Download Detection of Single Microtubules in Living Cells

Detection of Single Microtubules in Living Cells:
Particle Transport Can Occur
in Both Directions along the Same Microtubule
I. H. HAYDEN and R. D. ALLEN
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
ABSTRACT Video-enhanced contrast/differential interference-contrast microscopy was used
in conjunction with whole mount electron microscopy to study particle transport along linear
elements in fibroblasts. Keratocytes from the corneal stroma of Rana pipiens were grown on
gold indicator grids and examined with video microscopy. Video records were taken of the
linear elements and associated particle transport until lysis and/or fixation of the cells was
completed. The preparations were then processed for whole mount electron microscopy.
By combining these two methods, we demonstrated that linear elements detected in the
living cell could be identified as single microtubules, and that filaments as small as 10 nm
could be detected in lysed and fixed cells. The visibility of different cytoplasmic structures
changed after lysis with many more cellular components becoming visible. Microtubules
became more difficult to detect after lysis while bundles of microfilaments became more
prominent. All particle translocations were observed to take place along linear elements
composed of one or more microtubules. Furthermore, particles were observed to translocate
in one or both directions on the same microtubule.
Cytoplasmic transport is a process of ubiquitous occurrence
in eucaryotic cells and of fundamental importance to cell
function. One manifestation of cytoplasmic transport, the
translocation of optically detectable organdies and vesicles
(collectively called particles), has been extensively studied (20,
27). Microtubules have long been believed to play an important role in particle transport in such diverse cells as chromatophores (4, 22), neurons (15), tissue culture cells (13), and
in numerous protists, e.g., the foraminefer, Allogromia (34,
35). These cells possess impressive arrays of cytoplasmic microtubules, the presence and orientation of which have been
shown by electron (4, 5, 13, 34, 36) and immunofluorescence
(9) microscopy to be consistent with the hypothesis that
particle motion occurs along them. Furthermore, microtubule
depolymerizing drugs have been shown to inhibit particle
motion in all these cells (13, 17, 25, 33).
Until recently, the major limitation of particle transport
studies had been the inability to detect and identify cytoplasmic microtubules in the living cell. Video-enhanced microscopy has now made it possible to observe microtubules
in vitro in a slurry (1) or in a living cell (18). In a previous
study, using Allen video-enhanced contrast/differential interTHE JOURNAL OF CELL BIOLOGY • VOLUME 99 NOVEMBER 1984 1785-1793
© The Rockefeller University Press • 0021-9525/84/11/1785/09 $1.00
ference-contrast (AVEC-DIC) t microscopy in conjunction
with immunofluorescence microscopy, we demonstrated directly that particles move in keratocytes along linear elements
composed of microtubules (18). The particles displayed a
number of interesting behaviors including motion in either
direction along the same microtubular linear element. However, this study was limited in that the number of microtubules or other cytoskeletal elements making up a linear element could not be determined. Thus, it was possible that a
particle could move in one direction along one microtubule
in a linear element composed of microtubules and on another
microtubule in the reverse direction.
In the present study, we observed particles moving along
linear elements in keratocytes preceding fixation and preparation for whole mount electron microscopy. We show that
by comparing the video record to the corresponding whole
mount electron micrographs, one can determine the numbers
and kinds of cytoskeletal elements that make up a light
~Abbreviation used in this paper: AVEC-DIC microscopy, Allen
video-enhanced contrast/differential interference-contrast microscopy.
1 785
microscopic linear element. We demonstrate that A V E C - D I C
can detect (a) single microtubules in living cells, a n d (b)
filaments as small as 10 n m in cells that have been lysed a n d
fixed. Furthermore, particle translocations can occur in one
or both directions along the same microtubule.
