question whether a localized increment in calcium precedes (and causes),
follows (and is the result of), or merely accompanies (and possibly is only
correlated with) phagocytosis. To clarify this important issue, the authors
treated neutorphils with the microfilament inhibitor, cytochalasin, which
inhibits phagocytosis and the rapid changes in cell shape that accompany this
process. The drug had no effect on the calcium wave, however, consistent with
calcium triggering the phagocytic response to opsonin binding at the
neutrophil surface (rather than being a secondary response to
phagocytosis).
Opsonized particles trigger phagocytosis through their binding with
ß2 integrin, an integral membrane protein of the neutrophil
plasma membrane. The authors document the nondiffusible nature of
opson–integrin interaction by touching a pipette coated with opsin to a
neutrophil, in an impressive set of three video movies obtained simultaneously
from a single experiment, as illustrated in
Figure 2.
Advanced students may want to explore additional data in the paper
concerning the possible intermediacy of calpain, a calcium-stimulated
protease, in activating these integrins and triggering phagocytosis. They
might also enjoy speculating how the phagocytosis occurred in 3 instances (of
a total of 36) where no changes in calcium could be detected and presumably
integrin binding was absent. Unfortunately, no video records of these
additional experiments are provided.
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ROLE OF CALCIUM SIGNALING IN PLATELET AGGREGATION
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Calcium signaling is ubiquitous, it seems, and one of the earliest
signaling roles documented for this divalent ion is its part in triggering
platelet aggregation and blood clotting. Nesbitt et al.
(2003) have nicely modeled this
process by flowing platelets over a surface covered with fibrillar collagen
(Type I or III) and von Willebrand factor (vWf), both of which promote
clotting. They prelabeled the platelets with calcium-sensitive, fluorescing
dyes (Oregon green linked with BAPTA-1 and Fura red) and then observed the
change in fluorescence with confocal microscopy as the platelets became
tethered to the substratum and/or linked with each other in the flow chamber
to form thromboses. These events are well documented in a strikingly colored
video, as depicted in Figure
3.
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Figure 3. Platelets exhibiting low intracellular levels of calcium appear blue, and
as their calcium levels increase, their color changes from red to yellow to
white. A region—T1—is depicted where aggregation apparently
triggers simultaneous elevations in calcium when platelets make contact. The
platelets in this field are flowing upward at a speed magnified about sixfold
by time-lapse recording; a and b were recorded about 4 sec apart.
http://www.jcb.org/cgi/content/full/jcb.200207119/DC1/1.
Reproduced from The Journal of Cell Biology, 2003, vol. 160, pp.
1151–1161, by copyright permission of The Rockefeller University
Press.
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Introductory students will quickly appreciate the colorful bursts of
intracellular calcium that accompany platelet aggregation (evident as episodes
T1 and T2 in the video), and the more observant among them will also
appreciate the periodic calcium "flashes" (or oscillations) that
appear in individually tethered platelets ("S" in
Figure 3a). The video would
benefit from close scrutiny and repeated viewing, however, because not all
platelet aggregates exhibit calcium flashes during the time course of the
video. Moreover, intermediate students and their teachers may find the video
sequence a good starting point for discussing the source of the calcium in
these flashes and how the calcium levels can rise and fall in an oscillatory
manner. Indeed, they might find the authors' use of "intercellular
calcium communication" (ICC) in the title and elsewhere in the text
somewhat ambiguous: Does ICC refer to a direct flow of calcium among
contacting platelets, as the term would apply to cells in an epithelium, or to
a cascade of calcium elevations that is mediated by surface contacts among
aggregating platelets. The Nesbitt et al. paper describes a series of
experiments that investigate these questions and the intermediacy of the
purinergic ADP receptor. A journal club session might well be devoted to
examining the data in some detail.
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ACTIN POLYMERIZATION, PARTICLE STREAMING AND LAMELLIPODIAL LOCOMOTION
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Some of us have a difficult time appreciating how the assembly of
cytoskeletal subunits can generate a lamellipodial form of cellular motility
or the intracellular movement of such pathogens as Shigella or
Listeria (see Alberts et
al., 2002, p. 1446 ff). In part, this difficulty stems from
our failure to appreciate that this form of locomotion occurs much more slowly
than a naive viewing of time-lapse images would indicate. Also, the process
has no ready mechanistic counterpart in our everyday experience.
I was delighted, therefore, to see the recent investigation by Wiesner
et al. (2003) of an
in vitro model of particle motility using polystyrene microspheres
and the controlled assembly of monomeric actin (G-actin). Briefly, the authors
recorded the behavior of particles coated with the cell-signaling protein,
N-WASP (the activator of the Arp complex) in a defined medium containing ATP,
various cytoplasmic ions, and various purified proteins thought to be
necessary and sufficient for actin polymerization in situ: fibrous
actin (F-Actin), profilin, gelsolin, Arp 2/3 complex, and ADF
(actin-depolymerizing factor). Three phase contrast stills abstracted from the
video are presented in Figure
4, which depict the relative movements of several beads and their
accompanying actin "comet tails."
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Figure 4. The relative movement of 3-, 1-, and 0.5-mm beads in a defined medium
favoring the regulated assembly and disassembly of a branching network of
actin. The beads show a similar velocity, irrespective of their size, and the
actin tail of the large bead in the lower left of each frame continues to grow
away from the bead at the same rate, after the bead itself becomes immobilized
(at around 8 min).
http://www.jcb.org/cgi/content/full/jcb.200207148/DC1/1.
Reproduced from The Journal of Cell Biology, 2003, vol. 160, pp.
387–398, by copyright permission of The Rockefeller University
Press.
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The video is impressive, and it can readily form the basis for a comparison
by introductory students of particle velocity in this model with velocities
exhibited by intracellular parasites in situ and cells moving by
lamellipodia. Moreover, with suitable direction, intermediate students might
engage the other experimental results in this paper, specifically those
resulting from changes in gelsolin concentration or changes in media
viscosity. Do the rates of actin branching and capping, or actin nucleation,
limit the resulting motility? Finally, while these data and their explanation
provide more insight regarding the cytoplasmic movement of intracellular
parasites, it might be instructive for more advanced students to engage in the
extensive model building required to orchestrate the fractal nature of these
actin-based particle movements into the more complex behavior of lamellipodial
locomotion.
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FOOTNOTES
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Monitoring Editor: A. Malcolm Campbell
Corresponding author. E-mail address:
watters{at}middlebury.edu.
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REFERENCES
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Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and
Walter, P. (2002). Molecular Biology of the Cell,
4th ed., New York: Garland.Dewitt, S., and Hallett, M.B. (2002). Cytosolic free
calcium changes and calpain activation are required for
ß-integrin-accelerated phagocytosis by human neutrofils.J. Cell Biol.
159,181
–189.[Abstract/Free Full Text]
Nesbitt, W.S., Giuliano, S., Kulkarni, S., Dopheide, S.M., Harper,
I.S., and Jackson, S.P. (2003). Intercellular calcium
communication regulates platelet aggregation and thrombus growth. J.
Cell Biol. 160,1151
–1161.[Abstract/Free Full Text]
Wiesner, S., Helfer, E., Didry, D., Ducouret, G., Lafuma, F.,
Carlier, M.-F., and Pantaloni, D. (2003). A biomimetic motility
assay provides insight into the mechanism of actin-based motility. J.
Cell Biol. 160,387
–398.[Abstract/Free Full Text]
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