MATERIALS
AND
METHODS
Corneasfrom Rana pipiens were removedand the epitheliumand endothelium
were stripped away. The remaining stroma that contained the keratoeyteswas
placed between two number 0 coverslipsin petri dishes and grown at 24"C in
a 5% CO2 atmosphere, conditions under which the keratoeytesmigrated from
the stroma onto the coverslips.The cells were maintained in Eagle's minimal
essential medium supplemented to a final concentration of 10% fetal calf
serum, 0.I mM nonessential amino acids, 2.0 mM L-glutamine, and 1%
antibiotic-antimycoticsolution (100 U/ml penicillin, 100 #g/ml streptomycin,
0.5 ~,g/mlFungizone). Alt media components were supplied by Gibco Laboratories, Grand Island, NY. Gold London Finder grids (Ladd Research Industries, Inc., Burlington,VT) were Formvar- and carbon-coated,and sterilizedby
exposure to ultravioletlight. The keratocyteswere collectedfrom the coverslips
with 0.25% trypsin made up in Ca+* and Mg+* free Earle's balanced salt
solution (Gibco Laboratories) and seeded onto the prepared electron microscope grids.
In preparation for light microscopy,a coverslipwas fastenedto a slideholder
with vaseline-lanolin-paraffin(1:1:1), and the grids with attached cells were
placed in a drop of medium on the coverslip.A chamber was formed by laying
a clean coverslipon top of strips of Scotch tape that ran the length of two sides
of the bottom coverslip. These two sides were sealed with vaseline-lanolinparaffin (1:1:1). The preparations were examined with AVEC-DIC (1, 2)
microscopy using an inverted Zeiss Axiomat (Carl Zeiss, Inc., Oberkochen,
Wuerttenberg,West Germany) equipped with a 100 x/1.3 NA planapochromatic objective.The bias re'tardationwas set to h/9. The image was projected
either onto a C-10004)1 binary computer-compatiblechalnicon video camera
driven by a model 1440 Poly!aroeessorFrame Memory (Hamamatsu Systems
Inc., Waltham, MA), or onto a Chalmicon video camera driven by a newer
device, the C 1966 AVEC/VIM Photonic Microscope System Image Processor
(Photonic Microscopy,Inc., Chicago, IL).
Once the video records of the cells were taken, the preparations were
processed for whole mount electron microscopy.The cells were either lysed
and then fixed, lysed and fixed simultaneously,or fixed without lysis. Lysis
and/or fixationmedium was added to one open sideof the coverslippreparation
and drawn through by placing filter paper strips at the other open side. The
microtubulestabilizingbuffer used for lysisand/or fixationconsistedof 25 mM
PIPES (pH 6.9), 1.0 mM EGTA, 1% polyethyleneglycol 6000, and 0.5 mM
MgCI. This buffer contained 0.5% Triton X-100 and 1.0% glutaraldehydefor
lysis and fixation, respectively.Video records were taken of lysis and fixation.
After fixation for 30 min in glutaraldehyde, the preparations were removed
from the light microscope, washed with microtubule stabilizingbuffer, and
postfixed for 1 rain with 1.0% osmium in 100 mM Cacodylatebuffer (pH 6.0,
4"C). The preparations were then washed with distilled water, dehydrated
through a graded ethyl alcohol series, and critical point dried. At the 70%
alcohol dehydration stage, the preparations were stained for 1 rain with 0.5%
uranyl acetate in 70% alcohol.
The cellswere examinedwith a JEOL 100 CX electronmicroscopeoperating
at 100 kV, and the whole mount electron mierographs correspondingto the
video records were obtained.
RESULTS
All structures visible in the light micrographs of living cells
were referred to in general terms as either linear elements or
particles. After lysis a n d / o r fixation, the linear elements were
subsequently identified from the corresponding whole m o u n t
electron micrographs o n the basis of size a n d organization.
According to convention, the 6 - n m filaments were referred to
as microfilaments (MFs), a n d the 10-nm filaments as intermediate filaments (IFs). The microtubules (MTs) were identified both o n the basis o f their size 0 8 - 2 0 n m ) a n d their
characteristic pattern in the cell. (See figure legends for a more
detailed description of the results.)
In the flattened peripheral regions of these cells, various
linear elements could be seen (Fig. 1 a). In preparation for
whole m o u n t electron microscopy, the cells were lysed a n d
fixed while they r e m a i n e d in focus in the light microscope.
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THE IOURNAL OF CELL BIOLOGY- VOLUME 99, 1984
The living cell in Fig. I a is shown again in Fig. I b after lysis
a n d fixation in a microtubule stabilizing solution that contained 0.5% T r i t o n X-100 a n d 1.0% glutaraldehyde. The
replacement of the soluble c o m p o n e n t s of the cell with the
lysis m e d i u m led to a n increase in contrast for all linear
elements visible in the living cell, a n d caused m a n y other
cellular c o m p o n e n t s to b e c o m e visible for the first time. In
fact, one linear element became evident only after lysis. O n e
result of this increase in contrast was that some of the linear
elements pointed out in the living cell (Fig. 1 a) were more
difficult to detect. This is because the increased n u m b e r of
visible cellular c o m p o n e n t s after lysis tends to obscure, partially or totally, some linear elements.
Whole m o u n t electron miroscopy was used to identify the
linear elements seen in the living a n d lysed cells. Fig. 2 a is a
low magnification electron micrograph of the same region
shown in the light microscope in Fig. 1. The linear element
that b e c a m e apparent only u p o n lysis was a n intermediate
filament (Fig. 2, a a n d c), a n d two linear elements that became
more evident u p o n lysis corresponded to bundles of micro fila m e n t s (Fig. 2 a). O n e linear element, visible in the living cell
b u t totally obscured in the lysed cell, was composed of two
microtubules (Fig. 2 a). However, the two curvilinear elements
pointed out in the live a n d lysed cell (Fig. 1, a a n d b) are
single microtubules (Fig. 2, a a n d b). Thus, single microtubules are detected in the living cell by AVEC-DIC microscopy,
while a 10-nm filament could be detected only in the lysed
cell.
W e observed particles translocating along linear elements
composed of microtubules in living cells. In Fig. 3, a a n d b,
a particle is shown m o v i n g d o w n a p r o m i n e n t linear element.
This particular cell was then lysed for 30 s with 0.5% T r i t o n
X-100 in a microtubule stabilizing solution before fixation
with 1.0% glutaraldehyde. Once again, u p o n lysis m a n y more
cellular c o m p o n e n t s became apparent, thus obscuring the
linear element the particle m o v e d along (Fig. 3 c). Also, some
of the less evident linear elements in the living cell became
the most visually p r o m i n e n t elements in the lysed cell. The
corresponding whole m o u n t electron micrograph d e m o n strates these latter elements to be bundles of microfilaments
(Fig. 3, d a n d f ) , while the linear element along which particles
translocated in the living cell is composed of two microtubules
(Fig. 3, d a n d e).
Particles were also observed translocating along linear elements composed of single microtubules. In Fig. 4, a a n d b, a
particle is shown m o v i n g along one of three parallel linear
elements. A total of four particles translocated in the same
direction along the lower two linear elements. These linear
elements were identified as single microtubules in the corresponding whole m o u n t electron micrographs (Fig. 4, d a n d
e). I n this preparation, the cell was fixed without lysis in a
microtubule stabilizing solution that contained 1.0% glutaraldehyde. In this case, the microtubules only became obscured
by other cellular c o m p o n e n t s in a region of vesiculation;
otherwise, the microtubules appeared to retain their relationships to the rest o f the cell to the extent that they r e m a i n e d
the most p r o m i n e n t linear elements in the region.
Finally, in another preparation, a particle was observed to
translocate in both directions along a linear element (Fig. 5 a e). This preparation was lysed a n d fixed in a microtubule
stabilizing buffer that contained 0.5% T r i t o n X-100 a n d 1.0%
glutaraldehyde. The corresponding whole m o u n t electron micrograph shows that this linear element consisted of a single
microtubule (Fig. 6). This was one of two preparations in
FIGURE 1 AVEC-DIC images demonstrating linear elements in a living cell grown on a gold London Finder grid. (a) The living
cell with arrowheads and arrows pointing out various linear elements. (b) The same region after lysis and fixation. After lysis there
was an increase in contrast that caused many cellular components to become evident for the first time. In fact, a linear element
became visible (small arrows) that was not seen in the living cell. Also, the two linear elements pointed out in the living cell by
the large arrows gained in contrast to the extent that they became more evident after lysis. On the other hand, the increased
number of visible cellular components made it more difficult to detect some of the linear elements. For example, the curvilinear
elements (black arrowheads) and one linear element (white arrowheads) seen in the living cell became partially and totally
obscured, respectively, by the many cellular components made visible after lysis. Bar, 2.0 v,m. x 5,000.
which transport in both directions along a single microtubule
was documented. Thus, particles can not only translocate
along single microtubules, but they can also do so in either
direction.
DISCUSSION
Particle Transport
We have used video microscopy in conjunction with whole
mount electron microscopy to demonstrate that linear elements detected in the living cell can subsequently be identified
as single microtubules. Furthermore we detected and identified filaments as small as 10 nm in diameter in lysed cells.
The main significance of these observations is that some of
the functions of cytoplasmic microtubules can now be studied
directly. Using this approach to study the role ofmicrotubules
in particle transport, we have demonstrated that particles can
move in both directions on the same microtubule.
In a previous study with keratocytes (18), we showed using
AVEC-DIC microscopy in conjunction with immunofluorescence microscopy, that particles are transported exclusively
along linear elements composed of microtubules. Particles
were also observed to undergo a number of interesting behaviors including motion in either direction on the same microtubular linear element. However, it was not possible to determine the number of microtubules making up a linear element.
Particle motion in both directions along the same micro-
tubular linear element is of interest when one considers the
possibility that (a) the structural polarity of microtubules is
translated into a functional polarity, and (b) dynein, an ATPase mechanoenzyme, (14) is the mechanochemical transducer responsible for particle translocation.
Does the structural polarity of microtubules manifest itself
as a functional polarity? Some models for mitosis assume that
the direction of polarity of the microtubules plays an important part in chromosome motion (23, 24). Our work showing
particle motion in both directions along the same microtubule
indicates, at least for particle transport in keratocytes, that the
structural polarity of the microtubules does not dictate the
direction of motion. Also, recent studies with nerve axons (7,
11, 19), melanophores of the angel fish (10), and axopodia of
Echinosphaerium (10) have shown most of the microtubules
to be of the same polarity though bidirectional particle motion
occurs in these cells.
Whether dynein could be the mechanochemical transducer
responsible for particle translocation also hinges on questions
of polarity. Dynein is an asymmetric molecule that has been
used to determine the structural polarity of microtubules (16).
Ciliary dynein is attached to the A-subfiber by its A-end (32),
and it interacts with the B-subfiber via its B-end which contains the ATPase activity. In a simple microtubule-dyneinparticle hypothesis, the dynein could presumably be attached
to either the microtubule or particle by its A-end. Under the
proper conditions the dynein would then be able to generate
HAYOEN AND ALLEN
Particle Transport along Single Microtobules
I 787
FIGURE 2 Whole mount electron micrographs, corresponding to the region shown in Fig. 1, demonstrating that linear elements
detected in the living cell can be identified as single microtubules. In a the linear element that became evident only after lysis is
a 10-nm, filament (arrowheads, IF), while the two linear elements that became more evident after lysis are bundles of 6-nm
filaments (arrowheads, MFs). The two curvilinear elements seen in the live and lysed cell are single microtubules (arrowheads,
MT), and the linear element seen in the live cell but obscured in the lysed cell is composed of two microtubules (arrowheads, 2
MTs). A higher magnification of the microtubule defined by the small arrowheads (MT) in a is shown in b. A higher magnification
of the intermediate filament defined by the small arrowheads (IF) in a is shown in c. Bars: (a) 2.0 /~m; (b and c) 0.2 pro. (a)
x 11,000; (b and c) x 55,000.
movement by interacting with either the particle or microtubule via its B-end.
The evidence suggesting a possible dynein involvement in
particle transport includes work showing cross-bridges between synaptic vesicles and microtubules (21), and between
mitochondria and microtubules (26, 29, 30). The nature of
this cross-bridging material has not been determined, nor has
any ATPase activity been associated with it. Experiments with
permeabilized fibroblasts (12), neuroblastoma cells (31), and
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THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984
melanophores (8) have shown that vanadate inhibits particle
motion at concentrations that also inhibit dynein ATPase
activity. However, it also has been shown that vanadate does
not inhibit particle motion at concentrations that inhibit
ciliary beating in the same oviduct cell (6). Erythro-9-[3-(2hydroxymomyl)]adenine, another dynein ATPase inhibitor,
inhibits particle motion in intact erythrophores (3) and melanophores (8).
Our experiments raise the question of whether dynein is
FIGURE 3 Light micrographs and whole mount electron micrographs demonstrating particle motion along a linear element
composed of microtubules. A particle (large arrows) is moving down a prominent linear element (small arrows} in the live cell in
a and b. The lysed and fixed cell is shown in c. Here again, many cellular components became visible upon lysis; this tends to
obscure the linear element the particles moved upon. A portion of this linear element is still visible (small arrow), while some
linear elements (arrowheads in b and c) became much more prominent after lysis. The corresponding electron micrograph of this
cell region is shown in d. The linear element that the particle moved along is composed of two microtubules (large and small
arrows, MTs). A higher magnification micrograph of the region of these two microtubules defined by the small arrows in d is
shown in e. The microtubules in this region are still detectable in the light microscope after lysis (c) because the area immediately
surrounding them contains very little material. The linear elements that became more prominent after lysis are composed of
microfilaments (d and f, large and small arrowheads, MFs). A region of one of these bundles of filaments defined by the small
arrowheads in d is shown in/'. Bars: (c and d) 2.0/zm; (e and f) 0.2 #m. (c) × 4,300; (d) x 9,300; (e and f) x 60,000.
HAYDENAND ALLEN Particle Transport along Single Microtobules
1789
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THE JOURNAL OF CELL BIOLOGY - VOLUME 99, 1984
FIGURE 5 Light micrographs of a cell in which motion of a particle (white arrowheads, P) in either direction along the same
linear element (white arrows) was observed. The particle is moving down the linear element (a), pausing (b), and returning up the
same linear element (c-e). The cell is shown in e just before lysis and fixation. The particle is seen just before reaching the cell
mass (black arrowhead) present in both light and electron micrographs. The black arrow at the bottom points out a filopodium
that is also present in both light and electron micrographs. The cell is shown after lysis and fixation in f. The linear element that
the particle moved along (as well as the other linear elements) became much harder to detect after lysis. A section of the linear
element that the particle moved along is still visible at the arrow. Bar, 2.0 #m. x 12,000.
FIGURE 4 Light and whole mount electron micrographs demonstrating particle motion along a single microtubule. (a and b) A
particle (arrows) is shown moving along the lowermost of the three linear elements labeled by the black arrowheads. The same
region is shown in c after fixation without lysis. The particle shown in (a and b) had moved out of the field of view just before
fixation. Note that there was some vesiculation at the lower left (white arrowheads). The letter B (c and d) indicates holes in the
plastic that are useful as landmarks when comparing the light micrographs to the whole mount electron micrographs. The whole
mount electron micrograph corresponding to the light micrographs (a-c) is shown in d. Note that the three linear elements seen
in the live and fixed cell are single microtubules (black arrowheads). A higher magnification of the region of the lowermost
microtubule defined by the small arrowheads is shown in e. Other non-microtubular elements closely surround it and may run
parallel to it for a short distance. Some arrangements of cytoskeletal components may have the same width as a microtubule,
and so they appear to be microtubules at high magnification. In e, -1 cm to the left of the second arrowhead, the microtubule
appears to split in two and continue to the left as two microtubules. By using the lower magnification micrograph (d) to follow
the course of the microtubule, one sees that there is only one microtubule present and that another cytoskeletal component,
only a few tenths of a micrometer in length, happens to intersect the microtubule just to the left of the second arrowhead (e).
This demonstrates the importance of using both the microtubule width and pattern in the cell to identify microtubules. In the
living cell, two particles moved along the bottommost microtubule and two particles moved along the microtubule immediately
above it. In this preparation, the microtubules remained as very prominent elements after fixation without lysis. They only
became obscured in the region of vesiculation where an increase in contrast caused other cellular components to become visible.
Bars: (c and d) 2.0/zm; (e) 0.2 #m. (c) x 3,800; (d) x 20,000; (e) x 90,000.
HAYDENAND ALLEN Particle Transportalong Single Microtobules
1791
FIcueE 6 (a) Whole mount electron
micrograph, corresponding to the cell
region in Fig. 5, demonstrates that the
linear element that the particle moved
along in either direction is a single microtubule (white arrows). The letters ae correspond to the sequence of particle
positions seen in the living cell with the
white arrowheads showing the direction
of motion. The particle reversed directions at b and was observed at e just
before reaching the cell mass shown
here by the star (corresponding to the
area shown by the black arrowhead in
Fig. 5e). The filopodium noted in Fig. 5e
is shown at the lower right (black arrow).
The region of the cell in which the particle was last observed (Fig. 5e) is shown
at higher magnification in b. The star
labels the cell mass seen in Fig. 5 and
Fig. 6a. The arrows point out the microtubule that the particle moved along.
Note that other cytoskeletal components closely surround the microtubule.
Bars: (a) 1.0/~m; (b) 0.1 tLm. (a) x 22,000;
(b) x loo,0oo.
still a candidate for the mechanochemical transducer. If the
structural polarity of the dynein-microtubule interaction also
dictates a functional polarity, then our studies would appear
to rule out a dynein involvement in particle transport. However, there is some confusion on the point of dynein-microtubule polarity. In one study with microtubules assembled in
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THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984
vitro (16), dynein was shown to cross-bridge microtubules
only when they were of the same polarity; in another study
dynein was shown to cross-bridge ciliary microtubule doublets
of the same and opposite polarity (37). The doublets were still
capable of sliding relative to one another upon addition of
ATP. Resolution of this dynein-microtubule polarity question
is necessary to evaluate the significance of particle transport
in both directions along the same microtubule.
It is possible that a microtubule-dynein interaction may not
be involved in particle transport. It may be that microtubules
only serve to define the paths particles may follow through
the cytoplasmic matrix and that some other mechanochemical transducer is responsible for particle transport. We can
only say from our light microscopy studies that particles
always travel in optical contact with microtubules (i.e., within
200 nm). However, the corresponding electron micrographs
show that the microtubules are surrounded by as yet unidentiffed elements which are much closer than 200 nm to the
microtubules (Figs. 4 e and 6). It is possible that these elements
are also involved in particle transport.
Clues to the identity of any mechanoenzyme that may be
involved in particle transport remain elusive. We plan to
approach this problem by preserving the particles so that the
particle-microtubule interactions can be determined. We hope
that by correlating known particle behaviors at the time of
fixation to the corresponding whole mount electron micrographs, we will gain further insight into the problem of particle
transport.
Cytoplasmic Matrix
Some interesting observations of the present study came
from comparing the living and lysed cells to one another and
to the whole mount electron micrographs. Although the microtubules were the most prominent linear dements in the
living cell, linear elements composed of microfilaments were
more prominent in the lysed cell. All the linear elements
gained in contrast because the lysis removed the Triton Xsoluble components of the cell, thereby increasing the refractive index gradient between linear elements and background.
This increase in contrast caused many cellular elements to
become evident for the first time, thus obscuring the microtubular linear elements. However, this does not explain why
bundles of microfilaments, which were less obvious in the
living cell, became much more evident than the microtubules
in the lysed cell. After looking at the whole mount electron
micrographs, we predicted that the bundles of microfilaments
would be more prominent than the microtubules in the DIC
image, as indeed they are in the lysed cell. One possible
explanation for the low contrast of the microfilament bundles
in the living cells is that they are surrounded by a higher
concentration of Triton X-soluble material, such as G-actin,
than the microtubules. It has been shown that Triton X
removes 60-70% of the total cellular protein from tissue
culture cells, with actin as the major soluble component (28).
Removal of G-actin and any Triton X-soluble F-actin thus
would result in the great increase in contrast of the microfilament bundles we observe in the lysed cell.
In the future, we plan to expand this work by comparing
the optical properties, such as phase gradients (DIC microscopy) and birefringence (polarized light microscopy), of various components of the cytoplasmic matrix in both the riving
and/or lysed and fixed cell to the corresponding whole mount
micrographs. This type of study may help interpret the behaviors that the components of the cytoplasmic matrix exhibit
when their functions are studied with video microscopy.
The authors are grateful to Roger Sloboda and George Ruben for
their helpful discussion and criticism of the work. We particularly
thank Kenneth Orndorff for his photographic work.
This research was supported by a grant from the National Institute
of Neurological and Communicative Disorders and Stroke (NS
19962) to R. D. Allen.
Received for publication 8 May 1984, and in revised form 26 July
1984.
REFERENCES
1. Allen, R. D., and N. S. Allen. 1983. Video-enhanced microscopy with a computer frame
memory. J. Microsc. (Oxf.). 129:3-17.
2. Allen, R. D., N. S. Allen, and J. L. Travis. 1981. Video-enhanced constrast, differential
constrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubulerelated motility in the teticulopodial network of Allogromia laticollaris. Cell Motility.
1:291-302.
3. Bcckerle, M. C., and IC R. Porter. 1982. lnhibitors ofdyncin activity block intracelhilar
transport in erythropbores. Nature (Lond.). 295:701-703.
4. Bikle, D., L G. Tilney, and K. R. Porter. 1966. Microtubules and pigment migration
in the melanophores of Fundulus heteroclitus L. Protoplusma. 61:322-345.
5. Breuer, A. C., C. N. Christian, M. Henkart, and P. G. Nelson. 1975. Computer analysis
of organelle translocations in primary neuronal cultures and continuous cell fines. J.
Cell Biol. 65:562-576.
6. Buckley, I. K. 1983. Ciliary but not saltatory movements are inhibited by vanadate
microinjected into living cultured cells. Cell Motility. 3:167-185.
7. Burton, P. R., and J. L Paige. 1981. Polarity ofaxoplasmic microtubules in the olfactory
nerve of the frog. Proc. Natl. Acad. ScL USA. 78:3269-3273.
8. Clark, T. C., and J. L. Rosenhaum. 1982. Pigment particle translocation in dctergentpermeab'flized melanophores of Fundulus heterc~litus. Proc. Natl. Acad. Sci. USA.
79:4655--4659.
9. Couchman, J. R., and D. A. Rees. 1982. Organelle-cytoskeleton relationships in fibroblasts: mitochondria, Golgi apparatus and endoplasmic reticulum in phases of movement and growth. Eur. J. Cell Biol. 27:47-54.
10. Euteneuer, U.,and J. R. McIntosh, 1981. Polarity of some motility related microtubules.
Proc. Natl. Acad. Sci. USA. 78:372-376.
I 1. Filliatreau, G., and L. Di Giamberardina. 1981. Microtubules polarity in myelinated
axons as studied after decoration with tubulin. Biol. Cell. 42:69-72.
12. Forman, D. S. 1982. Vanadate inhibits saltatory organelle movement in a pcrmeab'dized
cell model. Exp. Cell Res. 141:139-147.
13. Freed, L J., and M. M. Lebowitz. 1970. The association of a class ofsultatory movements
with microtubules in cultured cells. J. Cell Biol. 45:334-354.
14. Gibbous, I. R., and A. J. Rowe. 1965. Dynein: a protein with ATPase activity from
(~lia. Science(Wash. DC). 149:424--426.
15. Grafstein, B., and D. S. Forman. 1980. Intracellular transport in neurons. Physiol. Rev.
60:1167-1283.
16. Haimo, L. T., B. R. Telzer, and J. L. Rosenbaum. 1979. Dynein binds to and crossbridges
cy~plasmic microtubules. Proc. Natl. Acad. Sci. USA. 76:5759-5763.
17. Hammond, G. R., and R. S. Smith. 1977. Inhibition of the rapid movement of optically
detectable axonal particles by colchicine and vinblastine. Brain Bes. 128:227-242.
18. Hayden, J. H., R. D. Goldman, and R. D. Allen. 1983. Cytoplasmic transport in
keratocyte: direct visualization of particle translocation along microtubules. Cell Motility. 3:1-19.
19. Heidemann, S. R., J, M. Landers, and M. A. Hamborg. 1981. Polarity of axonal
microtubules. J. Cell Biol. 91:661-665.
20. Hyams, J. S., and H. Stebbings. 1979. Microtubule associated cytoplasmic transport. In
Mierotubules. K. Roberts and J. S. Hyams, editors. Academic Press, Inc., London. 487530.
21. Jartfors, U., and D. S. Smith. 1969. Association between synaptic vesicles and neurotubules. Nature (Lond. ). 224:710-711.
22. Luby-Phelps, K. J., and M. Schliwa. 1982. Pigment transport in chromatophores: a
model system for intraceUular transport. In Axoplasmic Transport. D. G. Weiss, editor.
Springer-Verlag, Berfin Heidelberg. 15-26.
23. Margolis, R., L. Wilson, and B. Kiefer. 1978. Mitotic mechanism based on intrinsic
microtubule behavior. Nature (Lond.). 272:450-452.
24, Mcln~sh, J. R., P. K. Helpler, and D. G. Van Wie. 1969. Model for mitosis. Nature
(Lond.). 224:659-663.
25. Murphy, D. B., and L. G. Tilney. 1974. The role of microtubules in the movement of
pigment granules in telenst melanophores. J. Cell Biol. 61:757-779.
26, Raine, C. S., B. Getti, and M. L. Shelauski. 1971. On the association between microtubules and mitochondria within axons. Brain Res. 34:389-393.
27. Rebhun, L. I. 1972. Polarized intracellular transport: saltatory movements and cytoplasmic streaming. Int. Rev. CytoL 32:92-137.
28. Schliwa, M. S., J. van Blerkom, and K. R. Porter. 1981. Stabilization of the cytoplasmic
ground substance in detcrgent-opaned ceils and a structural and a biochemical analysis
of its composition. Proc. Natl. Acad. Sci. USA. 73:4329-4333.
29. Smith, D. S., U. Jarifors, and B. F. Cameron. 1975. Morphological evidence for the
participation of microtubules in axonal transport. Ann. N. E Acad. Sci. 253:472-506.
30. Smith, D. S., U. Jarlfors, and M. L. Cager. 1977. Structural ~ b r i d g e s between
microtubules and mitocbondria in central axons of an insect (Periplaneta americana).
J. Cell Sci. 27:235-272.
31. Stearns, M. E., and D. P. Bnggs. 198 I. Studies of axonal transport in digitonin permeated
neuroblastoma cells. J. Cell Biol. 91(2, Pt. 2):420a. (Abstr.)
32. Telzer, B. R., and L. T. Haimo. 1981. Decoration of spindle microtubules with dynein:
evidence for uniform polarity. J. Cell Biol. 89:373-378.
33. Travis, J. L. 1981. Motility of the reticulopodial network of Allogromia laticollaris.
Ph.D. thesis. Dartmouth College, Hanover, NH. 168 pp.
34. Travis, J. L, and R. D. Allen. 1981. Studies on the motility of the foraminifera. I.
URrastructure of the reticulopodial network of Allogromia latticollaris (Arnold). J. Cell
Biol. 90:211-221.
35. Travis, J. L., J. F. X. Kenealy, and R. D. Allen. 1983. Studies on the motility of the
foraminifera. IL The dynamic mierotubular cytoskelcton of the reticulopodial network
of Allogromia laticollaris. J. Cell Biol. 97:1668-1676.
36. Wang, E., and R. D. Goldman. 1978. Functions of cytoplasmic fibers in intracellular
movements in BHK-21 cells. J. Cell Biol. 79:708-726.
37. Warner, F. D., and D. R. Mitchell. 1981. Polarity of dynein-microtubule interaction in
vitro: cross-bridging between parallel and antiparallel microtubules. J. Cell Biol. 89:3544.
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